Regulation Studies of the Human Recombination Activating

Genes (RAGI and RAG-2)

Laurent Karl Verkoczy

A thesis submined in conformity with the requirements

for the degree of Master of Science

Graduate Department of Immunology

University of Toronto

O Copyright by Laurent Karl Verkoczy, 1995

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ABSTRACT

Regdation Studies of the Human Recombination Activating Genes (RAG-1 and RAG-2)

D e p z of Master of Science, 1995. Laurent Karl Verkoczy

Graduate Depamnent of Immunology University of Toronto

It has been suggested that a signal transduced through the Bceii antigen receptor complex (BCR) may be required for regulating the expression of the recombination activating genes. To study this possibility, we utilized a unique set of ce11 vanants fiom the human mature B ce11 line OC1 LYS wtiich Vary in expression of both the BCR and of the RAG-1 and RAG-2 genes. Two forms of stimulation were employed to generate a signal: either a soluble F(ab)'2 anti-p fragment or the combination of PMA

and ionomycin. Northem analysis demonstrated that cross-linking the BCRs of the RAG~OBCR+ parental ce11 line or its R A G ~ ~ B C R + variants with soluble anti-p results in the upregulation of RAG-1

mRNAs. This effect was found to be dose-dependent, time-dependent, specific, and reversible. Additionally, when secondary messengers of the BCR signaling pathway were directly activated with PMA and ionomycin, increases in RAG-1 vanscripts were detected in both BCR+ and BCR- clones. The finding that RAG expression can be upregulated by BCR ligation may be relevant in the context of its potential role in situations where BCR+ B cells may undergo secondary rearrangements for the purpose of "editing" their sIg receptors.

To better understand the molecular mechanisms that may be involved in this inducible upregulation as well as in the constitutively expressed increases in RAG gene expression in R A G ~ ~ OC1 LY8 variants (as compared to the parental R A G ~ ~ clone), we detennined to what extent various levels of reguiation were contributing to these effects. In both situations, it was found that increased transcription as well as message stabilization appeared to be involved. Furthemore, both the constitutive differences in RAG expression between OC1 LYS vanants and the signaling-mediated increases in RAG expression assessed in the parental BCR+ clone required de novo protein synthesis. Thus, the OC1 LY8 ce11 line and its variants may provide a valuable system for the identification and characterization of molecular factor(s) CO-expressed with RAG, including not only novel recombinase components CO-regulated with the RAG genes but also de novo synthesized elements involved in the

replation of RAG-1 and RAG-2 mRNAs.

1 am grateful to my supervisor, Dr. Neil Berinstein, for his support and encouragement, and to

the mernbers of my supervisory committee, Dr. Michael Julius and Dr. Gill Wu, for their sound criticism

and advice. 1 would also like to thank Niclas Stiemholm, Beata Kuzniar, and Li-Juan Duan, al1 of whom

have provided me with a F a t deal of valuable advice in the laboratory. 1 am indebted to my wonderful

fiancée, Victoria Chiao, and her son, Alexander Chiao, for their patience, understanding, and faith in me

through some difficult times. Recognition must also be given to my parents and brothers in Regina for

the love and friendship they have afforded me throughout the years. Finally, I would like to dedicate

this thesis to the single most important person responsible for cultivating rny interest in science, my

father- Dr. Bela Verkoczy.

PUBLICATION

An abridged version of this thesis has been accepted for publication in the Journal of

Irnrnunology and will appear M a y 15, 1995 as an original paper with the title If Upregulation of RAG

expression by signal transduction through the surface imrnunoglobulin receptorff by L.K.

Verkoczy, N.B J. Stiemholm, and N.L. Berinstein.

iii

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION

1. V(D)J tecombination

1. Cis-acting elements mediating V@)J recombination

2. Trans-acting factors mediating V(D)J recombination: the eukaryotic V(D)J recombinase

A. Lymphoid-specific recombinase components

i. Roducts of the RAG-1 and RAG-2 genes

Suucmral properties of RAG-1 and RAG-2 Function of RAG-1 and RAG-2 Rch-1: a protein that interacts with the RAG-1 protein The RAGdeficient background in lymphocyte development

ii. TdT (Terminal deoxynucleotidyl transferase)

B. Non-1 yrnphoid speci fic DS BR recombinase components

i . The murine scid (severe combined immunodeficiency) locus

ii. The xrs-6, XR- 1, and V-3 defects

iii. Molecular characterization of the xrs-6 and scid DSBR mutations

C. Other potential recombinase components defined biochemically

i. Factors that bind RSS

ii. Factors involved in VmIJ cuttina and lination

II. Repulation of RAG-I and RAG-2 expression 22

1. Pattern of RAG-1 and RAG-2 mRNA expression: lineage and developmental restrictions 22

2. Regulation of RAG-1 and RAG-2 expression by signal transduction

A. The BCR signaling cascade and its role in the regulation of B lymphocyte

gene expression

B. The regulation of RAG expression by signals uansduced through antigen

receptors

C. Regulation of RAG by signai transduction: implications for Iymphoid

progenitors

D. A mode1 for the signaiing-mediated regulation of RAG expression

in B lymphocyte development

E. Molecular aspects of signai transduction-mediated regulation of

RAG-1 and RAG-2

3. Transcriptional regulation of RAG-1 and RAG-2 expression

A. Mammalian gene expression: the imrnunoglobulin mode1

B. Inferences about RAG transcriptional convoI

4. Post-transcriptional regulation of the RAG genes

A. Conuol of message stability: implications for RAG mRNAs

B. Studies of RAG-2 protein regulation

5. Project Objectives

CHAPTER 2: MATERIALS AND METHODS

1. Ce11 variants and tissue culture

II. Antibodies and pharmacological agents used in signaling stud

III. Surface irnmuno fluorescence analysis

IV. Measurement of inuacellular ~ a * + and proüferation

V. Protein analysis

VI. RNA isolation and Northern blotting

VIL RT-PCR analysis

VIII. Nuclear run-on transcription assay

CHAPTER 3: RESULTS

ies

1. DifferentiaI RAG expression in clonally related variants undergoing secondary

Ig h gene rearrangernents

II. Upreeulation of RAG expression in OC1 LYS variants upon sIg cross-linking

III. Both increased uanscnptional and message stabilization contribute to the

upregulation of RAG mRNAs

IV. Both constitutive and inducible upregulation of RAG expression is

de novo protein synthesis dependent

CHAPTER 4: DISCUSSION

1. lnsighrs into the molecular replation of RAG-1 and RAG-2 expression in OC1 LY8

II. Upregulation of RAG expression in OCI LY8 through BCR ligation: implications

for receptor editing

III. The differential replation of RAG- 1 and M G - 2 by signai transduction in BCR + subsets 72

IV. The differential expression of RAG in OC1 LYS: a strategy for identifjmg novel genes 76

that CO-express with RAG-1 and RAG-2

REFERENCES 80

vii

LIST OF FIGURES

FIGURE 1.1

FIGURE 1.2 FIGURE 1.3 FIGURE 1.4

FIGURE 1.5

FIGURE 1.6

FIGURE 3.1

FIGURE 3.2

FIGURE 3.3

FIGURE 3.4

FIGURE 3.5

FIGURE 3.6

FIGURE 3.7

FIGURE 3.8

FIGURE 3.9

Structural features of the RAG locus and pmteins. The RAG defect in thymic development. The RAG defect in B lymphocyte development. The effect of mutations in various DSBR genes on V(D)J recombination. Mode1 of signaling-mediated regulation at various stages of B lymphocyte development . Hierarchy of putative transcription factors involved in regulation of RAG

transcription Comparison of RAG mRNA expression levels between OC1 LYS-C3P parental line and clonally related variants A84P and C3-A 1 IN. Representative immunofluorescence profiles of OC1 LYS-C3P, AS-6P, and C3-A 1 IN clones with respect to surface immunoglobulin phenotype. Schematic summary of clona1 relationship in OC1 LYS ceil culture system between RAG~o parental clone OC1 LY8C3P and R A G ~ variants C3-A 1 LN and AS-6P.

Time-dependence, dose-dependence, specificity, and reversibility of increases in OC1 LY8 RAG-1 transcripts as a result of ami-p cross-linking. Increased RAG- 1 expression in response to direct activation of BCR signaling- associated secondary messeneers with PMA and ionomycin. Effects of anti-p cross-linking on various signaling events in OC1 LY8-C3P and C3-A 1 IN. Quantitative nuclear run-on analysis of differences in RAG-1 and RAG- 2 transcription rates between unstimulated and anti-p cross-linked OC1 LYS-C3P, C3-A 1 IN, and AS-6P clones. Actinomycin D studies of RAG-1 mRNA half-lives in OC1 LYS variants. Cycloheximide studies of new protein synthesis dependence in OC1 LYS variants.

viii

LIST OF TABLES

TABLE 1.1 Putative factors involved in the regulation RAG- 1 and RAG-2 gene regulation 42

TABLE 3.1 Summary of effects o f signaling through the sIg receptor and the associated 57

PKC pathway on RAG-1 expression

TABLE 3.2 Summary of effects by various stimuli on proliferation, surface HLA-DR 59

expression, and inua-cellular Ca2+ levels

LIST OF ABBREVIATIONS

A-MuLV

BCR

bp cDNA D DNA DN DP DSBR EDTA

EMSA FACS Id

k J

kb

kD K

A

MAb MHC

mRNA N

PBS PCR

Abelson murine leu kemia virus

B ce11 antigen receptor complex base pair complementary DNA diversity gene segment deoxyribonucleic acid CD4 'CD8- double negative CD4+CD8 + double positive double strand break repair ethylenediamine tetra-acetic acid electrophoeresis mobility shi ft assay fluorescence activated ce11 soning idiotype immunoglobulin joining gene segment kilobase kilodaltons irnmunoglobulin kappa light chain imrnunoglobulin lambda iight chain monoclonal anti body major histocornpatibility complex messenger ribonucleic acid non-germline encoded nucleotides phosphate buffered saline polymerase chain reaction

PKC PMA PTK RAG RNA

RSS scid SDS SDS-PAGE sIg SSC SL TCR

TdT v

protein kinase C phorbol- 12-myristate- 13-acetate protein tyrosine kinase recombination activating gene ribonucleic acid recombination signal sequence severe combined immunodeficiency sodium dodecyl sulphate sodium dodecyl sulphate polyacrylamide gel electrophoresis surface immunoglobuli n sodium citrate/saline bu ffer surrogate Iight chain T ceU receptor terminai deoxynucleotidyl uansferase variable gene se-ent

CHAPTER 1: INTRODUCTION

The venebrate immune system is capable of specificaliy recognizing and responding to a large number of antigens. The interaction with an antigen is mediated by the immunoglobulin (Ig) and T ceU receptor (TCR) molecules expressed by T and B lymphocytes, respectively. Ig and TCR polypeptides consist of separate structural domains: a variable domain which forms the antigen binding site, plus one or more constant region domains which mediate various effector functions. The exons encoding the variable domains are assembled during lymphocyte development kom variable (V), diversity (D), and joining (J ) lymphocyte antigen receptor gene segments by a site-specific recombination process known as V(D)J recornbination. This process generates much of the required diversity in the binding specificities of Igs and TCRs.

1. Cis-acting elements mediating V(D) J recombinotion

The process of V(D)J recornbination (also known as TCR and Ig gene tearrangement), is the only site-specific recombination process in mammaiian ceus, and as such, involves several unique cis- and trans-actins elements. Cis-acting elements, both necessary and sufficient to mediate V(D)J recombination, have been identified and are referred to as Recombination Signal Sequences (RSS) [l-41. These sequences flank the recombining coding segment and consist of a palindromic heptamer (proximal to the coding segment) and an " A T rich nonamer. The two are separated by a spacer region of either 12 (+/-1) or 23 (+/-2) basepain. RSS are critical b a h in directing the recombination machinery to the proper site of recombination and regulating which p n e segments may be recombineci. Two gene segments with spacer regions of the same leiigth cannot recombine, but rather a gene segment with a 12 bp spacer region will only recombine with a gene segment that has a 23 bp spacer region (" 12/23" nile)

[1,51-

Experiments using extrachromasornal recombination subsuates fûrther elucidated the roles of the individual components of the RSS ([6]; reviewed in [73). These substrates are designed such that they can be introduced into rnammalian cells, recovered and transfected into bactena. The subsuates contain a drug-resistance gene which is only expressed after RSS-mediated recornbination has taken place in mammalian cells. Following recovery of the plasmid DNA from the marnrnaiian cells and the transfection of the bacteria, the number of h g resistant bacteria will offer a measure of recombination frequency. These substrates can also be recovered fiom the bacteria and analyzed in detail. Through the

alignment of available nucleotide sequences it has been possible to establish consensus RSS. Mutagenesis experiments showed that substitutions in the fmt three nucleotides of the heptamer, and to some extent the founh one, significantly decreased recombination efficiency. Although substitutions in nucleotides six and seven of the nonarner aiso decreased recombination efficiency, overall, the heptamer appeared to be more important than the nonamer [4,6]. In no instances did the efficiency of

recombination exceed that observed with consensus RSS. It was also demonstrated that the size of the

spacer re~ion , rather than the actual nucleotide sequence, is crucial.

2. Trans-acting factors mediating V@)J recombination: the eukaryotic V@)J recombinase system

As RSS are evolutionarily conserved across various venebrate antigen receptor loci they are therefore thought to be the target of a simiiarly evolutionarily conserved trans-acting recombinase system [8-101. This machinery can be assigned functions that can be subdivided into multiple distinct steps including the recognition and synapsis of RSS, introduction of double-break strands between the RSS, loss andlor addition of nucleotides at the coding junctions, deletion or inversion of DNA separating the codinrp sequences to be joined, and polymenzation-ligation activities to complete the joining process. The search for a single "recombinase" perfoming al1 these functions has been replaced by an appreciation that V(D)J joining appears to be carried out by a collection of enzymatic factors perfoming separate roles in the reaction. These factors, collectively referred to as the recombinase system, can be divided into lymphoid-specific components and non-lymphoid-specific components, some of the latter also having roles in double-strand break repair (DSBR).

A. Lymphoid-speciflc recombinase components

i. Products of the RAG-1 and RAG-2 genes

Using a molecular genetic suategy, two genes were identified as lymphoid-specific components (or at Ieast regulatory factors) of the V(D)J recombinase systern [11,12]. A iibroblastoid ce11 line with an integrated recombination substrate was selected for its ability to rearrange (thereby activating a dmg- resistance gene) upon transfection of genomic DNA. With recombination as the assay and through several rounds of uansfections ending in a genomic walk, a gene was isolated in 1989 by David Baltimore and his CO-workers which could potentiate authentic V(D)J joining when introduced by transfection into other non-iymphoid cells. By virtue of its specific rearranging function, the gene was termed "Recombination Activating Gene- 1" or RAG-1. One year later, a second gene- " Recornbination Activating gene-2" or RAG-2 was identified which, when transfected into non-lymphoid cells in combination with RAG-1, could increase recombination frequencies by more than 1000 fold. The essential and specific role that RAG-1 and RAG-2 play in V(D)J recombination is convincingly demonstrated not only by the ability of these genes to confer recombinase activity in non-lymphoid ceiis but also by the fact that mice which have had either their RAG-1 or RAG-2 genes dismpted are incapable of undergoing V(D)J recombination without any other apparent effect [13,14].

Structural propemses of RAG-I and RAG-2

The RAG genes, located on human chromosome 11 [15] and on mouse chromosome 2 [16], have an unusual structural organization for eukaryotic genes, and more closely resemble viral or fungai loci [8]. In both mouse and human, they lie in opposite transcriptional orientation, have an intergenic sequence within 10 kb of each other and have their entire coding regions contained within single exons. Funhermore RAG-1 and RAG-2 have no sequence homology to each other and are therefore not likely to have arisen from a gene duplication event (Figure 1.1a). Genomic probes for human RAG-1 and RAG-2 detect predominant mRNA species of - 6.6 and - 2.2 kb. However, by Northern blot anaiysis, several larger isoforms of RAG-2 mRNAs have aiso been detected in rnouse, human, and rabbit. For exarnple, human and murine RAG-2 vanscripts as large as 5 kb have been observed (N. Stiemholm and D. Schatz, persona1 communication). This suggests either the presence of multiple 5' upstream untranslateci RAG-2 exons or cross-hybridization to a gene with homology to RAG-2. In the human and murine RAG-1 genes, the intron between the coding exon and the first 5' untranslated exon in both species is - 5 kb (A. Zamn and D. Schatz, persona1 communication). Funhermore, in human RAG-2,

the intron between the coding exon and the first 5' untranslated exon is - 3.5 kb. Because transcriptional start sites for the human or murine RAG genes have not yet been determined, it is also possible that RAG-1 has more than one 5' untranslated exon. However, because only a single RAG-1 mRNA species is observed in Northern blots, it is unlikely that more than one 5' untranslated exon is present, unless these are exuemely small, and therefore produce multiple indistinguishable mRNA species.

At the protein level, the open reading frarne of murine RAG-1 and RAG-2 encode predicted translation products of 1041 and 527 amino acids (a.a.), respectively. (or - 119 and - 59 kD, respectively). Various recent structure-function mutational studies of the murine RAG proteins have provided information regarding the relative importance of various regions within the proteins important for V(D)J recombination in the context of extrachromasomal assays (Figure 1 b) [17-2 11. When deletions andior substitutions were inuoduced in various regions of the RAG-1 and RAG-2 proteins the only site absolutely necessary for V(D)J recombination appears to be a C-terminal active-site topoisornerase-like motif in RAG-1 (a.a 994-998). Truncations further C-texminal to this motif actually enhanced V(D)J recombination, suggesting that the RAG-1 C-terminus may have a negative regdatory function in recombination. Other regions, including a RAG-1 zinc finger-like cysteine-histidine domain (shared by various other putative transcription factors including the glucocorticoid receptor), a RAG-1 putative nuclear localization site, a RAG-2 acidic region representing a putative transcriptional domain, and the two pnnciple phosphorylation sites of RAG-2, senne 356 and threonine 490, were al1 found to be dis pensable in the sense that recombination was reduced, but not abolished. Interestingly, based on

a compilation of ail mutations performed in RAG-1 and RAG-2, the intemal 6056 core of RAG- 1 (a.a 38 1- 1009) can be defined as the region necessary for wild type exuachromasomal V@)J recombination but that the entire N-terminal region, from a.a 1-383 is dispensable (Figure 1. lb). In RAG-2, a.a 1-382 of the protein sequence appear to be important, but the last 145 a.a are dispensable.

At both the nucleotide and amino acid (a.a.) level, RAG-1 and RAG-2 are highly conserved across vertebrate species. For example, human RAG-1 is 95% homologous to mouse RAG-1 at the nucleotide level and the 1043 a.a. human RAG-1 protein is approximately 90% homologous with the 1040 a.a. mouse product. Moreover, transfection of 3T3 fibroblasts with the combination of rnurine RAG- 1 and human RAG-2 expression constructs confers comparable levels of recombinase activity as transfection with munne RAG-1 and RAG-2 consmcts [Il]. The RAG cDNA and protein sequences of chicken 1221, rabbit [23], and Xenopus 1241 have been determined and also exhibit high homology to both murine RAG genes and their products. Interestingly, the intemal region of RAG-1 shown to be essential for recombination is also the most highly conserved region in the RAG-1 sequence [19], whereas the 75% of the RAG-2 protein essential for recombination has less homology than the

dispensable 25% [20].

Funetion of RAG-l and MG-2

Two models have been proposed to account for how the RAG products confer recombinase activity: the RAG products could participate directly in the recombination reaction, or alternatively, they may be indirect components i.e. transcription factors in a pathway leading to the activation of other direct recombinase components. A third "hybridw model is that, although both required for V(D)J recombination, one of the RAG proteins may operate as a transcription factor, the other as a recombinase component. Several circumstantial arguments have been made in favor of the former model: 1) as with

RSS, both RAG genes have been highly conserved through vertebrate evolution, suggesting a direct cis- trans interaction between RSS and RAG. 2) V(D)J recombinase activity is the only lymphoid-specific property found in fibroblasts expressing RAG-1 and RAG-2. RAG-transfected fibroblasts have the morphology and growth requirements of typical fibroblasts, do not express B220, TdT, and fail to express their endogenous antigen receptor loci. If the RAG genes were regulatory factors, they might be expected to have more pleiotropic effects, including confemng other lymphoid-specific activities to non- lymphoid cells. 3) The recent structure-function studies show the dispensabiiity of putative transcription factor domains in the RAG proteins (the RAG-1 N-terminal zinc finger domain and RAG-2 C-terminal acidic region), but the necessity of the RAG- 1 topoisornerase-lik domain. However, the residue in this motif (tyrosine 998) analogous to the active-site tyrosine in type II topoisornerases was found not to be necessary for V(D)J recombination. Furthermore, one must use caution in interpreting the relative

importance of such regions in the context of extrachromasomal recombination substrates employed for

assaying recombination, since regions that rnay be important for endogenous V(D)J rearrangements would not be detected.

Although the precise roks of RAG-1 and RAG-2 are not known, we are beginning to understand at which steps in the V(D)J reaction these genes are involved in. Studies of RAG-1 and RAG-2 knockout mice demonstrate that ail antigen receptor loci are retained in gerrniine configuration [13,14].

Other experiments have assayed for the cutting of the V(D)J recombination signal sequences and have demonstrated this function to be dependent on RAG expression [25]. Furthermore, studies with an inducible RAG expression system have demonstrated the Ievels of V(D)J recombination are directly correlated wi th the levels of RAG expression. Taken together, these experiments provide strong evidence that the expression of both gene products are required for the initiation of the V(D)J reaction. RAG-1 and RAG-2, however, according to one study, rnay not always be sufficient for the initiation of V(D)J recombination [26]. Nevertheless, assuming that the recombinase components are directly involved in the V(D)J reaction, they could function in recombination initiation either by recognizing, synapsing, or cutting RSS (or any of a combination of these possibilities). One interesting recent study has suggested that in addition to initiation of recombination, RAG-1 rnay also be involved in a later step of V(D)J recombination: the resolution of coding ends [27]. In this study, two RAG-1 cDNAs appear to be expressed at differing levels when expressed in a reporter construct, the difference being attributed to a 45 base pair difference in the 5' untranslated region of these cDNAs. Funhermore, the high RAG expressing cDNA is able to resolve both signai and coding joints in extrachromasomal assays, whereas the low RAG-expressing cDNA (lacking the 45 base-pair region) has severely compromised coding joint resolution. This data therefore suggests the potential importance of the various 5' unuanslated regions of the RAG-1 and RAG-2 genes in regulating V(D)J recombination. Funhermore, it implies that the autonomous RAG activities of initiating recombination and resolving coding ends rnay be dependent on differential expression of these two genes. In this context, if RAG-1 functions as a transcription factor, the low expression of RAG-1 rnay be important in activating a gene involved in initiation of V(D)J recombination, whereas its high expression could activate a gene involved in coding joint resolution. Alternatively, if RAG-1 functions as a recombinase enzyme, its low higher expression rnay be required to provide sufficient catalytic activity for coding joint formation. In the section where I review the scid mutation, one possible specific role of the RAG-1 protein in coding joint resolution in relation to the

scid locus will be discussed.

RAG-I

RAG-I

dispensable ( 1 -383)

dispensable (388527)

Figure 1.1. Structure of the RAG locus and proteins A: The human rtcombination activating gene locus. Exons are teprexntcd as boxes, hmns as liaes, open iicading h m s as solid boxes, and other unpansiatcü regions as unfillecl boxes. Dashed arrows indicate the direction of traascription. RAG-2 may have mort than one 5' untranslated txon based on rnu1tipIe species of RAG-2 mRNAs that are dtltctbd in Northan blots (represented with question marks). The immn sizes betwan the coding cxon and the first 5' umransiatcd cxon for both RAG-land RAG-2 have ban dettnni#d (A.Zarrin, prsonaI coms'nunication) B: Mutationai analysis of ngions of miaiae RAG-1 and RAG-2 pmtcios critical for V@)J riccombinstion in the context of extrachromasomal subsuate assays. Data basad on compilation of deletional and substitution mutants h several independent -dies [17-2 11.

Rch-I : a protein thut interacts with the RAG-1 protein

It is likely that other proteins complex with the RAG products for proper recombinase activity. Using a yeast two-hybnd assay system, it has recently been found that while the RAG-I and RAG-2

proteins do not appear to associate with each other, a region N-terminal to the zinc finger domain in the RAG-1 protein specifically associates with four repeats of a non-lymphoid specific 58 k D nuclear product (Figure 1. lb, [28,29]). This protein represenu the human homologue of the yeast protein SRPl (supressor of a temperature-sensitive RNA polymerase 1 mutation) and the gene encoding this product has been termed Rch-1 (RAG Cohort-1). The region of RAG-1 that associates with Rch-1 is not requ ired for recombination [18-2 1 1. Furthermore, using extrachromasomal recombination assays, RAG-I in association with deletional mutant forms of this factor only slightly decrease recombinase activity as compared to associations with wild type Rch-1. However, because yeast SRPl has binds to the nuclear envelope and the Rch-1 binding region in RAG-1 contains a putative nuclear localization signal [18], it is possible that this interaction helps to localize RAG-1 (and possibly other rewmbinase components) to the nuclear envelope. It has been speculated that this localization may also ailow the proper s ynapsi ng of endogenous antigen receptor segments, which unli ke t hose in extrachromasomal plasmids, can be spaced apan at large distances. Consistent with the localization of RAG-1 is the predominant subcellulat localization pattern of this protein in the nuclear membrane as detennined by indirect immunofluorescence in early thyrnocytes (1183 and E. Spanopoulou and D. Baltimore, unpublished results). The importance of such a nuclear localization effect is observable in other cellular processes such as replication and transcription [30, 311. Interestingly, the RAG-2 product, which appears not to interact with RAG-1, appears to be found predominantly in the nuc~eoplasmic rather than the nuclear membrane protein fraction, suggesting it may serve an different function than RAG-1 in V(D)J recombination (C. Thompson, unpublished results).

The UA G -deficient background in lymphocyte development

In RAG- 1 or RAG-2 deficient mice, the arrest in lymphocyte development, although very early, is not as complete as the arrest in V(D)J rearrangement. Instead, RAG-deficient mice have a developrnental phenotype that is strikingly sirnilar to mice with targeted deletions in the A5, the JH locus,

or the IgH transmembrane region, al1 of which have impaired, but not complete blocks in V(D)J reamangements [32-341. Furthermore, the introduction of functionally assembleci antigen-receptor genes or other potentially relevant genes into the RAGdeficient background can circumvent the V(D)J rearrangement block and ailow diflerentiation to proceed [35]. Taken together, these observations suggesi that the RAG-1 and RAG-2 genes themselves do not play a signifiant regulatory role in early

1 ymp hoc yte development . Rather, i t appears t hat their speci fic ability to confer V(D)J rearrangement subsequently allows differentiation to proceed. However, a recent report argues that, independent of V(D)J recombination, the RAG genes may need to be properly regulated for the completion of lymphocyte development (at least T ce11 development). Specifically, mice in which RAG- 1 and RAG-2 were overexpressed while under the control of an lck promoter rapidly developed various abnormalities in the late stages of T ce11 development including lymphadenopathy, splenomegaly, and lymphocytic perivascular infiltrates [3q.

In the context of T ceii differentiation, RAGdeficient mice are specifically blocked at the Thy- 1 + , IL-2R+, C W -CD8 - double negative @N) stage in the thymus (Figure 1 -2) [ 13,141. Inuoduction of a Ig heav y chain transgene promotes thymocyte differentiation to a Thy- 1 +, IL-2R', CD4 +CDS+ double positive (DP) stage and increases thymic ce11 numbers to normal levels (100 fold) 137,381. introduction of a functional TCR a uansgene done does not promote any thyrnic differentiation or changes in thymic cellularity . However, introduction of both TCR $ and a uansgenes leads to the further differentiation of DP thymocytes to CD4+ or CDS+ single positive (SP) T cells in the thymus and the periphery. Additionally, the protein tyrosine kinase p5dck has been shown to be not only involved in the TCR signal transduction pathway and allelic exclusion of the TCR f.3 chah [39], but also in the progression of

nonnal T ce11 development, as demonstrated by p5dck-deficient mice in which 4 0 % of nonnal numbers of DP thymocytes and a complete absence of SP T cells. In this context, expression of an activated form of the kk gene can restore the number of DP thymocytes to normal levels [40].

Some interesting information has emerged from the RAGdeficient phenotype about early thymic signaling structures. Although DP thyrnocyte ce11 lines are able to express CD3 components oniy on the ce11 surface without any TCR polypeptides [4 11, DN thymocytes were previously thought to express CD3 components only in the cytoplasm. However, anti-CD3s treatment of RAG-1 or RAG-2 -/- mice (which do not express $ chain) induce the generation of DP cells and the downregulation of CD25

expression [42,43]. Thus, the CD3 complex is likely to be expressed on the DN thymocytes independently of TCR chains at a level below conventional detection methods. Transgenic TCR 0- chains are expressed on the surface of DP thymocytes in the RAG-deficient background and are likely disulfide linked to the uansmembrane protein pre-T receptor a (pTa), also known as gp33 [44]. pTa has previously been detected by surface biotinylation in TCRa-deficient mice, in association with CD3

components (the pre-T cell complex), and is capable of transducing physiological signals 1451. Therefore, while the CD3 complex in DN thymocytes appears to generate physiological signaling to the DP stage, the pre-T complex may generate signals that could lead to the cessation of f.3 chain rearrangements and the onset of TCR a chain rearrangements.

CD4 10 O - 10 +

CD8 - - - Io + c-kit + + 10 10 .. CD44 + + - O - CD25 - + + - -

Figure 1.2. The effect d the RAG dbruption m the progression d murine thymic development.

The RAG block in differentiation and subsequent restoration with TCR polypeptide tiansgenes is shown

with respect to the diffaentiation of TCR af3 lineage thyrnocytes in a normal balbk mouse-

Classification of thymic subpopulations is based on the scheme proposed in [lm, 1091.

With respect to B ce11 development, RAGdeficient mice are blocked at Randy Hardy's fraction C (the large, B220+HSA+BP-l+CW3+ pro-B ce11 stage in the bone marrow) (Figure 1.3 and[l3,14]). Analogous to the rescue of thymocyte maturation by the introduction of a TCR f3 transgene, introduction of a murine 6 + p transgene into the RAG-2 background or a human p transgene into the RAG-1

background promotes B ce11 development to the ffaction D stage (smali, B220+, CD43' pre-B cells in the bone marrow)[46,47]. This observation is also consistent with the previous finding that the expression of a p heavy chain is required for the pro-B to pre-B ce11 transition [34,48]. In conuast, the introduction of a functionally rearranged Ig light chah gene (u u A ) does not have any effect on B ce11

differentiation. Introduction of both rearranged heavy and iight chain genes leads to the generation of

surface Ig+ B cells that migrate to the periphery and populate secondary lymphoid organs in relatively normal numbers 146,471.

In mouse and human pre-B ce11 lines, 1 chains have previously been found to associate covalently to the A 5 polypeptide and non-covalently to the VpreB polypeptide, the two polypeptides together representing the surrogate light chain (SL) (reviewed in 1491). The p/SL complex also associates with the Ig-Mg-p heterodimers and has been demonstrated to elevate ~ a 2 + in response to cross-linking with either anti-p or anti-SL mAbs (but unlike the mature antigen receptor complex of sIg+

B cells, is incapable of initiating PIP2 hydrolysis), suggesting its potential ability to transduce developmental signals from the extemal environment [50-521. hterestingly, while SL chains have been found only on late stage pre-B ce11 lines [53,54j, recently, the surrogate iight chains in RAG-deficient mice have been found to be expressed detectably on fraction A pro-B cells in the absence of p heavy chain expression 1551. Moreover, pre-B ceils generated by complementing RAGdeficient mice with heavy chain transgenes express p heavy chains in the cytoplasm, but not detectably on their surface, at

least with respect to surface detection by FACS. These new findings suggest that SL expressed on pro- B cells, complexed with other undefined "early proteins" may in fact promote Ig heavy chain gene rearrangement. Additionally, it also implies that expression of the 1 heavy chain may, in fact, lead to

the downregulation of surrogate light chain expression. However, given the findings that surface CD3 complexes present at levels only detectable by surface biotinylation are capable of generating signals in DN thymocytes [42], it appears that analogously, SL chains may be expressed on the surface in pre-B cells (complexed with C( chain) but at levels detectable only by techniques more sensitive than FACS. Preliminary functional studies in C( transgene-complemented RAG-deficient mice have indirectly supported this possibility by demonstrating the induction of tyrosine phosphorylation mice following surface engagement of p protein [56].

B22O

CD43

HSA

BP- 1

- - (resti ng) pre-B

dull ddl dull dull + +

+ + dull ddl - - + + ++ +

large pre-BI1 srnall immature B mature B pte-BI1

% cells 20-30 70.80 ?CL90 - 5 GdGo (cycling) (cycl ing) (cycl ing) (resti ng) resting resting

Figure 1.3 Tk RAG d 6 n i p h in muiine B c d dcvclopmcnt. A: Stage at which B cell clifferentiation is bloclied in RAG deficient mice with respect to FACS analysis f a surface expression d the 8220. CD43. BP-land H S X

differentiation markers. Developrnental pogrtssion rescued by heavy and light chain aansgenes is represented bp

dashed arrows. The pariicular subfracticmation zicheme and assoa'ated nomendatue is Qrivedfrom [Er]. B: The RAG

block in B cell&velopmentwith respect to FACS analysis d srrfaœ and cytoplasmic expression of surrogare light chah

(SL) and p heavy chain ()rH) expession (adapted from [55j). The % cycling cells in e h subpopulation are based on

analysis cf normal 4week otd bdbk miœ in this study. Caresponding menclatuie classification fa each subpopuIation is based on [66].

ii. Terminal deoxyribonucieotidyl tramferase (TdT)

The enzyme TdT represents another lymphoid-specific component of the recombinase system. In mice, two fonns of functional TdT have been identified, each derived from an altematively spliced precunor RNA [57,58]. TdT mRNA of adult mice is found predorninantly in primary lymphoid organs, more specifically, in conical thymocytes and pro and pre-B cells in bone marrow. Lnterestingly, like the RAG and Rch-1 proteins, TdT appears to associate with the nuclear matrix [59,60] raising the possibility this factor may physically complex with the RAG- 1/Rch- 1 protein complex. The predominant protein species is a single polypeptide of 508 a.a. or 58 kD. TdT is a DNA polymerase which adds nucleotides to the 3'-OH end of DNA in the absence of a template. It has been implicated as the V(D)J recombinase component responsible for the addition of non-templated nucleotide insertions (N additions) at the junctions of Ig or TCR gene segment junctions, thereby increasing sequence diversity [61,62]. The e vidence supporting this observation is summarized below .

First of all, N regions are template-independent, G/Cenriched insertions found predominantly in the coding joints of V(D)J rearrangements. Because TdT adds deoxynucleotides in a non-templated fashion in vitro with a preference for adding G residues, this is consistent with the GC rich sequence found in most N regions [63]. However, while TdT is expressed in early B and T cells in adult mice, it is not expressed during fetal development 1641. This corresponds to the absence of N additions in the antigen receptors rearranged during fetal ontogeny 1651. Furthemore, in B ce11 development specifically, TdT expression appears to be highest in pro-B and pre-BI B cells i.e. up to heavy chain Ig rearrangements (using Melchers nomenclature) [Ml. This is consistent with the observation of a Low incidence of N region diversity in the light chain repertoire 1671. ThirdIy, the incidence of N regions in a pre-B ce11 line was found to be increased by expression of the cloned TdT gene [57]. Among several pre-B ce11 lines, there was also a strong correiation of insertions in signal joints with TdT activity, although coding joint insertions were common in al1 the cells. Further evidence cornes from the analysis of recombinants made in fibroblasts expressing RAG- 1 and RAG-2 [68]. These cells do not produce TdT, and coding junctions made there are devoid of random base insertions. Coexpression of TdT then results in coding joints with inserts like those found in lymphoid celis.

The proposition that TdT introduces N regions has recently been confinned by the observation that very few N additions are found in mice in which the TdT gene has been disrupted by homologous recombination 169, 701. Although mice deficient in expression of TdT lack N regions in their Ig and TCR gene rearrangements (the repertoire in an adult TdT-deficient mouse closely resembles that of the

neonate), they otherwise appear to function normally. Importantly, this study also unambiguously demonstrates that unlike the RAG gene products, participation of TdT in the V(D)J reaction is only optional.

Mutations in yeast that affect recombination also frequently affect the DNA repair process, particularly the double-strand DNA break repair (DSBR) pathway. Similarly, venebrate V(D)J recombination and double-suand break repair, in addition to involving double-strand breaks also appear to utilize common enzymatic activities such as exonuclease, polymerase, and ligase. Thus, it was hypothesized for some tirne that various non-lyrnphoid specific ubiquitous factors panicipating in double suand DNA repair may also be recmited by the V(D)J recombinase cornplex. The first clue revealing the necessity of ubiquitously expressed DNA repair activities in addition to lymphocyte- specific components in CO-ordinating the assembly of V(D)J segments was provided by somatic ce11 genetic studies of the factor mutated in scid mice.

i. The murine scid (severe combined imrnunodeficiency) locus

The scid mutation is a recessively inherited defect that maps to mouse chromosome 16 and is therefore distinct from the RAG factor [7 11. The locus encodes a ubiquitously-expressed DSBR activity that is also required for efficient joining of V(D)J segments. Lymphocyte precursors from scid mice have impaired V(D)J recombination of Igs and TCRs characterized by large deietions of junctional information, usually resulting in nonfunctional alleles. This defect in V(D)J recombination therefore leads to the arrest of T and B ce11 development at an early stage conferring a typical severe immunodeficiency phenotype i.e. the virtual ahsence of mature T and B lymphocytes ([72,73] reviewed in 1741). Despite its apparent specificity in the V(D)J recombination reaction, the scid factor is not lymphoid-ce11 specific, because the defect in DSBR conferred by the scid mutation results in hypersensitivity to ionizing radiation and other agents that introduce double svand breaks (DSB) in ail ce11 types, including non-lymphoid tissues 175-771.

The wild type scid locus is not absolutely required for DNA repair. Chromosomal DSB in scid cells can be repaired, albeit to a lesser extent than in wild-type cells, and DSB in extra-chromosomal substrates are repaired as effkiently as in wild-type cells. [78]. Furthemore, in the context of V(D)J recombination, scid mice have been temed as "leaky" [79-8 11. In other words, scid mice, unlike RAG-

deficient mice, develop populations of peripheral mature lymphocytes in a time- and strain-dependent fashion. In addition, recent experiments have shown the restoration of recombination in the TCR f3

chain locus after treatment of newborn scid mice with ionizing radiation [82]. The Iack of leakiness in the RAG mutation as compared to the scid mutation can be explained in simplest terms by the nature of the affected activities (Figure 1.4). Observations that scid mice and scidderived ceU lines are able to introduce DSB at RSSkoding sequence interfaces and to form relatively normal RSS joints, but impaired coding junction formation (impaired both in terms of aberrant junctions and reduced

normal recmbination

recognition and cuîting

scid v-3

Figure 1.4 . The effcct of mutations in various DSBR recombinase componcnts on V@)J rccombination. RSS are indicated by aiangles an4 coding scquences by boxes. During normal recombination, doublestrand breaks are intmduced between the RSS and adja~ent codhg scqucnces, foliowed by proper Ligation of these sequences. in the scid V-3, xn4, and XR-1 mutations, double-stcanà breaks appcar to k properly introduced, but in the scid and V-3 mutations, there is a defect in the ability to propcrly join c W g

sequences (although RSS arc mmaliy joined), whaeas in the xrs4 and XR-1 mutations, there is a defact in the ability to properly join botb RSS and d n g sequences. in RAGdeficitnt mice, RSS or coding sequences rcmain in germli# configuration as dwble-strand brtaks canaot k introduced. Diagram adapted from [a, 951.

formation) have more precisely characterized the activity at the scid locus to be important in the formation of coding junctions 183-851. Signal joint formation was much less affected, although there was uncharacteristic base loss in 40% of the recombinants. It has therefore been suggested that much of the leakiness results from the rescue of the iiberated coding joints in scid pre-lymphocytes by an illegitimate recombination mechanism that occasionally restores what appear to be normal joints [86, 871. In contrast, mice with either their RAG-1 and RAG-2 genes dismpted have al1 antigen receptor loci retained in germline configuration and no mature lymphocytes can be found [13,14]. This profound block in lymphocyte development in which the V(D)J recombination process cannot even be initiated suggests that the RAG products are involved in earlier steps of the V(D)J reaction than the wild type scid product i.e. the RSS recognition, synapsis, or cleavage steps.

One possible function for the wild-type scid gene in the "later steps" of V(D)J recombination has been inferred from analysis of recornbination intexmediate products. In scid mice, it has been possible to detect site-specific cleavage products from the TCR 6 locus of thymus DNA [88-901. Detailed

characterization of the cut RSS species revealed that the signal ends were blunt and 5' phosphorylated, but the coding ends had hairpin temini. In contrast, such "hairpin intermediates" did not accumulate in wild-type tissue samples. These studies, coupled with the observations that scid mice and scid4erived ce11 lines are able to introduce DSB at RSSkoding sequence interfaces and to f o m relatively nomal RSS joints, but impaired coding junction formation, suggested that scid defect resulted in the inability to nick hairpin intermediate products. ParadoxicaUy, when the ability of scid cells to nick open hairpin ends was tested with a hairpin linear recircularization assay, the yield of recombinant circles from scid and wild-type cells was very similar [91]. However, because this study makes use of synthetic hairpins and those in the normal context may exist as DNA-protein complexes and because other nucleases required for opening of hairpin intemediates may not be expressed in the cells in which the artificial hairpins have been introduced, the possibility that the scid defect affects the processing of hairpin ends cannot be excluded. Other direct roles for the scid locus in DNA repair and V(D)J recombination (such as the protection or processing of free DNA ends) as well as indirect roles (such as controiling ce11 cycle checkpoints) have also been suggested and will be discussed further in the context of the molecular characterization of this locus. How would the earlier study in which RAG-1 may also have a role in resolving coding joints fit in with the similar role for the wild type scid locus? Since the RAG-1 protein has a topoisornerase-like domain in its C-terminus, it may function in coding joint resolution by functioning as a topoismerase. Al1 topoisornerases operate by DNA breakage, protein attachment to one broken end, and rejoining DNA to a different partner DNA via energy transfer from the transient active- site DNA protein 1921. Therefore while the scid defect may be involved in such steps as the nicking of the hairpin intemediate or processing of nicked hairpin intermediates, RAG-1, as a topoisornerase, may be involved in the formation of hairpin intermediates. The precise relationships of these two genes in

coding resolution is intriguing and shouId be better understood once the structures of RSS i n cells uansfected with low expressing RAG constnicts are more precisely analyzed.

ii. The Xr-1, xisd, and V-3 d e f w

It could be argued that virtually al1 activity (except for TdT) participating in V(D)J recombination downstrearn of site-specific recognition and cleavage may play a part in a more general DNA repair mechanism. Thus, in an effort to find other ubiquitously expressed DNA-repair factors recruited by the V(D)J recombinase in addition to the scid factor, a large array of Chinese hamster ovary (CHO) ce11 lines having mutations rendering them sensitive to vanous DNA darnaging agents were tested for their ability to rearrange introduced V(D)I recornbination substrates following the introduction of RAG expression vectors [93-97. This panel of mutants included ce11 lines with defects in UV- induced excision repair, X-ray induced double stranded break repair, impairecl ligase 1 or 3 activity, and cells from patients with Ataxia Telangiectasia. Of the various lines tested, the only ones to show any impainnent in the ability to undergo V(D)J recombination were those that belonged to X-ray-sensitive double strand repair complementation groups such as the xrs-6, XR-1, and V3 mutants which belong to ionizing radiation (iR) complementation groups 4, 5, and 7, respectively (reviewed in [9]). However, two (US-6 and XR-1) demonstrated a marked dectease in abiiity to f o m both coding and signal V(D)J joins, while another, V-3, showed a preferential impairment in the joininp of coding sequences, reminiscent of the mouse scid defect (Fig. 4) [94]. Furthemore, fusion studies in which defective lines were fused with murine scid celi lines have s h o w that while the V3 mutant can prduce revenants with normal V(D)J activity and X-ray resistance, the other two mutations were unable to do likewise [95]. Revenants of xrs-6, XR-1 and V3 mutants were also obtained by direct introduction of distinct human chromosomes: chromosomes 2, 5, and 8, respectively. The murine scid defect can also be complemented by human chromosome 8, suggesting that it belongs to the same complernentation group as V3. Given the manifestation of the defects (Figure 1.4) and the evidence from complementation studies, it seemed likely that the xrs-6 and XR-1 mutant cell lines encoded separate genes and that the

xrs-6 and XR-1 mutations affect activities other than the mouse scid mutation. This notion has k e n confimed by the very recent molecular identification of two separate genes for two of the three DSBR cornplementation groups. A fascinating finding that the products of these two genes appear to represent different subunits of the same nuclear enzyme, DNA-PK.

iii. Molecular characterization of complementation group 5 and 7 DSBR mutations

The above-mentioned hamster ce11 line mutants with weli-characterized defects in DSBR and V(D)J recombination have provided the required complementation and genetic rnapping systems in which to identiw the genes that restore wild-type DNA repair. In this context, a human DNA repair szene, XRCCS, mapping to chromosome 2q33-35, has k e n show to complement IR group 5 mutants C

and has been identified as the 86 kD subunit of the Ku complex, a 70/86 kD heterodimer that binds ikee DNA ends, nicks and hairpins [98- 1011. Specifically, the 86 kD subunit of the Ku70/86 heterodimer, a nuclear protein originally identified as an autoantigen recognized by sera from various autoimmune patients, was already known to localize to 2433-35. Group 5 mutants (including xrs-6) were found to lack expression of Ku86. Furthermore, upon transfection of Ku86 cDNA back into the defective xrs-6 mutants DNA end-binding activity, x-ray resistance, and V (D)J recombination activity was rescued.

Secondly, vety recent investigations have provided evidence that the human complementation homologue of the munne scid and V3 mutants, the XRCC7 DNA-repair gene, CO-localizes to human chromosome 8qll with the gene encoding the large catalytic polypeptide (the p350 subunit) of the DNA-dependent protein kinase (DNA-PK) [102-1041. Chromosomal fragments encoding p350 complement the scid phenotype. p350 protein Ievels are greatly reduced in defective complementation group 7 cells (including celis derived h m both scid mice and V 3 ) as compared to cells h m wild-type mice. Furthermore, scid and V3 cells appear to lack in vitro DNA-PK kinase capabilities. Cloning and sequence analysis of the human 14.5 kb p35O cDNA is apparently underway in several laboratories, but precise function of DNA-PK in murine wild-type or scid cells has not been evaluated. DNA-PK was first studied as a possible regulator of transcription (reviewed in [105]), but recently has been proposed to also have roles in DNA replication, repair, and cell-cycle regulation [106]. Pnor to the identification of DNA-PK p350 as the scid gene homologue, the in vivo function of this ubiquitously expressed serinehhreonine kinase was obscure. Because it has not k e n possible to monitor DNA-PK activation in

vivo, its substrate specificity and activation requirements have oniy been studied in vitro. Studies utilizing synthetic peptides have identified a consensus phosphorylation site invoiving a serine or threonine followed by a glutamine residue (SQRQ). Several molecules containing the SQ/TQ consensus site have been show to be phosphorylated by DNA-PK in virro. These include the Ku 70/86 complex, the transcription factors Sp-1, c-jun, c-fos, and Oct-1, the tumor suppressor genes p53 and retinoblastoma (Rb), the carboxy-terminal domain of RNA polyrnerase II, as well as topoisomerase 11. DNA-PK rnay play an important role in regulating the cell-cycle response to DNA damage, consistent with the observation that scid T ce11 precursors are susceptible to radiation-induced lymphomagenesis [83]. Addi tionally, because V(D)J rearrangernent appears to be resmcted to GdG 1 [lO7- 1091, the failure

to regulate the cycling of precursors undergoing V(D)J recombination rnay enhance the amount of site- specific DNA damage in this context. The wild type DNA-PK rnay therefore be important in ensuring the proper regulztion of a cell-cycle conuoliing transcription factor such as p53 (the mutant DNA-PK potentially having a defective catalytic domain that cannot properly phosphorylate p53) which rnay prevent translocation-inducing events. Consistent with this notion are the high frequency of lymphoid tumours seen in p53-deficient mice [110]. Altematively, p350 rnay associate with enzymes that repair DNA damage, such as polymerase or ligase or rnay contain a structural domain that changes the chromatin structure around a damaged DNA region that aiiows the repair complex to join DNA ends.

Interestingly, since it has been previously found that the Ku and p350 proteins are subunits of DNA-PK, it appears that the wild-type products of the xrs-6 and scid loci function cooperatively as part of a DSBR protein complex that is required for normal V(D)J recombination. Specifically, biochemical studies in human ce11 lines have demonstrated that p350 is tethered to DNA by interactions with the p70/p86 Ku heterodimer, which functions as the DNA-targeting subunit of this kinase by specifically binding to free double stranded DNA ends (reviewed in [ I l l ] ) . Previous studies have aiso s h o w that the active f o m of the DNA-PK complex (which has ATP-dependent phosphotransferase function) requires both the presence of fiee DNA ends and the Ku proteins [l 1 11. There am thus various possible explanations for how the xrs-6 and scid defects affect DNA repair and V(D)J recombination. With respect to the xrs-6 defect, because Ku protein not only binds to free double stranded ends of DNA but also translocates along the DNA, coating it with evenly spaced multimeric complexes [112], it rnay act in both DNA repair and V(D)J recombination by preventing nuclease digestion of DNA double-strand breaks or by holding the DNA ends in a requùed conformation. Alternatively, it rnay be involved in activating other downstream activities. With respect to the scid defect, one can speculate upon a number of possibilities. For example, the defective p350 factor rnay differ from the wild-type protein in the extent or stabiiity of association with the Ku proteins. Another possibility is that because the activity of DNA-PK is defective, the phosphorylation-mediated control of a protein important in the resolution of hairpin-structure intermediates is affected. Two examples of proteins that rnay be affected in this manner are topoisornerase II which has been demonstrated to nick hairpins [98] and the Ku proteins themselves. DNA-PK rnay also be more generally involved in the phosphorylation-mediated control of other recombinase components involved in early steps of recombination. In this context, it is interesting that consensus DNA-PK recognition motifs have been reponed within the RAG-I and RAG-2 proteins [104]. Finally, the large size of p350 rnay provide a frarnework around which other repair and recombination enzymes are recruited to the cornplex. The defective p350 protein, whose expression is reduced, rnay not ailow this recruitment.

iv. Other potentid recombinase components defined bhehemically

Other efforts to find facton essential for V(D)J rearrangewnt have focused on predicted biochemical activities involved in V(D)J recombination such as binding, cutting, and ligation of RSS. None of these factors can yet be assigned a defined role in V(D)J recombination since the specificity of such identitied facton in general is not very high and only preliminary work on most has b a n reponeû. Several of these proteins wili be briefly summarized.

Factors that bind RSS

About 10 different signai-binding proteins have been described. The first to be reported was called "nonamer-binding protein" (NBP) and was identified on its ability to complex stably with a 23 - base pair spacer signal [113]. Afier further purification and characterization, NBP, a 53kD protein found to be expressed only in nuclear lymphoid extracts, was demonstrated to be very specific for the nonamer sequence, but did not possess any nucleolytic activity. A second protein identified upon binding to a 12-spacer signal was found to specifically interact with a heptamer again only in lymphoid extracts [114]. However, as with NBP, no enzyrnatic activity was subsequently reponed. Another protein was purified from an A-MuLV extract on the basis of its ability to bind a 23-signal DNA probe [Ils]. This 60 kD protein, called "Recombination Signal Binding Protein" or RBP-JK has attracted interest primarily because its amino acid sequence has some homology to the integrase family of bacterial recombination enzymes. However, expression of RBP-JK is not specific to lymphoid cells and genes with closely related sequences have been found in organisrns as remote as Drosophila and yeast. A founh protein was isolated as a cDNA clone by screening a pre-B cell library with a 12-spacer signal probe [116]. This clone, T-160, was specific for 12-spacer signals and failed to bind a sequence with a mutation in the heptamer's third position. The role of the T-160 protein in V(D)J joining was put into question because its binding properties as determined by Southwestern blot analysis could not be reproduced in electrophoresis mobility s hi ft assays (EMSA) and DNA footprinting assays. Another protein "Rc" was also isolated from a cDNA, in this case a thymocyte cDNA library screened with a probe containing both 12 and 23 base pair signals 11 171. Rc was found not only to bind to an isolated heptamer but also to an unrelated sequence motif. Perhaps the most promising candidate among the proteins identified on the basis of bi nding has been found in murine thymocytes [ 1 181. This 30 kD pmtein, called "recognition protein" or RP, is the only factor to date found specific for joining signals while binding to both heptamer and nonamer targets in both 12 and 23 spacer targets; it binds non-functional joining signals poorly, and it appears to be confine. to cells and tissues that exhibit recombination activity.

Factors implicated in V(D) J cutncutnng and ligaîion

For several years, a number of groups have attempted to detect a site-specific endonuclease activity in 1 yrnphoid celi extracts. None of these activities demonstrated themselves to be stnngentl y site-specific and little has k e n reponed beyond the initial characterizations. The search for such factors may now be renewed using recent evidence that intexmediate products of V@)J recombination occur as hairpins structures. With respect to Ligation activity, an interesting product was isolated based on its nonarner-binding propenies [119]. This 47 kD clone, called "V(D)J joining protein" or VDJP has a domain homologous to bacterial ligases, although it does not contain the ïigase active site. When VDJP was expressed bacterially, a joining signaldependent activity was observed and was greatly reduced upon deletion of either signal heptamer. This activity also seemed to correlate with expression in pre-B ce11 lines, as did the Lack of activity in fibroblasts. However, despite this joining signal-dependence, the products do not resemble signal junctions. The potential importance of a recombinase-specific factor such as VDlP is supported by tests of V(D)J joining in a human DNA ligase 1-negative ceil line 1119). In these cells, V(D)J rearrangement is normal, indicating that DNA ligase 1 is not required for the reaction.

II. Regdation of RAG-1 and RAG-2 expression

The expression of RAG-1 and RAG-2 in non-lymphoid cells confers the ability of these celis to reamange antigen receptor genes in extrachromasomal recombination substrates, yet does not allow endogenous Ig rearrangements to occur. This suggests that the RAG genes are necessary, but not sufficient for generating endogencus antigen receptor rearrangements. In this context, other lymphoid- specific influences have been show to be important in regulating the "accessibility" of V(D)J antigen receptor loci to the common recombinase system, such as the methylation or transcription status of these loci when in gemiline configuration, or the quality of RSS (reviewed in [120,121]). However, while the

majority of the literamre to date bas focused on such mechanisms, much less emphasis has been placed on understanding how the RAG genes themselves are regulated. A number of recent rcprts have contributed to a preliminary understanding of factors which appear to influence their expression, both at the RNA and protein level. However, little is known about the precise cellular and rnolecular mechanisms responsible for shutting off expression of the RAG genes at various stages of lymphocyte development. Even less is known about what is responsible for inducing their expression.

A. Pattern of RAG-1 and RAG-2 mRNA expression: lineage and deveelopmentaï restrktiom

At the cellular level, RAG- 1 and RAG-2 rnRNAs are in general, expressed concordantly, and in a lineage-specific fashion i-e. T and B lymphocytes [8]. Without this concordant expression in developing lymphocytes, recombinase activity is reduced several thousand fold [9]. Interestingly, RAG- 1 mRNAs are generally expressed 10-100 fold more abundantly than RAG-2 mRNAs. Some notable exceptions to the concordant and lineage-restricted pattern of expression exist: RAG-1 alone has been reported to be expressed in neuronal tissue of the central nervous system [122] as well as in a transitional ce11 type during thymic differentiation [123]. Furthemore, RAG-2 alone has been found to be expressed in avian bursal B cells [16].

Extrachromasomal substrate assays performed both in lymphocyte ce11 lines and RAG-

transfected non-lymphoid ce11 lines (murine and human) have aimost invanably demonstrated the amount of RAG gene expression to strongly correlate with the level of recombinase activity 191. Only one study to date has found this not to be the case [25]. In vivo, RAG mRNAs, similar to TdT, are expressed at the highest levels in primary lymphoid organs, especially in the bone mmow and thymic cortex 11241. More specifically, the developmental stage at which RAG expression is first observed in mouse corresponds to lymphocytes which are undergoing their first series of V(D)J rearrangements: CD44+IL-ZR- DN (CD&CD8-) thymocytes and fraction A pro-B cells (large B22O+CD43+WA-BP-1- )[125- 1271. Additionally, and consistent with their critical fûnction in V(D)J recombination, the highest

level of RAG expression, in vivo, appears to comspond to lymphocyte populations undergoing active rearrangernents at thyrnocyte 0 and a loci and B ceil heavy and iight chain loci (i.e DN and DP TCR'

thymocyte populations and pro- and pre-B ce11 fractions B through D), after which developmental downregulation of RAGs then occurs.

Recent studies have suggested that the developmental window in which RAG expression occurs in pre-B cells and DN thymocytes may be more complicated than initiaiiy thought. This is suggested by

recent reports in which RAG downmodulation has been further specified to two "waves" of RAG expression in precursor stages of lymphocyte development. Specifically it appears that RAG expression peaks when B ce11 heavy chains are rearranging and subsequently downregulated, after which another peak occurs again when light chains are rearranging 11281. This is consistent with recent observations that there is a sharp decrease in RAG-1 and RAG-2 mRNAs in the predominantly cycüng, HSA bright, Fraction C' pro-B cells (R. Hardy, unpublished results). A similar pattern occurs in a study of thymic subpopulations where upregulated RAG expression is observed to coincide with C D W DN thymocytes undergoing rearrangement at the f3 chain locus, downregulation occurs in the intemediate CD25' DN population, and finaiiy RAG transcripts are again upregulated in DP thymocytes undergoing a chain rearrangement 11261. Additionally, within the context of the heavy chain rearrangement program of

pre-B cells, it has k e n found that Abelson murine virus pre-B ce11 lines with DJ rearrangements have more recombinase activity than do those with VDJ rearrangements [129].

In vitro, RAG mRNA expression is also seen primarily in pre-T and pre-B ce11 lines [Il]. However, in these lines, RAG rnRNAs are penerally expressed at significantly reduced levels and in a more unstable fashion as compareci to in vivo primary tissue samples [124]. For example, in AMuLV (Abelson murine leukemia virus)-transfomed pre-B ce11 lines, RAG expression was found to Vary 1000- fold (even within subclones of the same ce11 line) and decrease in a tirnedependent fashion, rendering in

vitro replation studies of these two genes quite difficult [130]. In such situations, it has been speculated that this loss of expression occurs because the persistent expression of the RAG genes in ce11 lines frozen in a developmental stage (i.e: a stage in which RAG would be transiently expressed and then shut off in vivo) rnay in fact be detrimental to cellular metabolism, thereby conferring a selective pressure for clones with lower RAG expression. Supponing this notion is the deleterious effect of RAG

overexpression (including incomplete thymopoiesis, compromised cellular immuni ty, and increased rates of mortality) in uansgenic mice that carry RAG- 1 and RAG-2 under the contol of the proximal kk promoter [23].

B. Regulation of RAG-1 and RAG-2 expression by signal trduct ion

Because this thesis is concernai with the cellular and molecular study of how the BCR signal transduction pathway influences the replation of the RAG-1 and RAG-2 genes in a dg+ B ce11 line, 1 have introduced this section by providing an overview of the current understanding of the BCR signaling cascade from surface to nucleus.

i. The BCR signaiing cascade and its role in the reguiation of B ceU gene expression

The most distinctive surface structure of the mature B lymphocyte is the B-ceil antigen complex or BCR (reviewed in 11 3 1-1 331). Like the T cell antigen receptor (TCR), the BCR is a complex hetero- oligomenc structure in which separate receptor subunits are responsible for ligand binding and signal transduction. While the antigen-binding component of the BCR is comprised of membrane immunoglobulin (SI@ in the form of a tetrarneric complex of two Ig heavy chains and two light chains, the structure implicated in transducing signals is comprised of one (or more) disulfide-bonded heterodimen of the m(>-1 and B29 gene products, Ig-a (CD79a) and Ig-0 (CD79b). Cross-linking the

BCR complex may result in numerous distinct biological responses including activation, antigen presentation, proliferation, induction of Ig secretion, cellular differentiation, growth arrest, inhibition of Ig production, or induction of prograrnmd ceil death (PCD) (reviewed in [134]). The stage of B ceii differentiation may be one aspect influencing this outcorne. For exarnple, in immature B cells, antigen binding results in long-lasting non-responsiveness or deletion of cells bearing BCR with high affinity for that antigen. In contrast, cross-linking of the BCR on mature resting B cells results in cell-cycle entry, and often proliferation. Besides the differentiation and activation stage of the B cell, the nature, concentration and affinity/avidity of the crosslinking agent, as well as additional signals simultaneously delivered to the ceil via receptors other than the BCR (such as receptors for cytokines, bacterial products, mauix proteins and cell surface deteminants of neighbounng cells) have also been shown to determine the various possible outcomes in response to BCR signaling. Nevenheless, such biological responses represent the net products of a cornplex cascade of signal transduction events whose more proximal events include the activation of protein tyrosine kinases (PTK) and whose later events include the activation of various "early response" genes, most of which represent transcription factors involved in regulating gene expression and ce11 cycle events. The current understanding of these signaling events will be bnefly reviewed.

Although the process by which the BCR induces the above mentioned cellular responses is only partially understood, a number of early signaling events following sIg ligation are well defined (reviewed in [135]). First, it is weii established that rapid structural changes occur including the rapid association of sIg molecules with the cytoskeleton, a rapid and transient increase in polymerized actin,

the aggreggation of sIg molecules (patching and capping), and slg endocytosis. Secondly, Ligation of the BCR induces various biochemical alterations, the most rapid of these (within seconds) involving the induction of protein tyrosine kinase (PTK) activity and the subsequent tyrosine phosphorylation of various target s ignahg proteins. Tyrosine phosphorylation of such proteins facilitates their interaction with other proteins via src-homology 2 (SH2) domains that bind specific phosphotyrosine-containing sequences in other proteins with high affinity. The BCR activates the B lymphocyte-specific SRC famiiy kinases p ~ ~ b l k , p59m, p561ck, and p ~ ~ d ~ n . Although the data are controversial in tenns of elucidating the sequence of phosphorylation events, one possible mode1 based on genetic and biochemical studies is that the pnmary signal in this pathway may in fact be the activation of the slg- associated PTK p72s~k , resulting in the binding and activation of a src-related PTK such as p~3/561~n (132). This PTK is then presumed to tyrosine phosphorylate a conserved motif termed antigen receptor homology 1 (ARHI) which is not only found in the cytoplasmic domains of Ig-a and Ig-B. but also in components of other receptor complexes, including the CD3 polypeptides y, 8 , and s and the TCR accessory chahs and q . The phosphorylation of ARH! can then in mm induce hinher recmitment of

p72s~k, as well as the reonentation, enhanced binding, and activation of one or more ARHI-associated src-family PTKs present in B celis which include p55b4 p59fyn, p5dck, or p53/5d~n.

The activation of one or more of the BCR-associated PTK induces cm subsequently regulate key signal transduction pathways involving SH2 interactions, for instance the association of signal transducers such as Phospholipase C-y1 and y 2 with the BCR (reviewed in [136, 1371). Phospholipase C mediates the hydrolysis of phosphatidylinositol (4,s)-bisphosphate (PIP2) to generate inositol(1,4,5)- visphosphate (IP3) and diacylglycerol (DAG). Generaliy, within 15-30 seconds afier sIg ligation, IP3

stimulates the release of fke ca2+ ions firom the extraceiiular milieu as well as rnobilizing intracellular stores of calcium ([caZ+]), leading to the activation of calcium/calmodulindependent protein kinase II (CaM-KII). DAG is an activator of the a, 0, and 6 isofoms of protein kinase C (PKC). Although

homologous recornbination studies indicate that p72s~k, in cooperation with ~53/561~n, plays a pivotal role in the regulation of PLC-y, G-protein blocking and reconstitution studies indicate that PTK- mediated regulation of PLCy may also be additionally and proximally regulated by a pertussis-toxin- sensitive G protein. Other downstream events of PTK activity include the activation of phosphoinositide 3-kinase (PU-kinase). PU-kinase produces inositol phospholipids that activate a unique PKC isoform, PKC-c, which is essential for growth factor-induced proliferation in fibroblasts. The p 2 l m

oncoprotein, a potent regulator of cell growth, is also activated by BCR. Severai proteins involved in the activation and down-stream effects of RAS via SH2, SH3, and N-terminal domain interactions are tyrosine phosphorylated after BCR cross-linking. These include the SH2domain containing adaptor protein (SHC), the rasGTPase activating protein (GAP), the GAP-associated proteins p62 and p 190, and the p95Vav protein (a haematopoietic GTP-exchange factor that is rapidly tyrosine phosphorylated

following ligation of the BCR). Finally, BCR crosslinking results in tyrosine phosphorylation and activation of the p42 mitogen-activated protein (MAP) kinase, a serine-threonine protein kinase that can be activated by both tyrosine and threonine phosphorylations.

The exact relationship of each step of the protein phosphorylation and second messenger generation cascades with downsueam events concerning gene transcription remain elusive. However, there are several examples of candidate transcription factors k i n g such downstream targets of signaling cascades (reviewed in [ 1351). For example, the proto-oncopne c-jwi must undergo two modifications in order to serve as an active transcription factor, first a dephosphorylation step regulated by the PKC activation pathway that allows for DNA binding and second, a phosphorylation step at N-terminal residues that is regulated via the MAP kinase pathway and ultimately activates c-jun. MAP kinase also

has been shown to phosphorylate p62TCF, thus stimulating c-fos transcription of c-fus, another transcription factor that dimenzes with c-jun to form the AP-1 leucine-zipper dimer uanscnptional complex. Additionally, both CaM-KI1 and various isofonns of PKC have been implicated in the modulation of Ers-1 and Egr-1 transcription factors. PKC activation can also result in the induction of the transcription factor elk and the early response oncogene c-myc. Furthemore, PKCC has been demonstrated to regulate the B-ce11 transcription factor NF-KB, which will be discussed in more detail

further on. More recently, two additionaf putative transcription factors, nur77 and nup475, have also been found to be induced upon anti-IgM stimulation or directiy activating the PKC pathway of B cells.

ii. The regdation of RAG expression by signais transduced through antigen receptois

The developmental restriction of RAG expression to early stages of lymphocyte differentiation may not be as cornpiete as onginally thought. Recently, several groups have described in vivo and in

viîro situations in which lymphocytes with mature surface antigen receptors (BCR and TCRs) can still continue to express RAG mRNAs, albeit at reduced levels [125, 127, 138-1421. For example, in situ

hybridization by Ma et al. has estimated that 5% of sIgM + B cells in a normal BALB/c mouse continue to express RAG mRNAs and Randy Hardy's group by RT-PCR has detected RAG expression in fraction E (sIgM+). The fact that RAG expression stiii persists at these later stages of lymphocyte development suggests that at least in certain situations, the presence o f expressed V(D)J recombination products is in itself not adequate for terminating RAG expression, but rather a signal transduced through these products is required.

A number of reports have provided evidence for the decrease of RAG expression upon cross- linking of mature antigen receptors. For example, Ma et al. found that cross-linking the BCR of s1g +RAG+ B ce11 lines from Eu -N-myc uansgenic mice with F(ab)'2 anti-p fkagments down-regulated

RAG-1 and RAG-2 mRNAs [140J. This downregulation effect was rapid ( ~ 1 0 fold within one hour),

specific (in the sense that no other B celi-specific genes assayed were simultaneously down-regulated), and could be reversed when the cells were nmoved fiom anti-p cross-linking. Similar observations have been observed when signals are delivered through the TCR of thymocytes. For example, Turka rr al. showed that cross-linking the TCR of DP (CD4+CD8+) thymocytes in vitro with an ami-CD3 resulted in a signal which rapidly terminates the steady state leveb of RAG-1 and RAG-2 mRNAs 11251. This effect appeared to be specifically generated through a TCR-rnediated pathway, as cross-linking of other T-ce11 surface antigens such as CD4, CDS, or HLA class 1 had no effect. A similar downregulation of RAG-1 and RAG-2 mRNA was also reponed in C D 4 thyrnocytes by Takahama and Singer upon cross-linking with a polystyramine plate-bound MAb to TCR $ [143]. Furthemore, stimulation of thymocytes in these two reports with agents that directly activate second messengers such as the phorbol ester PMA (a potent translocater and activator of PKC) in combination with the calcium ionophore ionomycin (which aiiows extracellular ca2+ fluxes) also led to significant decreases in RAG transcripts [125, 1431. Severai recent reports in transgenic systems have strengthened the observation of Turka et al. by providing evidence that in vivo TCR ligation of conical thymocytes with self-MHC dunng positive selection results in the dowmeguiation of RAG expression [144, 1451. In one such study, both in si& hybridization of the thymic cortex of mice bearing transgenes for a and f3 TCR alleles as well as MHC class 1 H-2b molecules (necessary for positive selection of the transgenic TCR) displayed a striking reduction in RAG-1 transcripts [145]. Funhemore, Nonhern blotting of those thymic populations which had undergone positive selection (DP T C R ~ ~ thymocytes) in this system demonstrated greatly reduced levels of RAG expression compared to those which had not @P T C R ~ thymocytes). In conuast, thymi of transgenic mice also carrying the same TCR a and alieles but expressing MHC transgenes (H-2d) that did not positively select the transgenic TCR continued to display high levels of R AG- I transcripts.

On the other hand, there is one intriguing example of an instance in which RAG mRNAs can be upregulated in response to anti-p ligation. Tiegs et al. have provided evidence in a transgenic system that RAG expression of a sIg+ bone marrow B cell sub-population already expressing RAG mRNAs is further induced upon autoantigen encounter 11461. Specifically, heavy and light chain transgenes encoding an H - 2 ~ k b antibody specificity (3-83) in this study were introduced into the germline of mice with either a H - 2 ~ k (or H-2Kb) background present in al1 tissues or into mice with a H - ~ K ~ background (non-deleting mice). Bone manow cells with the 3-83 idiotype were sorted by FACS and their mRNA levels were quantitated by reverse-transcriptase polymerase chain reaction (RT-KR). It was found that the 3-83 ceils in cenually deleting mice had significantly higher levels of RAG mRNA than the 3-83 cells of the non-delet hg mice. Funhermore, cenually deleting mice bearing the higher affinity ligand KL appeared to express higher levels of RAG-1 and RAG-2 mRNA than those expressing the low

affinity Ligand Kb, suggesting that affinity of the antigen-receptor interaction rnay quantitatively influence the degree of RAG upregulation.

The observation that autoreactive clones can respond to autoantigen by increasing their level of RAG mRNAs in a transgenic system implies that the RAG genes in sIg+ B cells can be differentiaily regulated. The has prompted Tiegs et ai. to suggest the "receptor editing" model ([146]; reviewed in [147]). In this model, non-autoreactive clones would promptly have their recombinase shut off so that no further rearrangements could occur. However, in situations where a B ceiî is *autoreactivem, upregulation of the recombinase genes could allow funher Ig heavy or light chain secondary rearrangements. Such secondary rearrangements in autoreactive B cells could then generate new receptor specificities replacing or "editing" the autoreactive one. The Tiegs et ai. transgenic system further supports this model, as RAG-upregulated 3-83 B cells in this system appear to also be specificaily replacing their anti-H-2k specific K transgenes with endogenous A chains. At this point, the physiological relevance (if any) of this process as an alternative tolerance mechanism to the well- established mechanisms of clonal deletion and clona1 anergy is not known. Nevertheless, the factors in sIg+ populations that result in the upregulation of RAG expression in some instances, and downregulation in others, remains to be elucidated. This specific point wiU be discussed in more detail in Chapter 4 with respect to data presented in Chapter 3 as well as future experirnents.

E Replation of RAG by signai transduction: implications for lymphoid progenitors

By vinue of directly controlling the essential lymphocyte-specific recombinase components, signals that are transduced through antigen receptors represent a novel, signaiing-responsive method of regulating the V(D)J reaction. This level of regulation rnay be considerably more relevant if one considers that it rnay not be solely restricted to more advanced stages of lymphocyte development in which surface antigen receptors are expressed, but to developing lymphocytes with pre-antigen receptor- like complexes as well. For example, the downregulation of RAG-1 and RAG-2 in lymphocytes could occur at the stage pnor to sIgMC expression and rnay involve equal downregulation of aU developing lymphocytes by signals generared through the pre-T or pre-B receptor complexes. In this context, the dowmeguiation observed in antigen-receptor positive populations rnay occur only after antigen-receptor expression and the remaining RAG expression detected is frorn a transient sIgM+ subpopulation which either has not yet had a signal transduced to shut RAG gene expression off completely, or which has responded to antigen-receptor ligation by upregulating RAG mRNAs. Alternatively, this downregulation rnay be due to differential expression of regulatory factors of RAG expression intrinsic to the differentiation plan and independent of signaling altogether. However, it is just as possible that signaling through a pre-B cell p/surrogate light chain receptor cornplex of pro-B (and pre-B ceiis) or in analogous pre-T ce11 receptor complexes of DN thymocytes (both of which have been demonstrated to

be comptent signal transducen) could also be responsible. Furthemore, the recent reports describing two waves of RAG expression in developing lymphocytes demonstrate that RAG rnay not just be downregulated in mature antigen receptor-expressing lymphocyte populations, but in CD25' DN

thymocytes and pre-B cells that have just finished heavy chain rearrangements [126, 1281. This rnay therefore be another situation in which signaling through pre-antigen recepton rnay be mediating the downregulation of RAG- 1 and RAG-2.

Currentfy, it has not yet been assessed if and how signais transduced through the surrogate light chain complexes have an effect on RAG-1 and RAG-2 expression. However, indirect evidence that pre- antigen-receptor-asstrciated signals can alter RAG expression cornes from observations that agents which directly activate second messengers of antigen receptor signaling pathways can do u, in pre-B lines, pre-T ce11 lines, and in DN thpocytes 1125, 126, 148, 1491. In al1 such instances, activation of secondary messengers by treannent with PMA in combination with Ionomycin has demonstrated, as with the majority of studies with mature antigen receptors, significant reductions in RAG-1 and RAG-2 transcripts. One caution in interpreting these data, however, is the qualitatively âifferent signaling pathways that may be generated by pre- versus mature antigen receptors 11321. For example, while the mature BCR responds to anti-p cross-linking by increasing intracellular and extracellular ~ a 2 ' levels and IP3 production, the SL complex responds to ligation with AS or p MAbs by increasing ca2*, but

with no detectable increases in IP3. More direct evidence will therefore have to be obtained by direct ligation of SL complexes.

Of considerable interest are studies assessing what signals induce, rather than shut off RAG expression in pro-B and DN thymocytes. There are Iikely at least two instances in which RAG induction rnay be required. The first is the initial induction of RAG that presumably occurs very won after T or B lineage cornmitment. Secondly, in the two waves of RAG expression that have k e n observed, RAG would then have to be re-expressed in the second "wave" of expression [126, 1281. Although induction or upregulation of RAG expression rnay be mediated through pre-antigen receptor complexes, it rnay also be via other receptors and associated signaling pathways. For example, it has recently been observed that the addition of uiterleukin 7 (IL-7) to early mouse thymocytes in vitro can specifically induce RAG expression [LSO, 1511. However, this study does not rule out the alternative explanation that IL-7, a requisite for the survival of early T ce11 progenitors in vizro, simply maintains RAG expression by allowing these cells to expand while another factor may in fact be responsible for RAG induction. Alternatively the differential effect rnay be mediated by ligation of the pre-antigen receptor complexes. In the initial induction of RAG expression, this rnay result because the SL chains associate with proteins other than heavy chain. In the situation where RAG is shut off and is then re- expressed, differential replation rnay occur through the same SUp chain complex by changes in the

intracellular pathway associated with differentiation. Interestingly, incubation of the RAG- downregulated intennediate CD25- thymocyte population in medium alone upregulated RAG expression 10 fold, suggesting the down-regulation of RAG in vivo was mediated by a reversible negative signal that was not present in the medium. In contrast, the same effect could not be observed in the earlier, high RAG-expressing CD25+ population, suggesting differential regulation of RAG expression in these two subpopu!ations [1263. A similar upregulatory effect was also found upon incubation of day 19 CD4- fetal thymocytes in medium alone 11431. Regardless of what signaling factors or surface receptors are required for elevatinp RAG expression in developing thymocytes, the upregulation of RAG expression may be mediated through a cyclic AMP (cAMP)dependent Protein Kinase A (PKA) signaling pathway. One report in fact provides evidence for increased RAG mRNAs and recombinase activity in pre-B cells that are induced by CAMP-inducing agents such as caffeine and theophyiiine [148].

iv. A model for the signaiing-medisted regulation of RAG expression in B lymphocyte development

Taken together, the studies of signaling-mediated regulation of RAG mRNA expression do not yet accurately describe what cellular or molecular factors are involved at various stages of lymphocyte developrnent. However, they do suggest that RAG expression may be dynamicaily altered throughout the lymphocyte developmental program. Additionally, they sugpst that different types of signaling stimuli as weU as signaling at various stages of developrnent can difierentiaily regulate the expression of RAG-1 and RAG-2 mRNAs. In this context, in Figure 1.5, a mode1 of signaling-mediateci RAG regulation at various stages of B lymphocyte development is proposai.

lymphoid 8: G pmgenitor L: G

pro-B H: V-DJ L: G

II: open L: closed

H: open L: closed

preBII H: VDJ L: v-J

H: closcd L: open

I

"positive selection"

Figure 15. Model of sigding-meduted regdation of RAG expnssba at variairs stages of B Iymphocyt,

development In brief RAG expression miy fint be iriduced in pro-B ceils eitber by ligation of the surrogate iight chin (SL) / d y protein (such as pl30 or pS5) complex with ri@ dclivasd hm s ü m m l cek &or by iïytion of c y c o h e rraptorr sucb as the IL-7 tdccptor RAG expression may e i k be dowl~~gulted or uprcguhicd in p - 8 alis

by stromal ceil signal- SLIP chah compkx intuactions (m .id respedvely). F i d y . in immpwc BCR+ B ah still

expresshg RAG, depeadtog on rbe autoreactivity of the particulPr clone, RAG may be differmtially rcgukted by cross- iinking antigcn The 0 lympbocyte dcvelopment and V(D)J reunogemcnt ciassifications are bucd on tbc aomciulmuc

[661

v. Molecular aspects of signai transduction-mediated reguiation of RAG-1 and RAG2

Several recent reports have k g u n to assess the molecular mechanisms involved in the alterations of RAG by signal transduction pathways, and to date have yielded a complicated picture of the mechanisms involved. One report by Neale et ai. has demonstrated that the downregulation of RAG mRNAs in DN thymocytes and pre-B cells by treatment with direct activators of second messengers occurs by both decreased transcription and RAG mRNA destabilization [149]. The Takahama and Singer study also argues for a strong contribution by a mRNA destabilization mechanism in the downregulation of RAG mRNAs [143]. Funhermore, in the Neale et al. report, the regulatory proteins involved do not a p p a r to be newly synthesized since pre-ueament with the protein synthesis inhibitor cyclohexamide has no effect on the observed RAG-1 or RAG-2 mRNA downregulation implying that such a mechanism occurs via a post-translational pathway, such as by tyrosine phosphorylation- mediated activation (or repression) of a constitutively expressed PTK. In this context, the abelson tyrosine kinase has k e n shown to have a negative regulatory effect on M G - 1 and RAG-2 mRNA and protein expression in a temperature sensitive A-MuLV transformed pre-B ceil line. Specifically, the heat-permissive activation o f the abelson tyrosine kinase in this line shuts off RAG-1 and RAG-2 mRNA whereas inactivation of the abelson tyrosine kinase by shifting to non-permissive temperatures results in the upregulation of RAG mRNAs 11521. Furthermore, the increases in RAG expression in this report are ce11 cycle-independent and occur in the absence of protein synthesis. Unlike the Neale et ai.

snidy, however, in which downregulation is achieved by altenng transcriptional rates and message stability, the upregulatory effect in this study appears to occur pnrnarily at the transcriptional level, as the increases in RAG-1 and RAG-2 were not found to be due to differences in RAG mRNA stability.

While the two above mentioned reports argue for a protein synthesis independent pathway of RAG mRNA regulation, Takahama and Singer have reponed that the downregulation of RAG rnRNAs upon TCR anti-6 cross-linking or ueatment with PMA in CW-CDS~ fetal thymocytes can be reversed

in situations where there is CO-incubation with cycloheximide, thereby demonstrating the dependence of de novo protein synthesis [143]. A requirement for newly synthesized proteins has also been observed in the downregulation of RAG-2 protein in DN and DP thymocytes upon stimulation with PMA and ionomycin [153]. Furthermore, other mechanisms of RAG regulation besides in vitro signaling- mediated alterations may also require de novo synthesis. For example, cycloheximide treatment of thymocytes alone has been found to increase RAG expression 6-8 fold without affecting expression of other genes such c-fos, c-mye. or TCR-0, suggesting the presence o f a RAG-specific regulatory element

constitutively expressed in pre-T cells that may have to be continually synthesized for maintenance of steady state expression because of its very rapid turnover rate [149].

The fact that de novo protein synthesis rnay or rnay not be required in different situations of RAG mRNA regulation coupled with evidence that RAG mRNAs can be regulated by controlling b a h transcription and mRNA stability suggest that RAG-1 and RAG-2 are under numerous regulatory controls. The apparent complexity of RAG regulation at the molecular level is not surprising given the apparently complex developmental expression of these genes. It is also consistent with the view that the

tight regulation of V(D)J recombinase activity is required, since lower expression rnay result in inefficient recombination of antigen receptor genes whereas increased expression rnay lead to recombination errors that are deleterious to the ceU. Additionaiîy, one might speculate that these genes rnay be transcriptionaily regulated in a manner similar to other tightly developmentally regulated lymphocyte-specific genes. In this context, the immunoglobulin loci, like the RAG genes display a tight cell-type specificity, being active only in the B ce11 lineage, and only at various stages of B-ceIl deveIopment. Furthemore, the 18 promoter-enhancer systems are among the most extensively studied and best understood models of cell-specific eukaryotic transcription. Therefore, before discussing the currently very limited understanding of RAG transcription, the cis and trans-acting elements in Ig transcription will be briefly reviewed.

i. Mammallan gene transcription: the immunogïobiilin mode1

Al1 eukaryotic mRNAs are synthesized by the multisubunit enzyme RNA polymerase II [154]. In order to reach maximal rates of transcription, RNA polymerase II requires two types of DNA sequences, termed promoters and enhancers. Promoters are located 5' of the transcriptional start site (TSS), and are required for transcription initiation. The promoters of most eukaryotic genes are associated with a "CA" dinucleotide cap site surrounding the TSS and a AT-nch conserved TATA-box

promoter elernent, located approximately 30 base pairs (bp) upstream of the transcriptional start site [155]. The TATA box, which is important in directing the RNA polymerase II complex to the TSS, rnay work most efficiently with other upstream promoter elements such as the CCAAT box and a GC rich sequence located - 40 and 110 bp upstream of the transcription start site, respectively [156, 1571. Promoters required for the transcription of immunoglobulin (Tg) genes are located 5' of al1 variable region segments, are expressed specifically in B cells, and in general require fewer than 150 bp 5' from the TSS. The most suiking feature of immunoglobulin promoters is that they contain a highly conserved octamer motif, ATi'TGCAT for iight chains, and the reverse complement, ATGCAAAT for heavy chains [158]. The octamer motif is necessary both for the efficient activation of Ig gene transcription and in confemng tissue specificity to the process, since deletion of the octamer results in complete loss of Ig promoter activity whereas addition of the octamer confers activity in non-Ig specific

promoters [159]. in addition to the octamer, other Ig promoter elements have also k e n identified. For example, a conserved heptamer motif and a pyrimidine-rich sequence, both located upstream of the octamer, and vanous "E box" motifs have been identified in the heavy chains and an octamer-proximal pentanucleotide sequence has been identified in the u chain locus [160, 1611.

Enhancers function to maximize the rate of transcription for promoters, and unlike promoters, can be located at substantial distances h m the gene k ing tranxribed. Additionally, enhancers, unlike promoters, are capable of functioning in an orientation-independent rnanner and are often tissue specific. The first eukaryotic enhancers to be identified were the Ig heavy and K chah enhancers (reviewed in

[162]). Ig enhancers are located in the introns that separate the J and C segments of these genes, a particularly strategic location considering they activate transcription hom the V region promoters that are broupht into proxirnity by gene rearrangement. The IgH locus enhancer is located in the J-Cp intron,

upstream of the switch signal, and therefore is conserved dunng heavy chain class-switching. DNA- binding repions in this enhancer include 6 "E box" motifs which share the consensus sequence GCAGXTG, three core regions and the same octamer motif present in Light chain promoten. Mutations or deletions in any of these individual regions decrease, but do not eliminate enhancer activity, suggesting functional redundancy. In the K locus, one enhancer has been identified within the intron separating the J K and C K region, and a second approximately 7 fold stronger than the intronic enhancer has been detected 9 kb 3' of the CK gene. DNA-binding regions in the intronic enhancer include the ICB

site, and three E boxes.

While the transcriptional activity of Ig loci are mediated by promoters and enhancers, the function of these regions are controlled by DNA-binding proteins hown as transcription factors. These include seven general factors required by al1 rnammalian systems for proper RNA polymerase II activity: TFIIA, IIB, IID, HE, IIH, and IIJ 11541. However, in order to achieve the required tissue and developmental specificity of Ig transcription, numerous families of transcription factors including the octarner-binding protein family, the basic-helix-loop-helix (bHLH) family (which bind to the large array of E box elements in Ig promoters and enhancers) the ets family, and the fei/hiF-~B family are required to interact with the various DNA-binding elements present in Ig regulatory regions (reviewed in [163- 1661). There are multiple levels of complexity involving interactions between these trans-acting factors and cis-acting regulatory elements that are ultimately responsible for determining the developmental and lymphocyte-restricted nature of Ig transcription. For example, within Ig promoters and enhancers both the ubiquitously-expressed Oct-1 protein and the B-ce11 specific Oct-2 protein can bind the octamer motif 11671. Secondly, a single transcription factor, such as NF-KB, may bind to multiple regulatory elements with little sequence homology to each other, both within and outside the Ig regions [165]. Thirdly, transcription factors rnay have different expression andior activity within either different tissues

or developmental stages. Again, using the octamer binding family of proteins as an example, while earlier studies had impiicated the role of Oct-1-mediated octamer activity, the recent targeted disruption of the oa-2 locus in mice suggest that Oct-2 confers octamer activity at late stages of differentiation [ 1681. These differences may not be oniy due to differences in transcription, but can occur as a result of modifications by differential splicing. For example, at least 6 Oct-2 mRNA species have been detected [169]. However, such differences can also result as a result of pst-translational modification/activation mechanism. For example, a key property of NF-K B, which is one of the most ubiquitous and pleiotropic

transcriptional activators known, is that its activation can be effected through a signal-transduction pathway ultimately resulting in the phosphorylation and consequent disruptiùn of an inactive complex consisting of NF-KB and a repressor protein designated IuB [166]. Fourthly, the tendency of numerous Ig DNA-binding proteins to f o m homo- or hetero-dimers, such as the bHLH E-motif binding factors provides even more possibilities for differential activation. In this context, a family of inhibitory proteins, the Id proteins inhibit members of the E motif-binding bHLH transcription factors family by forming inactive heterodimers with these proteins via their helix-bop-helix motifs, thereby abolishing their DNA-binding activity [170]. Finally, because Ig promoters and enhancers have sites for both negative and positive regulating factors, the net result in transcription represents the additive contributions of a large array of individual factors at a given time.

ii. Inferences about RAG transcriptionaï control

It is somewhat surprising that the RAG-I and RAG-2 genes were cloned over 4 years ago, but so little is known about what factors regulate these genes at a molecular level. To date there are no reports which have characterized RAG cis-acting elements. However, a number of inferences can be made fiom the interesting genomic structure of the RAG locus. First, because the genes are convergently transcribed, it is likely that RAG-1 and RAG-2 are under the control of independent promoters. Additionally, while the multiple species of RAG-2 mRNA may be due to alternative splicing, they may also result from multiple independent TSS and hence separate promoters 5' of each upstream untranslateci RAG-2 exon. If so, the choice as to what promoters to use would Iikely depend on unique DNA-binding elements within each promoter that confer these qualitative differences in transcription. While RAG-1 and RAG-2 rnay contain multiple promoter regions, their coexpression and CO-regulation suggests however that they are under the control of the same enhancer element(s) or may have separate enhancer regions but with common DNA-binding elements. Although enhaiicers can act over large distances, in the case of Ig transcription (for example the intronic heavy chain enhancer), it appean to work efficiently only after it has been placed in proximity to an Ig promoter following a remangement event. Analogously, if there is a common enhancer for both RAG genes, an ideal location for it would be in the 8 kb intergenic sequence. Differences in transcription, however, rnay not only be regulated

within the RAG introns, but also in untranslated exon regions. For example, a study by Gallo et. al

demonstrated that two distinct RAG-1 cDNAs (a 5.8 kb cDNA, RAG-lA, and a 6.8 kb cDNA, RAG-1B) differed in gene expression by 15 fold in luciferase assays [2q. Vanous RAG-1AB chimeras were conswcted to identify the region responsible for these differences, and interestingly, it appeared that the lower expression of the RAG-1 cDNA clone, RAG-1B could be attributed to the absence of 45 base pairs in the 5' untranslated region of this consuuct.

While the RAG promoters and enhancers structures have not yet k e n elucidated, several recent studies in which targeted dismption of transcription factorencoding genes have predicted direct o r indirect roles for their products in the transcriptional regulation of RAG-1 and RAG-2 (reviewed in [ 17 11). One candidate is the paired-domain containing transcription factor BSAP (B-cell lineage- specific activator protein) which has binding sites in the promoters of other B-ce11 specific genes such as CD 19, VpreB, and A 5 [167]. Like in RAGdeficient mice, rnice with targeted disruptions of the gene encoding for BSAP, Pax-5, are developmentally blocked at the transition fkom the large CD43+ pro-B ce11 to small CD43' pre-B cell stages [l7 11. Additionally, Pax-5 mRNA expression appears to correlate strongly with expression of both RAG genes (M. Schiissel, unpublished results). However, if Pax4 does have a role in the transcnptional activation of RAG-1 and R4G-2, it is probably not the onîy factor involved since unlike the complete remangement block of RAG knockout mice, intact heavy chain rearrangernents can be detected in Pax-Sdeficient mice 11721.

Another panicularly interesting candidate is the E lî/E47 protein complex which represents the bHLH-containing, dimer-associated, alternatively spliced products of the E2A gene. E2A-f mice have a block in B ce11 development earlier than RAG-1, RAG-2, and Paxddeficient mice, suggesting that this

factor is an important fontroi point in B-ce11 differentiation (173). Funher suggesting the importance of this gene in B ce11 development is the observation that E2A -/- mice cornpletely lack mRNA transcripts for other B-ce11 specific genes A5, CD 19, and have reduced expression of mb-1 mRNA 11741. Because E2A proteins have k e n s h o w to bind numerous sites in immunoglobulin genes (such as the E2 and ES boxes of the heavy chain enhancer and the E2 box of the K enhancer), they have k e n thought to be

critical in the regulation of antigen receptor gene expression [165]. E2A-deficient mice also lack or have reduced expression of various Ig germline transcripts [174, 1751. The role of ESA in transcnptional activation of not only the Ig loci but the RAG locus was also suggested in a study by Schlissel et al., in which a E2A cDNA clone transfected in a pre-T ce11 line significantly stimulated expression of not only endogenous gemiine heavy chain genes, but also endogenous RAG- 1 and RAG- 2 while at the same time inducing V(D)J recombination at the Ig heavy chah locus 11761. However, evidence for a definitive role of the E2A products in the transcriptional activation of the RAG locus (at least RAG-1) is the fact that EZAdeficient mice completely lack RAG-1 transcripts as well 11751. This also suggests that the RAG-1 promoters and or enhancers have E box DNA-binding motifs for the E2A

proteins to confer transcriptional activation of this locus. Interestingly, E2A-deficient mice alsa have striking reductions in the B-ce11 specific transcription factor Pax-5 [175]. Therefore, the EZA proteins may either have a direct role in transcriptional activation of the CD19, AS, and RAG loci andor may work indirectly by activating Pax-5, which in turn activates transcription of RAG and other B t e l l specific genes.

Candidates for the repression of RAG transcription have also k e n recently dexribed. It is not surprising that a recent study in which transgenic mice overexpressing the Id1 gene not only have downregulated levels of E2A uanscripts, but also have downregulated expression of ail other genes that

E2A have been shown to affect, including strikingly reduced levels of RAG-1 and RAG-2 [177]. Thus the Id proteiris appear to have a definitive, albeit indirect role in repressing RAG transcription. This indirect negative regulatory effect o n RAG transcription further demonstrates that complex transcriptional networks, like those involved in Ig transcription, are involved in regulating the RAG genes. Furthemore, since expression of the RAG genes and Ig genes are both lymphoid restricted and developmentally restricted, it is not surprising that both of these loci share common transcription factors. However, while the E2A knockout experiment indicates that these products are crucial for RAG

activation (directly or indirectly) in early B cells, it also shows that the E2A products are not crucial for TCR rearrangements, since E2A-/' mice express TCRaRCR$. The fact that the MG-1 gene does not

require E2A to become activated in the thymus suggests a separate set of transcriptional factors are necessary for RAG transcription in developing thymocytes. Figure 1.6 s u m a r i z e s the hierarchical relationship of the various putative transcription factors regulating the RAG genes with respect to the B lineage.

i. Control of message s&biîity:impiications for RAG mRNAs

The concentration of a given mRNA in the cytoplasm depends not only on its rate of synthesis, but also on its stability. By controlling the rate of mRNA turnover, cells have an additional opportunity to fine-tune mRNA levels. The range of mRNA stability in mamrnalian celis can Vary h m half lives of 20 min to over 24 h 11781. RAG-1 and RAG-2 mRNAs which have both been estimated to have half lives between 30-45 min, therefore represent mRNA species on the highly unstable end of this spectnun [140, 1521. The fact that RAG-2 mRNA has a similar stability as RAG-1 mRNA suggests that the

reduced levels of RAG-2 total RNA as compared to RAG-1 total RNA is mediated by transcriptional di fferences.

Pax-5 - VpreB - --A5

Figure 1.6. Hiemhy of pu l l i ve truiseription factors involved h the ~eylation of RAC transcription. Arrows indiate the transcriptional targets cf the E2A and Pax-5 ~ o d u c t s ,

which in addition to the RAG locus also include Bcell s p a f i c genes including V p e B , A5.

CD 19, and the Ig heavy and K chain loci. The Id proteins which function to inactivate bHLH

~ o t e i n s s uch as the E2A pducts , have been i ncluded in the diagram as indirect reprsscrs d

RAG îransaiption. Question marks mpresent the airrent uncertainty as to whether E2A

proteins function as indirect andor direct RAG transcriptional activators.

Searches for eukaryotic mRNA motifs that modulate decay rates have revealed several types of elements found throughout the length of the message (reviewed in 11791). Structures implicated in mRNA stabilization include the 5' cap structure and the 3' poly (A) tail, both of which appear to be

involveci in protecting the message fiom exonuclease digestion [179]. Various structures have also been implicated in mRNA destabilization, including 5' untranslated secondary structures, premature termination codons, open reading frame sequences and the 3' untranslated sequences. Of these sequences decreasing mRNA stability, the 3' untranslated mRNA regions have received the greatest attention.

Two well-studied examples of destabilizing elements in the 3' untranslated regions have been descnbed. The first, the bon Responsive Element (IRE), is found in the 3' untranslated region of the transfemn mRNA. The IRE contains five distinct stem-loop secondary structures capable of binding a trans-acting IRE-binding protein (reviewed in [180]). The binding affinity of this protein to the IRE sequence appears to be decreased by cellular iron, thereby destabilizing the transfemn mRNA. However, transferrin mRNA destabilization does not appear to be mediated by the IRE sequence itself, but is instead made unstable by an as yet uncharacterized destabilizing sequence. The LRE-binding protein is therefore thought to prevent association of a destabilizing factor with this undefined sequence. The second well-characterized 3' untranslated destabilizing motif is the A+U Rich Element (ARE) found in various genes exhibiting very short half-lives including the early response proto-oncogenes c-fis and c-jun and cytokine genes including human granulocyte-monocyte-stimulating factor (GM-CSF), tumor necrosis factor- f3 (TNF-p) and interleukin- 1 (IL- 1). These sequences are defined as the pentanucleotide AUUUA repeated once or several times, often within the U-rich area of the 3' unuanslated mRNA region. The initial observation that ARES conferred mRNA instability was made by Shaw and Kamen in 1986 when a 62 bp sequence of DNA representing the 3' unuanslated regions of GM-CSF mRNA were inserted into $ -$lobin DNA [18 11. The destabilizing effect of the AN-rich sequence was clearly demonstrated by a substantial decrease in half-life of the recombinant $-globin mRNA (-1 h) as compared to the wild-type 0-globin mRNA (- 10 h).

Consistent with an ARE mechanism of RAG mRNA destabilization is the presence of numerous copies of the AUUUA sequence in the 3' untranslated regions of RAG-1 and RAG-2 mRNA (Cl821 and A. Zarrin, persona1 communication). In this context, it is tempting to speculate that since signaiing- mediated destabilization has been implicated in RAG-1 and RAG-2 downregulation in the studies of Takahama and Singer and Neale et al., ARE-binding proteins may be either newly synthesized or post- translationally activated via a PKCdependent signaling pathway [143, 1491.

The RAG proteins may also be under numerous regulatory controls. For example, activity and stability of the RAG-2 protein appear to be controiied by two separate phosphorylation pathways. First, it has been found that phosphorylation at an acidic domain site of RAG-2, ~er356 by casein kinase II enhances RAG-2 activity in vitro [183]. Furthermore, a mutation at Ser3s6 decreases recombinase activity in vivo in the context of extrachromasomal recombination substrate assays, without affecting its nuclear localization or steady state protein levels. Secondly, phosphorylation of RAG-2 at wm results in rapid degradation of RAG-2 protein [183]. Subsequent ce11 cycle analysis of RAG-2 protein expression has demonstrated RAG-2 protein expression to be a Go/Gl resaicted phenomenon, suggesting that RAG-2 exists as an unstable phosphoprotein for a large portion of the celi cycle 11841. A similar ce11 cycledependent pathway of RAG-2 protein regulation has also been observai in avian thymocytes 11531. The G&l accumulation of the RAG-2 product is consistent with other reports demonstrating the restriction of RSS-double strand break products and V(D)J rearrangements in thymocytes to the CdG 1 phase of the ce11 cycle [107], and suggests that phosphorylation-dependent RAG-2 destabilization may be a mechanism by which V(D)J recombination is restricted to Gû/Gl. Furthermore, the Lin and Desiderio study suggests that RAG-2 protein may preferentially accumulate at this stage through regulation of a cell cycle related kinase such as the cyclin family kinase p34&* which has been shown to phosphorylate RAG-2 at ~ h # ~ ~ in vitro [184]. Imponantly, this report also demonstrated that ce11 cycle does not produce alterations in RAG-1 protein levels or in RAG-1 or RAG- 2 total RNA expression. One caveat with attributing the importance of RAG-2 destabilzation in restricting V(D)J recombination to Gû/Gi is that while the ~ e r ~ ~ ~ site appears to be required for recombination in extrachromasomal recombination assays, the MW site in this context does not have any significant effect on recombination [18, 20, 21, 1831. However, it can be argued that under conditions of transient transfection, RAG-2 protein expression is not limiting, but in lymphoid progenitor cells it is, both because the levels of RAG-1 and RAG-2 protein are lower than in transientiy transfected fibroblast cells (S. Desiderio, unpublished results) and because RAG-2 expression is significantly lower than RAG-1 at the mRNA level [9].

3. Project Objectives

The overall objective of this study was to contribute to the understanding of mechanisms influencing RAG-1 and RAG-2 mRNA expression. For two principle reasons, the OC1 LY8 human mature B ce11 culture system, which has previously been extensively used in our lab for the charactetization of secondary rearrangements at the human A locus, was chosen for such studies [142].

First, it continues to express RAG, both in the absence and the presence of surface immunoglobulin, thus allowing the detailed study of sIg signaling-mediated regulation of RAG-1 and RAG-2 expression. Secondly, unlike many RAG-expressing ce11 lines, the OC1 LYS cell culture system expresses RAG mRNAs stably both between variants and with respect to time in culture, thereby increasirig the interpret ability and reproducibility of experimental results.

In the previous section, 1 have described a number of reports, which cumulatively, ptovide evidence that RAG mRNA expression can be regulated by signals delivered through the BCR. Therefore, the fint specific objective o f this study was to identify and characterize the regulatory link between signaling through the surface immunoglobulin receptor and RAG expression in OC1 LY8 sIg+ variants. The generai approach to achieving this objective was first to generate signais either by anti-p- mediated BCR-ligation or by direct activation of BCR-associated second messengers with the combination of a phorbol ester and a calcium ionophore in three representative OC1 LY8 clones: a BCR+RAG" clone, a BCR+RA@ clone, and a BCR-RA@ clone. For each of these variants, RAG-1 and RAG-2 RNA expression was quantitatively compared between induced and uninduced clones by the combinat ion of Northem blotting and phosphorimage analysis.

Various reports have provided evidence that RAG gene expression is mediated by numerous levels of regulatory controls (reviewed in Table 1.1). Thus, the second objective of these studies was to characterize the contribution that various molecular regulatory mechanisms may have in regulating OC1 LY8, in particular the contribution that such mechanisms may have in the constitutive differences between RAG and R A G ~ clones and in differences between induced and uninduced sIg+ clones. In order to do this, nuclear nin-on transcription assays and Actinomycin D transcription inhibition studies were perfomed to assess the contribution by transcriptional and post-transcriptional mechanisms respectively. Funhermore, cyclohexamide experiments were perfomed to determine the requirement for factors involved in both inducible and constitutive differences in RAG to be newly synthesized.

In Chapter 3,1 present evidence that in sIg+ OC1 LYS variants, RAG expression is upregulated in a dosedependent, tirnedependent, specific, and revenible fashion. This effect can also be seen when second messengers in the BCR signaling pathway are directly activated. 1 also provide data supporting the role of both increased transcription and increased message stabilization in the upregulation of RAG (both induced and constitutive), and that in the constitutive differences, but not inducible ones, tbese effects are dependent on new protein synthesis. Chapter 4 contains a general discussion including suggestions for future experimentation based on the data presented in Chapter 3.

Table 1.1 Overview of putative factors involved

Factor - Eflect on RAG ex~cssion

1 .A nl i~en-recepfor Iiuation

BCR ligation

in vitro: anti-p cross-linking mRNA don nrcpu1atic.m il, viw: negatil-e sclwtion mRNA uprcgulation

TCR ligation

in vitro: anli-CD3 cross-l inkinp mRN A dotmrcgulation anti-TCR cross-iinking mRNA dot\-nrcpulaticin

in vivo positive xl<siion mRVA downrcgutar ion

2. Kirruses

abclson tj-rosinc kinasc mRNA do~nrcgulaiion

, ,MC~C~ knuc EWG-2 proccin dort-nrcgulation

3. Transcription faciors

E3A prduccs mRNA uprcgulatim

p h o r b l ester + calcium iunophorc

mRNA uprcguiatim

mRNA dot\ nrcgulaiion

mRNA uprcgulation

42 in RAG-1 and RAG-2 gene regulation

- protcin dcsr;ibilizition via 153. 1 8 3 . 1% phosphon lation or Thf13" -ccllcyclc&pcn&nt (prcfcrcntial dcgradariim in G2;S ) - RAG- 1. MG-? rnRNX, RAG- 1 proiein caprcssion

cc11 cyclc indcpcndcnt

truwxiplionril activation

inhibition of E2A (Jimcriiration \t ith bHLH)

PKC iranslocatim and cstncciluiar calcium flux

- PKC translocation - üanscripional rcprcsion - transcripi stabilimtion - de now indcpcndcnt - de IIOW dcpcndcni

CHAITER 2: MATERIALS AND METHODS

1. Ce11 variants and tissue caitwe

The cell line OC1 LY8-C3P (aih+) is a singleceil clone fiom the OC1 LY8 ce11 line (IgM+AgD-

/CD1OC/CD19+/C~20+/CD38+), a ce11 line originally established from a patient with a B lymphoid diffuse large ce11 lymphoma [185]. sIg clones were isolated fiom the OC1 LY8Ç3P ce11 line by fluorescence activated ce11 sorting (FACS) and plated in 96weli microtitre plates at limiting dilutions (0.1 cell/well) [ 1421 . Spontaneously arising slg+ld- clones were isolated nom these expanded sIg- populations by plating at limiting dilutions. Al1 cells were routinely cultivated at 37OC 15% CO2 in RPMI 1640 medium (ICN, St-Laurent, Quebec, Canada), supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 2mM L-glumine, 100 U/ml penicillin, and 100 p g m l streptomycin (Life

Technologies, Inc., Gaithersburg, MD).

II. Antibodies and pbarmaco1ogicaï agents used in signaiing stuclies

Antisera used for various cross-linking experiments included an affinity-punfied polyclonal goat anti-human p F(ab)'2 antibody or a control F(ab)'2 anti-u antibody (Tago, Buriingame, CA). Direct stimulation of second messengers PKC and Ca2+ was achieved with the combination of phorbol-12- myristate-13-acetate (PMA) and ionomycin (Calbiochem, LaJolla, CA). For experiments using actinomycin D, 5 p@ml actinomycin D (ICN) was added for various durations to unstirnulated cells or to cells that had been cross-linked with anti-p. For cycloheximide experiments, cells were incubated in 10 pgml cycloheximide (Sigma, St. Louis, MO) for various durations. Stock solutions of PMA and

ionomycin were solubiiized in dimethylsulfoxide. Actinomycin D was solubilized in ethanol.

m. Surface immunofluorescence analysis

After being washed in phosphate buffered saline (PBS), 1 X 106 cells were pelleted and stained with the appropriate pnmary monoclonal antibody (MAb). Following incubation on ice for 30 min, the cells were washed twice in serum-fiee medium, stained with a fluoresceinated secondary secondary goat anti-mnme (GAM) IgG 1 antibody (Jackson Immunoresearch Laboratories, West Grove, PA), and again incubated on ice for 30 minutes. Finally, the cells were washed twice in serum-free medium, resuspended in 0.5 ml of 2% paraformaldehyde fixative and analyzed within 24 h on an EPICS profile flowcytometer (Coulter Corporation, Hialeah, FL). For characterization of OC1 LY8 varianu, the primary MAb used included: 1.D.12. (anti-fi), 3.F.l. (anti-A), 22:7 (anti-Id), and 14.D.1. (anti-Oz'3gA

isotype). For assessing B ceU activation, the anti-HLA-DR and IOT14 (anti-CD25) prirnary MAbs were used (AMAC, Westbrook, ME). For assessing B cell proliferation, the IOA7 1 (anti-CD71) primary MAb was employed. The secondary goat anti-mouse IgG 1 FITC antibody (Jackson lmmunoresearch Laboratones) alone was used as a negative contml.

IV. Measurement of intraceMar ~ a 2 + and prolireration

For measurements of proliferation, ceiis were cultured in flat-bottomed 96-well rnicrotiue plates (Linbro, McLean, VA) at a ce11 density of 2 X 105 celis/ well, in the absence of stimuli or in the presence of either F(ab)'2 anti-p, F(ab)'z anti-K, or PMA and Ionomycin. Afier 66 h, 1.0 (ici of PH] thymidine (6.7 Ci/mmol; ICN) was added to the culture, and the cells were allowed to incubate for another 6 h. The cells were then harvested ont0 glass fiber mats (Skauon, Lier, Norway) using a Skatron 2000 ce11 harvester. [ 3 ~ ] thymidine uptake was determined on triplicate samples in a Waiïac 1410 liquid scintillation counter (Phannacia, Piscataway, NJ). Changes in intra-cellular ~ a * + were assessed by loading 5 X 106 cells with indo-1 dye. After an incubation with 2 p M of acetoxymethyl

ester for 20 min at 370C, cells were washed and suspended in ca2+tontaining buffer. Once a baseline for caZC had been established, F(ab)'2 anti-p or PMA and ionomycin were added and ca2+

concentration was calculated fkom Indo- 1 vio1et:blue ratio.

V. Protein analysis

Western blotting was carried out according to the protocol supplied with the ECLf kit (Amersham, Arlington Heights, IL). Briefly, 10' cells were lysed in 300 pl lysis buffer containing 10

mM Tris-Hcl (pH 7.4), 150 mM NaC1, 5 mM EDTA, 1% Triton X- 100, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.23 U/ml aprotinin. The lysate was incubated on ice for 10 min, spun down in a microfige and the supernatant was recovered. 10 p l of the sample was size fractionated on a 15% SDS-

PAGE gel and electroblotted ont0 a nitrocellulose membrane (Schleicher & SchueU, Keene, NH). The membrane was blocked according to the instructions from Amersham, hybndized for 1 h to the polyclonal anti-human phosphotyrosine antibody (Transduction Laboratories, Lexington, KY), washed according to the suppiied protocol and hybridized to a secondary goat anti-rabbit, IgGl HRPanjugated MAb. The membrane was developed using the ECLm system, with exposure times ranging h m 15 sec to 1 minute.

VL RNA isolation and Northern BIotting

Cells in log-phase growth (5 X 106 celldml) were incubated with various reagents and then harvested at specific tirne points for isolation of total RNA. Total RNA was extracted by the single-step guanidium thiocyanate phenol-chloroform extraction procedure [lw. 10-20 p g of RNA was resuspended in 50% formamide, 6% paraformaldeyde, 0.02% XC-bromophenylblue, 1X 3-(N- morpholino)propanesulfonic acid (MOPS), and e1ectrophoretically size fractionated in a 6% formaldehyde, 1X MOPS, 1% agarose gel. The RNA was transferred ont0 a Zeta-probe nylon membrane (Bio-Rad. Hercules, CA) by ovemight capillary transfer in 10X SSC. Membranes were then cross-linked in a UV stratalinker (Stratagene, LaJolla, CA), pre-hybridized, hybridized to the appropriate probe, washed and exposed to X-ray film (Kodak, Rochester, NY) at -70°C. Al1 technical procedures were according to the protocol supplie. by the manufacturer of Zeta-probe. The intensity of the hybridization signals were quantitated by scanning densitometry (LKB scanning densitometer, Stockhoim, Sweden). The RAG densitometric values were nonnalized to actin levels to control for variations in sample loading. For probing of Nonhern blots, the foilowing [ a - 3 2 ~ ] d ~ ~ ~ - l a b e l e d cDNA

probes were used: 1) a 0.9 kb RAG-1 fragment generated by XhoI and HindIII digestion of the full-

length 6.6 kb cDNA clone supplied by Dr. D. Schatz, Section of ïmmunobiology, Yale University School of Medicinell 11 ; 2) a 0.6 kb RAG-2 fragment generated by NotI and SaiI digestion of the 2.1 kb RAG-2 cDNA provided by Dr. L. Turka, Department of Medicine, University of Michigan 11 181; 3) a 0.7 kb human c-fos fiagment generated by NcoI and Pst1 digestion of a PC-fus (human)-1 clone obtained from the American Tissue and Culture Collection; and 4) a 1.0 kb cDNA probe specific for the human 6-actin gene, obtained from Dr. N. Lassam, Department of Medicine, University of Toronto.

VIL RT-PCR analysis

To remove contaminating chromosoma1 DNA, 50 pg total RNA was incubated for 30 min at 37OC with 20 U of human placental ribonucease inhibitor (RNAsin) (Phamacia), 20 U of RNAse-fkee DNAsel (Pharmacia) in 10 mM Tris-CI, pH 8.3, 50 mM KCI, 1.5 mM MgCl2. After extraction once with phenol/chlorofonn/isoamyl alcohol(25:24: 1) and once with chloroformlisoamyl alcohol(24: l), the supernatant was precipitated in ethanol in the presence of 0.3 M NaOAc and RNA was dissolved in diethyl pyrocarbonate-treated water. cDNA was prepared usine the "First-Strand cDNA Synthesis Kit"

(Pharmacia). PCR amplification of five fold dilutions of cDNAs involved the following: a 5 min., 9 4 O ~

initial denaturation step followed by 29 cycles of 1 min at 9 4 * ~ , 1 min. at SOC, 1 min at 7 2 O ~ , and a 7

min., 7 2 ' ~ final extension step. The primers used for PCR amplification are as foliows: RAG-1; RAG- 1A = 5'- CAG CGT TIT GCT GAG CTC CT -3 ' , RAG-1B = 5'- GGC TIT CCA GAG AGT CCT CA -3', RAG-2; hR2A = 5'- 'ITC TTG GCA TAC CAG CAG -3', hR2C = 5'- CTA TIT GCT TCT GCA CTG -3' , fhbulin; Tub-5' = 5'- CAG GCT GGT CAA TGT GGC AAC CAG ATC GGT -3', Tub-3' =

5'- GGC GCC CTC TGT GTA GTG GCC GGC CCA -3'. The following [y*?-labeled oligonucleotides were used for detection of RAG-1, RAG-2, and $-tubulin PCR producü, respectively:

RAGlT = 5'- AAG TAT AGG TAT GAG GGA A -3', hR2B = 5'- GAG TCT TCA AAG GGA CTC G -3', and TubP = 5'- ACC TGA GCG AAC AGA GTC CAT G -3'. The sequences of above mentioned oligonucleotides are derived nom [187]. Using these primes, PCR amplification products for human RAG-1, RAG-2 and 0-tubulin are 364, 192, and 302 bp, respectively. Controls included: no reverse transcriptase added in the cDNA synthesis step, fbbulin as an intemal standard, and dilutions of the 6.6

kb human RAG-1 and 2.1 kb human RAG-2 cDNA fragments, both previously described.

VIII. Nuclear =-on transcriptional d y s i s

Approximately 2 X 107 cells in log phase were washed once in PBS and resuspended in 1 ml buffer solution, containing 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, and 0.3 M sucrose.

Cells were lysed by adding 1 ml of the same buffer containing 0.04% Nonidet P-40 (Sigma) and incubated on ice for 5 min. Nuclei were isolated by centrifugation at 400 g for 5 min at 40 C, resuspension, incubation on ice for 10 min, and pelleted through a 30% sucrose cushion by another round of centrifugation at 400 g for 5 min at 40C. The nuclei were resuspended in 125 p l storage buffer,

containing 25 mM Tris-HCl pH 8.0, 125 KCl, 6 mM MgC12, 2.5 mM dithiothreitol and 30% glycerol, and kept at -700C for up to 2 weeks before k i n g used. For in vitro transcription, nuclei from each sample were incubated with continuous mixing for 30 min at 270C in 75 pl of a reaction solution containing transcription buffer (300 mM (NH4)2S04, 100 m M Tris-HCl pH 8.0, 4 mM MgCl2, 4 mM C12,200 mM NaCl, 400 PM EDTA, 100 pM PMSF), 1 mM each of ATP, CTP, and GTP, 1 mM Dm, 10 mM creatine phosphate, 40 U RNasin (Pharmacia), and 50 pCi [aJ2~]-labeled UTP (300 Ci/ mmol; ICN). Transcription was stopped by adding 24 p g of RNAse-free DNAse 1 (Phamacia) for 10

min at 370C. Samples were adjusted to 1% SDS, 5 mM EDTA, and 10 mM EDTA and were incubated with 300 @ml Proteinase K for 30 min at 420C. Labeled RNA was then isolated by the single step

method [21], denatured, and hybridized to 5 pg of denatured plasmid DNA, which was immobilized

ont0 nitroceilulose membranes (Schleicher and Schueli) usine a dot-blot apparatus (Schleicher and Schuell). Hybridization was camed out in a solution containing 40% formamide, 1X Denhardt's solution, 4X SSC, 5 mM EDTA, 0.4% SDS, and 200 puml salmon spem DNA for 3648 h at 42%.

Membranes were sequentially washed with IX SSC and 1.0% SDS for 15 min at 5S°C, 0.2X SSC and

0.2% SDS for 15 min at 55W, 2X SSC and 20 j@ni RNAseA for 15 min at 370C. and 2X SSC for 15 min at 37OC. Blots were then visualized by autoradiography as previously described. Quantitauon of RAG-1 and RAG-2 signals was performed on a ~ h o s ~ h o r ~ m a ~ e r @ (Molecular Dynamics. Inc., Sunnyvale, CA ) with the ha8eQuant@ software package provided by the manufacturer. The foiiowing lineanzed plasmids were used as probes for nuclear run-on analysis: 1) the phagemid pBluescript SK- (Stratagene) containing the 6.6 kb RAG-1 cDNA f iapent ; 2) the phagemid pBluescript SK- containhg the 0.6 kb RAG-2 cDNA fragment; 3) the pUCl l8 plasmid containing the 1.0 kb 0-aain cDNA fragment; and 4) the pBluescript SK- and pUC118 vectors as negative controls for non-specific hybridization.

CHAPTER 3: RESULTS

1. Dwerential RAG expression in c iod iy related variants undergohg secondary IgA gcne rearrangements

We have previously documented that the parental ce11 line OC1 LY8-C3P undergoes secondary non-producave Igh gene rearrangements on its productively rearranged IgA ailele, and thereby gives nse to s I g variants at a rate of 1.3 X 10-%ell/generation [142]. In contrast to the

parental ce11 line, which stably maintains low levels of RAG expression, these s I g variants have significantly upregulated levels of both RAG-1 and RAG-2 expression. Cenain sIg' variants, such as ASN, continue to undergo yet additional Ig h gene rearrangements through which they are able to regenerate sIg expression (sIg+ld3 at a rate of 2.7 X 10-4/cell/generation. in contrast to the SI?+ parental ce11 line, aU sIg+ki- clones maintain elevated levels of RAG expression, similar to those observed in the sIg- vanants. These characteristics make this ce11 culture system suitable for studying the effects of signals transduced through the slg receptor on the expression of the

RAG genes as well as for studying the molecular basis for the differential expression of RAG mRNAs between variants within the OC1 LY8 cell line.

In order to study the regulation of RAG expression in this ce11 culture system, 1 chose three representative OC1 LY8 variants for further analysis: the sIg+/RAGb parental cell line, OC1 LYS-C3P, an s ~ g + / R A ~ h i clone (A8-6P) and an S I ~ - / R A G ~ clone (C3-AL LN). Figures 3.la and 3. l b show representative Northern and RT-PCR analysis, respectively, of RAG mRNA levels in these variants. In al1 such experiments, both the C3-A 1 IN and A8-6P variants almost invariably expressed 15-20 fold more RAG-1 and RAG-2 mRNAs than the parental clone, as determined by phosphorimaging. The C3-Al IN clone was selected instead of the C3-A8N variant because it exhaustively rearranged both its alleles and therefore cannot undergo funher I g A rearrangements in culture, whereas C3-A8N continuously generates sIg+ki- cells, thereby

creating a heterogeneous population. Representative surface immunofluorexence profdes of the

clones with respect to sIg phenotype is depicted in Fip. 3.2. A summary of the clonal relationship between the variants used in this study is depicted in Fig. 3.3.

Figure 3.1. Comparison of RAG mRNA expression levels between OC1 LYB-

C3P parental cell line and clonally related variants A8-6P and C3-AIIN. A :

Northem hybridization o f 10 pg totai RNA ernploying a 0.9 kb human RAG- 1 cDNA fragment

and a 1.0 kb human p-actin cDNA fragment as probes. The increases in RAG-1 mRNA

signals in C3-A1 IN and A8-6P as compared to the parental d o n c was 20.8 fold and 16.4 fold.

respectively, a s quantitated by Phosphorimager analysis. The T lymphoblastoid ce11 line CEM

is a negative control for RAG expression. B: RT-PCR analysis of RAG-1 and RAG-2

expression in OC1 LY8-C3P and C3-A 1 1 N. The amplitïca~ion product sizes Cor RAG- 1.

RAG-2. and P-tubulin are indicaied to the left Convols included: no reverse iranscriptase

added in the cDNA synthesis step (-Rn. P-tubulin as the internat standard, and dilutions of

human RAG- 1 and RAG-2 plasmid cDNA fragments as positivc controls.

Figure 33. Schematic summory of clonal relationship in OC1 LYS cd1 culture system

between RAG~O parental clone OC1 LY&C3P and R A G ~ ~ variants C3-AllN and AS-

6P. Arrows point to clonal variants generated through secondaiy rearrangements at the h Iight chain in culture (For detaiis, see 11421). The C3-A8N variant is included because it

cives rise to the A8-6P variant. C3-A8N was originally thoughc to be dg-. but hÿs recently C

k e n shown to surface express a receptor termeci sIgACL comprised of a truncated h chain

in association with p chah a low IeveIs 11921. Unlike C3-A8N, the C3-Al LN variant

cannot undergo further rearrangernents.

II. Upreguiation of RAG expression in OC1 LY8 variants upon sIg cross-iinking

The sIg+ variant clone A8-6P continued to express high levels of RAG expression despite regeneration of sIg expression (Fig. 3.la). This suggested that expression of surface Ig alone was not sufficient to terminate RAG expression. 1 hypothesized that a signal transduced through the sIg receptor complex was required. To test this hypothesis, an F(ab')2 ami-p antibody was used to cross-

link the surface immunoglobulin receptors on these cells. 1 found that cross-linking the antigen receptor of OC1 LY8-C3P or A&6P results in the upregulation of RAG-1 mRNAs. This increase peaked by 12 h of antibody ueatment in OC1 LY8-C3P and withîn 6 h in AS-6P (Fig. 3.4a). This effect was dose-dependent and, usine an incubation period of 12 h, was maximal at 10 of anti- p antibody (Fig. 3.4b). The F(ab)'2 ami-p antibody mediated its effect specifically through the sIg receptor, as cross-linking the sIg' variant A 1 1N did not affect RAG expression (Figs. 3.4a, b).

The induced increases in RAG- I mRNA may have represented an irreversible differentiation event. In order to test if upregulation of RAG-1 was a reversible event, cells were cross-linked for 12 h, after which they were washed and re-plated in fresh medium for various periods of tirne. RAG-1 expression remained increased after 24 h, but returned to basal levels by 36 h (Fig. 3.4~).

However, the reversibility of the effect was dependent on the absence of antigen receptor ligation since increased RAG expression persisted even after incubation with anti-p for 48 h.

To assess whether or not these effects on RAG expression were mediated by ca2+ mobilization and/or PKC activation, the cells were incubated with either PMA, ionomycin, or the combination of PMA and ionomycin for various times. Stimulation with either PMA or ionomycin alone at various concentrations was not enough to upregulate RAG expression (data not shown). However, stimulation with the combination of PMA and Ionomycin resulted in increased RAG-1

mRNA levels in al1 variants, including the sIg- variant C3-A 11N (Fig. 3.5). The effects were found to be time-dependent, with maximal increases detected at 6 h. Concentrations less than 5 nM PMA and 500 nM ionomycin did not mediate significant changes in RAG expression (data not shown). These results suggest that both PKC translocation and increased ca2+ levels are required intermediate events in the increase of RAG-1 expression by signal transduction through sIg cross- linking.

The kinetics of the increases in RAG-1 expression as quantitated by phosphonmaging are shown in Table 3.1. In most situations, peak increases are modest: 4 fold in response to anti-p noss- linking and 7 fold in response to activation of secondary messengers with PMA and ionomycin, but appear to be reproducible.

Figure 3.4. Time-dependence, dose-dependence, specificity, and reversibility

of increases in OC1 LYS RAG-1 transcripts as a result of anti-p cross-linking.

Northern blot analysis of A: OC1 LY8-C3P, C3-A11N. and A8-6P variants that have k e n

cross-linked with 10 p d m l soluble mu-p for various times (h). B: OC1 LYS-C3P, C3-A11 N,

and AS-6P variants that have been cross-iinked at various conceiiwations (pg) of soluble anti-p

for 12 h. C: OC1 LY 8-C3P parentai clone either cross-linked with 10 j@ml anti-p for 12 or

48 h. or washed and replated in tiesh medium alter having becn cross-linkcd for 12 h. Ail

figures are representative of at Ieast three independent experirncnts. In al1 cases IO pg of total

RNA was used. Exposure times for RAG-1 are 12 h for A and C and 36 h for B. whereas

exposure times for P-actin are 6 h for A and B and 12 h for C.

Actin

Figure 3.5. Increased RAG-1 expression in response to direct activation of

BC R signaling-associated second messengers. Representri tive Northern blot of 10 pg

total RNA from OC1 LYS-C3P, C3-A1 IN, and A8-6P variants that have k e n either left

unstimulated, or have k e n stimulated with 5 nM PMA and 500 nM ionomycin for 3 or 6 h.

Exposurc tirnes for RAG- L and bactin are 18 and 6 h. respectively.

Table 3.1 Swnnuay of efects of signaling hrough the d g receptor anà the arsociated PKCpmhway on RAG-1 expression

Fold increase in RAG- 1 mRNA a

sIg+ sIg- Signaling methW StimuIus Duration of Stimulus ern~loved Concentration S r n a t i o n fil OC1 I .Y 8-C3P A8-6P C3-A11N

sIg cross-linking 10 ~ @ d 6 1.52 0.7 3.1 2 1.1 1.1 2 0.1 soluble F(ab) > anti-p 12 3.820.9 3 . 9 2 0 . 4 1.020.1

24 4.0 f 1.1 3.7 2 0.5 0.9 2 0.2

-

10 P!W 12 3.9 f 1.1 ND^ 1.120.1 48 3.4 2 0.4 M) 1.0 2 0.1

12 + 24 h wash 3.3 2 0.8 ND 1.0 20.1 12 + 36 h wash 0.9 2 0.2 ND 0.9 2 0.2

PKC, ca2+ activation 5 nM + 500 nM 3 3.220.7 3.420.0 3.620.3 PMA + ionomycin 6 5.621.3 6.921.2 7.121.4

12 4.6 2 0.9 5.4 2 1.1 5.5 2 0.8

a Values are densitmetric ratios of RAG-1 message norrnalized to actin levels and in aii instances represent the mean 2 S.E.M. of at least 2 independent experiments.

ND, not determined

Because of the unexpected result that RAG mRNA levels were further induced instead of shut off upon sIg engagement, we were interested in deterrnining what effect cross-linking the

antigen receptor of OC1 LY8 had on various defined signaling outcomes. To do this, the effects of cross-linking on several events associated with B cell signal transduction were assessed. These events included proximal events (ca2+ mobilization and tyrosine phosphorylation), intermediate signaling events (c-fos mRNA induction), and distal signaling events (DNA synthesis, CD25, CD71, and HLA-DR expression). Table 3.2 shows that when F(ab)'~ anti- p

was used to cross-link the sIg+ OC1 LYS variants OC1 LY8-C3P and A8-6P, increases were observed both in ca2+ and in surface HLA-DR levels, whereas decreases were observed in DNA synthesis. These effects were not seen in the sIg' clone C3-A 1 IN, confirming the specificity of the effects of the anti-p antibody ernployed. When the combination of PMA and Ionomycin was used, similar effects were seen except that increases were also observed in C3-A11N.

Importantly, the greatest effects in al1 three parameters were obsemed in the sIg+ variants OC1 LY8-C3P and A8-6P when 10 pgml of Ab was used (Table 3.2). Furthemore, the combination of 5 nM PMA and 500 nM ionomycin generated the most significant changes in the signaling events being assessed (Table 3.2). Thus, concentrations of F(ab)'2 anti-p and of PMA and

ionomycin that yielded the maximal effects on RAG upregulation also yielded the maximal effects on these sisnaling events.

Cross-linking OC1 LYS-C3P with 10 pgml anti-p also produced increases in other parameters such as tyrosine phosphorylation by 1 min. (Fig. 3.6a), c-fos mRNA induction between 15 and 30 min. (Fig. 3.6b), an increase in the activation marker CD25 (the a chain of

the IL-2 receptor) at 36 h (Fig. 3.6~) and a decrease in the proliferation marker CD71 (transferrin receptor) at 36 h.

Table 3.2 Summary of Nects by various stimuli on proliferation, su@uce HLA-DR expression, a d intra-ceüub w+ leueh

Parameter Stimulus

OC1 LY8 variant

s Ig + sIg-

OC1 LYS-C3P A8-6P C3-A11N

Proli fer ationa 10 nM PMA 1 pM Iono 1 nM PMA, 100 nM Iono 5 nM PMA, 500 nM Iono 0.1 pg/ml ami-CI 1 pg/mi anti-p 10 p@ni anti-p 10 pghi anti-K

surface HLA-DR media alone expressionb 5 nM PMA, 500 nM Iono

1 puml anti-p 10 pgml anti-p 10 pg/ml anti-ic

invacellular CaZ+c 1 pM Iono 1 p@mi ami-p 10 pg/d anti-p

-

a Results represent the averages of three independent experiments and are expressed as %

growth inhibition, which is 100% X 1- [(cpmew)/(cpmmedU The standard emr mean (S.E.M.) for each data point was in a11 cases 2 1 1% of the mean. b Results are shown as mean fluorescence intensity (MF9 2 S.E.M of the fluorescence histogrms and in ail cases represent the averages of at least nkro independent experiments. c data are shown as the increase in [ ~ a * + ] (nM) h m baseline levels and is IzprWnted as the mean 2 S.E.M. of two independent experiments. d ND, not detemiineù

Figure 3.6. Effects of anti-p cross-linking on various signaling events in OC1

LYS-C3P and C3-A11N. A: Western blot analysis of total cellular protcins incubated with

a polyclonal anti-phosphotyrosine antibody. Cells were either Ieli unstirnulated, or stimulated

with anti-p for 1.3, or 5 min pnor to protein extraction. B: Northern blot of ç-fos expression.

10 pg of total RNA was exuacted from OC1 LY8-C3P and C3-AI I N cells that were eithèr

unstimulated or anti-p cross-linked for 15, 30, or 60 min. Afiér hybridizütion with a 0.6 kb

human egos probe, the membrane was stripped and re-hybridizcd with a human P-actin probe

to adjust for loading differences. Exposure tirnes for c-/os and p-actin are 72 and 6 h.

respectively. C: surface detection of CD25 and CD7 1 expression before and aîter anti-p cross-

linking for 36 h. In al1 instances, the optimal concentration (10 pg/ml) of anti-p was

em ployed.

c-fos

fiilcd 2. CAM-FITC alone - unstiinulatcd - stimulated

III. Both transcriptional and ans message stabililntioa contribute to the upregulation of RAG expression

Nuclear run-on assays were performed to determine whether the increased levels of RAG

mRNA seen after antigen receptor cross-linking was due to an enhanced transcription rate. Fig.

3.7a shows that significant increases in RAG-1 and RAG-2 transcription were seen after antigen

receptor cross-linking in the sIg+ ce11 variants. However, an elevated rate of transcription was

not seen after cross-linking the sIg- C3-A1 LN variant. The various increases in RAG-1 and

RAG-2 transcription as quantitated by phosphorimaging are represented in relation to the

unstimulated parental clone OC1 LYS-C3P in Fig. 3.7b. 1 also assessed whether antigen receptor

cross-linking would alter the stability of RAG transcripts. Fig. 3.8a shows that in the presence of

the transcriptional inhibitor actinomycin D, RAG-1 transcripts in OC1 LY8-C3P persisted for a

Ionger period of time after antigen receptor cross-linking as compared to unstimulated cells.

Specifically, the M G 4 rnRNA t1/2 increased approximately 2 fold (from 15 min to 30 min)

after antigen receptor cross-linking as quantitated by phosphonmaging and subsequently

graphically represented in Fig. 3.8b. Similar increases in transcript stability were seen for the

dg+ A8-6P variant (data not shown).

Because RAG rnRNAs were constitutively upregulated in the RAG" variants C3-A 1 1 N and A8-8P as compared to the RAG~O parental clone OC1 LY8-C3P, 1 was interested, as in the

signaling-induced upregulation of RAG, in determining what regulatory mechanisms mediated

this effect. Interestingly, both increased transcription and increased message stability also appear

to be involved in the increased M G - 1 and RAG-2 steady state RNA levels present in C3-A 1 1 N and A8-6P. In particular, >5 fold increases in transcnption rates (Fig. 3.8b) and >3 fold

increases in M G - 1 half-lives can be seen in both C3-Al1N and AS-6P (Fig. 3.7b). In overall

terms. the quantitative phosphorimaging analysis appean to suggest that in both types of

upregulation (induced and constitutive), increased transcription in general plays a Iarger role than

message stabilization.

Figure 3.7. Quantitative nuclear run-on analysis of di fferences in RAG- 1 and

RAG-2 transcription rates between unstimulated and anti-p cross-linked OC1

LYS-CJP, C3-AllN, and AS-6P clones. A : Unstimulated cells are denoted by (-)

wheteas 12 h anti-p (10 pg/rnl) cross-linked cells prior to nuclei extraction are denoted by (+).

B: Quantitation of differences in transcription rates was performed by caiculating RAG-1/k

actin and RAG-2@-act.h ratios and then normalizing these values to the unstimuiaied OC1 LY8-

C3P RAG- llp-acPn and RAG-2P-actin ratios. respectively. Quantitation of RAG-1 and

RAG-2 signals was performed on a Molecular Dynamiçs phosphorimager using thc

ImageQuantT" software program. Results are repmntative of t h e independent exprriments.

+ RAGE

RAG-2

0 Actin

Bluescri pt pLTCll8

mu- p

variant

Figure 3.8. Actinomycin D studies of RAG-1 mRNA half-lives in OC1 LY8

variants. A: Companson of mRNA hall'-lives in unstimulaied RAG~o parental clone OC1

LY 8-C3P (-). OC1 LY8-C3P that had b e n cross-linked with 10 p g m l for 12 h. OC1 LY8-C3P

(+). and unstirnulated R A G ~ ~ varïants. C3-A11N (-) and A8-6P (-). Both unstimulated and

cross-linked cells were then subjected to the 5 p g m l of the transcriptional inhibitor actinomycin

D for specific tirne points (min) pnor to RNA extraction. Time of exposure for RACL 1 in OC1

LY8 (-) and OC1 LY8 (+) was 8 days whereas in C3-A11N and A8-6P it was 1 0 h (delikrate

ovemxposure was nccessary in the parenial RAG1° clone in ordcr io visualize the RAG- 1 half-

life pattern. Northern blots in al1 situations are loaded with 15 pg total RNA. B: Graphical

representation of RAG-1 mRNA half-life data- RAG-1 mRNA hdf-lives were calculated by

lirst deterrnining RAG- ilp-actin ratios for each time point and Lhcn norrnalizing al1 time poins

to the O minute time points. Using this methodology, the data w u thcn repressnted as the

fraction of initial RAG- l/B-actin ratio.

O 10 20 30 40 50 60 70 80 90 100 110 120

time (min)

IV Both constitutive and inducible upreguiation of RAG expression is de novo protein synthesis depenàent

The constitutive as well as signaling-mediated increases in RAG-1 message observed in OC1 LY8 could either require the de novo synthesis of regulatory proteins or could be due to a post-translational modification of already existing factors. To determine whether constitutive expression was mediated by newly synthesized molecules, cells were incubated in the presence of the translation inhibitor cycloheximide. For al1 experimena, the effects of the amount and duration of cycloheximide usage was assessed. In ail cases, >90% of cells were viable post- treatment, as determined by m a n blue exclusion. After incubation of the parental clone OC1 LY8-C3P and the R A G ~ ~ variant C3-Al IN with 10 mgml of cycloheximide for 3 h. RAG-1

mRNA was found to be increased in both variants, indicating that either a de novo transcriptional repressive factor or a factor promoting mRNA destabilization may be present in the system (Fig. 3.9a). Alternatively, it is possible that the inhibition of RAG-1 translation in itself may be involved in RAG-1 mRNA stabilization, a mechanism that has been observed for other eukaryotic genes [179], and may be independent of the de novo synthesis of regdatory factors. However, cycloheximide-mediated increases in RAG-1 observed in the RAG~O parental clone are more suiking than the increases observed in the R A G ~ ~ variant C3-A 1 IN, this suggesrs that the

constitutive differences in RAG-1 mRNA between these variants is partly mediated by the preferential de novo expression of either a transcriptional repressor or an mRNA destabilizing protein in the R A G ~ ~ parental clone.

Because of the relatively slow kinetics of RAG mRNA upregdation following either BCR ligation (peak increases observed between 6-12 h) or PMA + ionomycin (peak increases observed at 6 h), 1 was also interested in detennining whether this upregulatory effect also required de novo protein synthesis. A preliminary experiment was performed in which the parental clone OC1 LYS was either incubated alone with optimal arnounts of PMA + ionomycin or cycloheximide for 6 h, or pre-incubated with cyclohexirnide for 3 h prior to the addition of PMA and ionomycin (Fig. 3.9b). This result demonstrates that while RAG-1 mRNA can be increased individually by PMA + ionomycin or cycloheximide, there are no funher increases in RAG-1 mRNA upon pre-incubation with cycloheximide. Consistent with the kinetics of upregulation in this situation, this result suggests that signaling-mediated increases in RAG-1

also require newly synthesized molecules. However, this does not rule out the possibility that

both the constitutive and signaling-mediated upregulation of RAG- 1 in OC1 LY8 is mediated by the same repressing factor that in the former situation needs to be newly synthesized, but in the

latter situation rnay be pst-translationally modified.

Figure 3.9. Cycloheximide studies of de novo protein synthesis dependence

in OC1 LYS variants. A: Northem blot of 10 pg total RNA from RA@ OC1 LY8-C3P

and RAGhi C3-Al IN cells that were either left untreated (-) o r incubated in 10 pglml

cyclohexamide for 3 h prior to RNA extraction (+). 8: Northem blot of 15 jtg total RNA from

OC1 LY8-C3P that were either left untreated (-), stimulated with 5 nM PMA + 500 nM

ionomycin for 6 h (6h P+I), incubated with 10 p@ml cyclohrximide for 9 h (9h cyclo). or

preincubated with 10 pg/ml cycloheximide p n o r to addition of 5 nM PMA + 500 nM

ionomycin for 6 h (3h cyclo. 6h P+I). The fold increases in RAG-1 mRNA cornpared to the

unstimulated cells as detennined by Phosphorimage andysis are indicated at the bottom of the

various lanes. Exposure times for RAG-1 for A and B an: 48 and 24 h. respct ivdy.

CHAPTER 4: DISCUSSION

1. Insights into the molecular reguiation d RAG-1 and RAG-2 in OC1 LYS

1 have made the observation that cross-linking the sIgM receptor of OC1 LYS variants with a soluble, polyclonal anti-p F(ab)'z fragment resulu in increased RAG expression. It is unlikely that the

upregulation in this system be due to selection or differentiation rather than a transient signai-mediated effect for several reasons. First, with respect to selective pressures accounting for this effect, the kinetics of the inducible upregulation argues against this possibility. Specifically, the doubling times of the sIg+ clones used in these studies, OC1 LY8-C3P and AMP, have been estimated to be 18 h and 22 h, respectively (N. Stiernholm, personal communication) whereas detectable increases in RAG-1 c m be

observed by 3 h and peak by 12 h (Fig. 3.4). This renders it unlikely that the observed increases in RAG-1 expression are due to a selective outgrowth of a variant subppulation expressing higher levels of RAG mRNA. Secondly, because the increases can be reverseci when the source of cross-linking ligand is removed, it does not appear likely that the observed increased RAG expression represents a sIg-induced differentiation event. AdditionaUy, the effect occurs independently of the initial amounts of RAG expression and rearrangement status of the variant, as relative increases are seen not only in the rearranging R A G ~ parental clone OC1 LY8-C3P but also in the non-rearranging R A G ~ clone C3-A 11N (Fig. 3.4). Although 1 have not assessed the contribution of cell cycle to this effect, its role in the upregulation of RAG through BCR ligation in OC1 LY8 is unlikely for two reasons. First, changes in ceIl cycle distribution could not be detected when RAG was upregulated by temperature-mediated inactivation of v-ab1 [152]. Secondly, ce11 cycle studies of RAG mRNA and protein expression by Desiderio's group demonstrate that while RAG-2 protein expression appears to accumulate preferentially in GdG 1, the expression of RAG-1 pmtein or RAG-1 and RAG-2 rnRNAs are not found to fluctuate at any stage of the ce11 cycle, both in thymocyte and pre-B cell populations [lW].

In both constitutive and signaling-mediated upregulation of M G - 1 in OC1 LY8,I have observed that transcriptional as well as pst-transcriptional mechanisms appear to be invoIved (Figs. 3.7 and 3.8). This suggests that the regulation of the RAG genes in OC1 LY8 at the molecular level is complex and likely involves several regulatory components. The combination of altered gene transcription and message stabilization is uncommon [188], although it has been observed with the early response genes c- fos [189], c-myc [190] and with the IL-2 gene [191]. Of the two mechanisms involved in upregulation of RAG expression in our system however, it is Iikely that transcription plays a larger role both in inducible and constitutive increases. For example, constitutive increases in RAG expression can be atîributed to modest stabilization effects (- 3-fold) whereas the contribution by increased rates of transcription appear to be more striking (Fig 3.7a, Fig 3.8b). Furthemore, while increased transcription was demonstrated for both RAG-1 and RAG-2, message stabilization assays were done only with respect to RAG-I

mRNA. Therefore, these studies cannot rule out the possibility that unlike RAG-1, RAG-2 gene expression may be controlled only at the transcriptional level. However, it could be argued that because the increases in the rates of transcription (both in constitutive increases and iigation-mediated increases of BCR" clones) are comparable between RAG-1 and RAG-2 (Fig. 1.4b) and RAG-1 and RAG-2 mRNA levels are also elevated proponionaily in OC1 LYS RA@ variants ([142] and Fig. l.lb), there is likely an mRNG stabilization effect occumng with RAG-2 mRNA as weli. The finding that RAG-1 mRNAs are controlled in Our system by message stabilization and transcription mechanisms is consistent with the report by Neale et ai. and the estimateci range of short RAG-1 mRNA half-lives in ail variants (- 30 and - 45 min., respectively) appear to be comparable to the ranges observed by others using B lineage cells [140, 1521. However, Our conclusion that message stability is involved in the upregulation of RAG-1 expression conflicts with that of Chen et al., who reponed no significant differences in RAG-1 or RAG-2 half-lives between non-permissive (high RAG-expressing) and permissive (low RAG-expressing) conditions [l52]. The different results could be attributed to a different set of regulatory mechanisms present in the independent experimental systems, but could also be due to differences in interpretation, since the stabilization effects are not striking. For example, Chen et al. also observe a 2-fold increase in stability for RAG-2 (and no differences for RAG-1) but do not consider this difference significant.

At least one of the regulatory proteins responsible for mediating the constitutive increases in RAG-1 expression in the R A G ~ OC1 LY8 variants appears to be newly synthesized in culture, as inhibiting protein synthesis by cycloheximide treatment appears to upregulate RAG expression in the RAG" parental clone OC1 LYS-C3P. (Fig. 3.9a). The fact that this increase takes place largely in OC1 LY8-C3P suggests that the parental clone constitutively expresses a negative regulatory element at higher levels than in C3-A11N. This selective increase is probably not due to a limiting quantity of RAG mRNA that can be synthesized in C3-A 1 IN since RAG expression can be upregulated by PMA +

ionomycin stimulation in this variant (Fig. 3.5). Because cycloheximide treatment does not appear to upregulate OC1 LY8-C3P RAG expression to levels observed in the R A G ~ variant C3-A 1 IN, this suggests that this de novo synthesized factor does not completely account for the differential expression of RAG in these vanants. Therefore, other negative regulatory factors present in OC1 LYS-C3P (or positive regulatory factors in C3-Al IN) that are not de novo synthesized may also be present. Nevenheless, the presence of a constitutively expressed negative regulator of RAG expression is consistent with increased RAG expression observed afier cycloheximide ueatment of thymocytes and a pre-B ce11 line in the study of Neale et al. 11491. In tenns of the inducible increases in RAG expression, preliminary results indicate that pre-incubation of either C3-AllN or OC1 LY8-C3P with cyclohexamide before stimulation with PMA + ionomycin blocks increases in RAG expression (Fig. 3.9b). This preliminary result supports the study of Takahama and Singer in which signaling-mediated

alterations in RAG expression were affected by translational inhibitors [143], but is not consistent with observations that cycloheximide pre-treatment does not affect signaling-induced RAG downregulation 11491 or upregulation [152] in other systems. The apparently distinct mechanisms between reports are consistent with the diffenng kinetics of the observed effects. For example, the PMAhonornycin-induced upregulation of RAG mRNA in OC1 LY8 did not peak until 6 h (Fig. 3.4), while protein synthesis- independent alterations in RAG expression in other reports were observed to peak within 1 h. This is consistent with the rapid kinetics of phosphorylation-repulated phenornena such as the phosphorylation- dependent mechanisrn in which NF-uB is activated in pre-B cells by PMA and which is typically

measured in minutes rather than hours [1663. One interpretation for the discrepancies in the above mentioned reports is that different modes of signaling-mediated RAG regulation may be present in different developmental subsets. If so, this would imply that signaling-rnediated mechanisms of RAG- 1

and RAG-2 mRNA regulation are themselves developmentally regulated.

With respect to the constitutive differences in RAG expression between the R A G ~ parental variant and the R A G ~ variants, it is not clear what mechanism is responsible for the striking increases in RAG expression observe:! in slg- vanants. We initially hypothesized that RAG expression was upregulated in an attempt to correct a non-functional (and apparently non-expressed) CA6

rearrangement. However, upon more careful examination of the V-J-CM rearrangement produa of a

panicular R A G ~ ~ s l g variant, C3-A8N, we have recently dixovered that, although lacking the terminal end of the C region, this product is in fact expressed on the ceU surface at low levels in conjunction with p heavy chain 11921. Furthemore, upon cross-linking this receptor cornplex, which we have termed

sIgACL, we detect distinct tyrosine phosphorylation, increased CD25 expression, and decreased transferrin expression similas to that seen in the dg+ parental clone, but not in a "tnie" sIg- clone such as C3-AI IN. This suggests that this receptor behaves like a conventional sIg receptor in its abiiity to transduce regulatory signais. Thenfore, because C3-A8N is derived from the parental R A G ~ clone, yet still upregulates its RAG expression, it seems unlikely that the constitutive increases in RAG expression in OC1 LYS are due to the loss of sIg expression. It could therefore be speculated that the difierential expression observed between RA@ vanants and the parental line is due to one or more mutations i ntroduced du ring clona1 propagation. Because a number of inde penden tly-den ved variants O ther than C3-A8N have also been found to express elevated RAG-1 and RAG-2 mRNAs 11421, it is difficult to imagine how increased RAG expression in this system is due to spontaneous mutation alone. Perhaps there is some type of selective pressure for cells with such mutations because increased RAG expression in this panicular situation confers an unknown benefit to such cells in culture. Nevertheless, such mutations rnay be specifically introduced in RAG-regulating factor($) dunng clona1 propagation, in turn hindenng proper expression or function of such factors. Alternatively, the differential expression of RAG-1 and RAG-2 could be explained by the occurrence of such mutations within a cis-acting

regulatory component (such as the promoters and enhancers) of the RAG locus itself. However, because differential expression stemming fbm mutation(s) in the RAG-locus would be de mvo protein synthesis independent and cycloheximide treatment appean to preferentiaiiy increase RAG expression in the R A G ~ parental clone, this argues in favor of such differences being due to mutation(s) that affect expression of RAG regulatory factor(s).

IL Upreguiation of RAG expression in OC1 LYS by cr06s-linking the BCR: impiicstions for receptor edSting

What is the function of increasing RAG mRNAs in response to signals delivered through the BCR? The most obvious answer is to allow the formation of "secondary" rearrangements that

completely or panially replace the initiai or "pnmary" rearrangements of germiine aileles. Secondary rearrangements have been shown to occur in human and murine B ceil lines in the processes of V gene replacement of rearranged heavy chain aileles [193,194] and ongoing rearrangement of the u light chain

genes [195- 1991. Three potential roles for secondary rearrangements have been suggested. First, they may conuibute in nonnalizing V gene usage in the expressed repertoire. A second function would be to provide cells which have made non-productive primary rearrangements with a "corrective mechanism". This concept is not difficult to accept, because by definition, a non-productive rearrangement is one that is not expressed, and this lack of expression would presumably not allow the sIg-mediated downregulation of RAG expression to occur. In mm, the lack of RAG downmodulation would then allow productive rearrangements to proceed. Thirdly, secondary rearrangements may provide the immature B ce11 with an alternative tolerance mechanism to anergy and deletion, in which such rearrangements would attempt to replace or " d i t " a functional, but autoreac tive primary rearrangement product. What is the evidence that supports a receptor editing mechanism of tolerance? As previously mentioned, the autoreactive transgenic model of David Nemazee and CO-workers has correlated auto- antigen-slgM receptor interactions in immature bone rnamow B cells with increased levels of RAG expression as well as with the replacement of autoreactive K light chain uansgenes by newly-formeci endogenous À chains 11461. Manin Wiegen and colleagues have also provided similar evidence for

receptor editing in a transgenic anti-DNA model [200, 2011. In this system, B ceils with antidsDNA- specific sIg receptors were not only absent from the periphery (Iike in the Nemazee system), but also from bone rnarrow. While young mice had smkingly reduced levels of B cells, adult mice had almost normal levels of mature B lymphocytes which had replaced their transgenic light chains with endogenous light chains. Finally, receptor editing in B ceU transgenic systerns is analogous to the T-cell transgenic systems of Borgulya et al. and Brandie et d., in which DP thymocytes are observed to continue expressing high levels of RAG-1 and RAG-2 mRNAs and undergo secondary rearrangements at the TCR a locus until positive selection occurs [144, 1451. The caveat with the above-mentioned

evidence for receptor editing is that it al1 stems h m transgenic systems. This leads one to ask: can such an effect be mimicked in vitro as well as normal ih vivo situations?

In this context, there are two principle observations about OC1 LY8 which suggest that it may represent an sIg+ B ce11 captured in the process of receptor editing. The fint is that RAG expression in this celi line is constitutively expressed and can be funher increased upon slg engagement. Secondly, the parental ce11 line undergoes secondary rearrangements which replaces the functional V-J-CA3 primary rearrangement to a V-J-CA6 rearrangement. With the recent observation that the V-J-Ch6

rearrangement likely represents a functionai (albeit tmncated) rearranpment product [192] and that this

product is nevenheless subsequently replaced by a V - K A 7 rearrangement product, it could be argued that this cell line goes through not one, but two rounds of "editing" functional recepton. A difficulty with relating the process of receptor editing to our model is that it has been proposed to occur in immature B cells which have yet to leave the bone marrow. However, for several reasons OC1 LY8 does not likely represent an immature stage of differentiation, but has features which more closely resemble those of germinal centre B cells. For example, the cell surface phenotype, in particular, the absence of slgD, TdT, and the pre-B associated proteins 14.1, 16.1 combined with the presence of CD10, CD 19, CD20, and CD38 are distinguishing characteristics of a mature B cell. Furthexmore, the

extensive clustering of mutations in the CDR1, CDR2, and CDR3 regions of this line (as determined by comparing VH and DH germline sequences to published germline sequences) suggests that hypemutation in this cell Line has already occumd [202]. Finally, OC1 LY8 is a large cell lymphoma, and as such, is believed to represent a more mature stage of differentiation than foüicular lymphomas. This is supported by the fact that a significant portion of follicular lymphomas (which themselves by morphology, phenotype, and ongoing hypemutation, represent a germinal centre B cell) progress into more aggessive large cel) lymphomas (reviewed in [203]). Therefore, if OC1 LY 8 does in fact represent an in vitro mode1 of receptor editing while also representing an in vitro counterpart of a germinal centre B ce11 which has already hypermutated, it must be argued that receptor editing occurs in germinal centres, presumably in cells that have generated autoreactive receptors as a result of hypemutation. Funhemore, in order for a peripheral B ce11 to undergo receptor editing, one would have to argue that RAG expression persists in the germinal centres. In this context, Guy-Grand er al. by in s i t u

hybndization and PCR analysis clairn that 10% of sIg+ B cells in peripheral lyrnph nodes contain RAG- 1 mRNAs 11381.

As an aItemative to OC1 LYS representing an in vitro model of receptor editing in a peripheral B cell, it is just as possible that this ceil line represents a deregulated RAG phenotype resulting fimm the

malignant transformation of this cell. It is also possible that OC1 LY8 represents an immature ce11 captured in the process of receptor editing that normally would have shut off RAG expression pnor to

hypetmutation, but that instead has had its RAG deregulated at this stage, yet was allowed to continue differentiation to a mature B celi. Evidence to support a deregulated RAG phenotype cornes nom our laboratory's failure to detect RAG expression in several other lymphoma ce11 lines with similar phenotypes as OC1 LY8. However, 1 would have to argue that the RAG+ phenotype of OC1 LY8 is not due to complete disregulation because we can alter expression through signaling, and then reverse the effect (Fig. 3.5).

III. The differentiai expression and regulotion of RAG-1 and RAG-2 mRNA in slg+ su-

Increased RAG expression in response to sIg ligation in OC1 LY8 is consistent with the autoreactive transgenic system of Tiegs et al. 11461, but is in direct contrast with the results of others includinp Ma et al., who upon cross-linking of a sIgM+ murine B ce11 line observed significant decreases in RAG-1 and RAG-2 mRNA [MO]. Therefore, an important issue that arises is understanding what factors determine how RAG expression is regulated upon sIg engagement. 1 would like to discuss three principle possibilities that may account for this differential regulation based on how certain factors have been shown to differentially regulate other signaling parameters such as proli feration and tyrosine phosphory lation: 1) stage of di fferentiation at whic h sIg engagement occurred 2) the type of receptor engagement that occurred and 3) the presence or absence of CO-stimulation.

The first possibility is that there are intrinsic developmental differences (either structural differences in the BCR or differential expression ~Vassociation with signal transduction intermediates) in various subsets of RAG+sIg+ B cells influencing the way RAG expression is modulated in response to BCR cross-linking. For example, several reports have provided evidence that the distinct isotypes of "immature" and "mature" sIg + B cells (immature sIg + B cells express IgM only on their surface, whereas mature sIg+ B cells, as a result of class switching , express both IgM and IgD) may mediate differential signaling. In al1 studies of this son, cross-linking sIgM+ B cells or sIgM+sIgD+ with anti-p delivers apoptotic and or anti-pmliferative signals, whereas cross-linking with anti-b results in signals that either do not affect or enhance proliferation 1204-2071. Certain studies have suggested that these different signai-transducing properties are due to differences in specific structural portions of the isotype. For example, the transfection of chimeric sIgG/sIgM genes in an immature ce11 line reveal that the membrane region ~f slgM is not sufficient for transducing growth inhibitory signals and suggests that the extracelIular portions (such as the hinge or the spacer) of the p chain is required for this purpose 12081. Other differences in signaiing may also be due to their differential association with, or expression of, signal-mediating proteins. For example, in a human B lymphoma line, cross-linking of either sIgM or sIgD appean to phosphorylate only a partially overlapping set of proteins [209]. Additionaily, a tyrosine-phosphorylated protein (pp4l) has been found to be associated with IgM, but not IgD [209].

Although a difference in isotype could potentiaily mediate differential RAG regulation ktween RAG+ immature and mature B cells, it cannot resolve out observation with that of Ma et al., since both lines are sIgM+. However, even within the same isotype, B ceiis have been demonstrated to produce distinct signaling outcornes when expressing and/or signaiing through different PTKs. in this context, a recent study implicates the PTK b ü is an integral part of the growth inhibitory pathway leading to growth arrest and apoptosis [210]. in this system, IgM+ apoptosis-sensitive îines express significantly higher levels of b&, whereas IgM' apoptosis-resistant ce11 lines expres significantly higher levels of fyn . Addif : xally, anti-p cross-linking causes increases in bk phosphorylation and kinase activity only

in the apoptosis-sensitive cell lines and in these iines antisense oligonucleotides for bik inhibit negative sipnaiing. In this context, it is interesting that the activation of the abelson tyrosine kinase (presumably by phosphorylation) appears to have a negative regulatory effect on RAG expression [152]. Perhaps in Our ce11 line, the signaling cascade does not involve this kinase but does in situations where RAG

downregulation is observed, such as the ce11 line Ma et al. employed. Altematively, downstream regulatory elements may also be differentially expressed or regulated within sIgM+ cells according to the developmental stage of the cell. For example, it has k e n found that apoptosis-resistant B cells expressed the transcription factor egr- 1 after sIgM cross-linking or PMA stimulation, whereas apoptosis - sensitive bone marrow B cells and B ce11 lines fail to transcribe this gene upon the same type of stimulation due to the specific methylation of egr-1 121 11. Analogously, one could therefore imagine a scenario where Pax-5 or E2A are developmentally regulated (or developmentally inhibited by the Id proteins) in response to upsueam signaiing events in B cells which downregulate RAG expression and the converse in B ceUs that upregulate their expression.

To address whether the differential expression and regulation of the RAG genes in sIg+ B celis can be influenced by the developmental stage of those cells, it will be necessary to isolate phenotypic subsets of sIg+ B cells from primary and secondary lymphoid organs. Recently, Pascual et al. were able to distinguish five distinct subsets of human B cclls tenned Bml-BmS from human tonsil based on reactivity with several monoclonal antibodies 12121. After depletion of T cells using sheep red blood cells, germinal centre cells from tonsillar lymph nodes could therefore be sorted by FACS on the basis of IgD, CD23, CD38, and CD77 expression according to the Pascual et al. subfractionation scheme. Furthemore, sIg+ bone marrow could be fractionated on the basis of IgM and IgD expression, using F(ab) monomeric fragments in order to minimize cross-linking potential. RNA from these various subsets couId then be extracted and RAG expression could be assessed by RT-PCR, taking into account the small arnounts of RNA that are likely to be obtained from sorted populations (-100 ng in 1 X 10' cells). In this context, I have show the ability of the RAG-1 and RAG-2 primers to amplify cDNA from as linle as 5 ng of the R A G ~ ~ variant C3-A1lN and 100 ng of RA@O variant OC1 LYS-C3P, demonstrating that RAG may potentially be detected from small amounts of subsets that express low

levels of these genes (Fig. 3.1). Additionally, within these subsets, it would then be interesting to see if sIg engagement could alter RAG expression, and if so, if differential expression could be observed among subsets. The sorted populations could then be transferred to ce11 culture medium and either left unstimulated or cross-linked with anti-p for a short duration prior to RNA extraction. Differences in

RAG mRNAs between unstimulated and anti-~i cross-linked ceils, like in the studies in OC1 LY8, could

be quantitatively assessed by phosphorimaging. Imponantly, this series of experiments would also be informative with respect to another salient issue: whether there exists an in vivo developmental, RAG- expressing counterpart for the OC1 LY8 line, both in ternis of constitutive RAG expression and upregulation of RAG upon sIg engagement. Funhermore, if such a subpopulation actually exists, it would be interesting to detennine whether this ceîl line represents a pst-hypemutation germinal centre B cell. In temis of the Pascual et al. subî3actionation scheme, this would correspond to Bm4 centrocytes (mature B cells with an IgD- , CD38 +, CD77- phenotype).

Because the anti-p reagent used to cross-link the Ep-N-myc and OC1 LY8 ceil lines in vitro

were sirnilar (polyclonal, soluble F(ab)'2 fragments), this likely cannot account for the differential regulation of RAG expression in these two studies. However, in vivo, even within the same developmental subset, the type and degree of receptor cross-linking may be important for differential RAG expression. There is precedence that varying the concentration, the affinity/avidity of the relevant antigen (monoclonal versus polyclonal), as well as its nature (soluble versus membrane-bound), may in fact generate qualitatively different cellular responses. For example, in one study of human clonal B cell populations, the net avidity of sIg-ligand interactions was found to dictate the proliferative response 12 131. Speci fically, bivalent, soluble anti-p was found to inhibit proliferation, whereas mauix-bound antibodies were stimulatory. Additionaily, low doses of the same soluble anti-p was found to suppress DNA synthesis, whereas high doses were stimulatory. Additionally, in the context of B celi tolerance, several studies involving transgenic systems indicate that the distinct mechanisms of clona1 deletion or functional inactivation (anergy) appear to be mediated by the degree of cross-linking, such that monomeric soluble antigen induces anergy, whereas highly multivalent membrane-bound antigen mediates deletion [2 141.

In the context of receptor editing then, if varying the sIg-ligand interaction really can differentially regulate expression of RAG-1 and RAG-2, this may be important in defining what constitutes an "autoreactive" B ce11 clone versus one which is "non-autoreactive". As mentioned in the

previous section, one of the possible factors that may influence differential regulation of RAG-1 and RAG-2 is in fact the type and degree of cross-linking taking place. In this context, the receptor editing mode1 proposeci by Gustav Nossal and David Nemazee suggests three mechanisms of B ceU tolerance induction in the bone mmow: deletion, anergy, and editing is explained in terms of the relative strengths

of dg-ligand interactions. According to this suggestion, three types of sIg-ligand interactions may occur: 1) no interaction (ignorance), in which RAG expression is downregulated or shut off, and the immature B ce11 proceeds to differentiate into a mature B ce11 2) a weak interaction, in which the ce11 is anergized and 3) a strong interaction, in which case differentiation is arrested, the RAG genes are upregulated, and the B ceiî is given an opportunity to edit its self-reactive receptor through secondary rearrangements, and if unsuccessful at doing so, will die. However, the first type of interaction suggested in such a mode1 is not consistent with the apparent requirement for an antigen-receptor mediated signal to downregulate RAG expression, nor with the view that sIg+ B cells are positively selected through an sIg-ligand interaction in germinal centres for swvival (reviewed in [215]). More plausible then is a situation in which a low-avidity interaction may downregulate RAG and a high- avidity interaction may upregulate RAG.

Another related pcissibility for differential RAG regulation is that signaling through co- stimulatory molecules such as CD19, CD40, or CD32 could also alter how sIg-mediated signaling ultimately affects RAG expression either at separate stages of development or even within a given deveIoprnenta1 d g + B ce11 subset or ceU Line. CD19, has been found to form a complex with the complement receptor CD21 (CR2) and with TAPA-1 (CD81). It also appears to be functionaiiy and stnicturally associated with sIgM, as anti-IgM induces tyrosine phosphorylation of CD19 and CD19 co- modulates with the BCR 12161. In mature B cells, ligation of CD19 alone renders B ceUs refractory to siibsequent stimulation through sIgM, which correlates with the ability of ami-CD19 to suppress the increase in intracellular ~ a * + induced by ami-IgM cross-linicing. On the other hand, CO-ligation of CD19 and surface IgM can reduce the B-ce11 activation threshold by two orders of magnitude. This type of interaction would therefore allow low-affinity receptors on sIg+ B cells to be triggered more readily, thus potentially allowing the upregulation of RAG expression even if antigen-binding is relatively inefficient. In addition, ligation of CD40 either with a soluble anti-CD40 mAb or with the natural CD40 ligand (gp39) can rescue centrocytes in the germinal centres from programmeci cell death (reviewed in [217]). Additionally, simultaneous engagement of FC-yRII receptor (CD32) either prior to or simultaneously with anti-IgM cross-iinking can inhibit sIg-mediated proliferative responses 12161.

To address whether RAG can be differentially regulated within the same sIg+ B cell, OC1 LY8 variants could be subjected to varying degrees of cross-ünking stimuli other than a soluble anti-p . In particular, RAG expression could be assessed afier cross-linking the antigen receptor with monoclonal anti- p (anti-idiotype), polyclonal anti-p coated to polystyramine plates, anti-p followed by cross-linking with a second-step antibody, or cross-linking either with anti-CD40 (or -gp39) or antiCD 19 either alone or in conjunction with anti-p cross-linking. An alternative possibility, however, is that the inducible

upregulation we see in OC1 LY8 represents a deregulated signaling pathway, such that no matter what

alterations are made either with respect to cross-linking agent o r CO-stimulation, increased RAG will always result. However, in al1 parameters assessed including ~ a 2 + flux, tyrosine phosphorylation, c-fos

induction, gtowth arrest (measured by decreased thymidine uptake and decreased transferrin expression), and activation (measureù by CD25 induction), it appears that OC1 LY8 can function in the capacity of a signaling-cornpetent sIg* B cell (Fi?. 3.5). Assuming that the cross-linking induced upregulation of RAG expression in our cell fine does not represerrt a dereplated event, it would be interesting to then study the molecular basis for this differential regulation. In this context, the fact that both PMNionomycin stimulation and sIgcross-linking upregulate RAG suggests that if differential regulation can occur in this ce11 line (either by CO-stimulation or altering cross-linking agent), the molecular factors involved in mediating this differential effect would either be downsueam or independent h m ca2+ and PKC.

IV. The differential expression of RAG in OC1 LYS: a strategy for identifyiiig novel genes thst CO- express with RAG-1 and RAG-2

For numerous reasons, the OC1 LY8 ceil culture system represents a valuable tool for identimng potentially novel genes that are CO-replated with RAG-1 and RAG-2 at the level of RNA expression. The finding that differential constitutive RAG expression is observed between clonal OC1 LY8 variants as well as between induced and uninduced sIg + variants provides two excellent systems in which to isolate such genes for several reasons. In both systems, differentially expressed genes are being compared in clonally related vanants in which the only apparent difference is in RAG expression itself. The constitutive differences between R A G ~ and RAGY variants in particular may be a good system to

isolate CO-expressed genes because the differences in RAG mRNA are suiking (Fig. 3- 1). Furthemore, the differences in RAG expression between the parental clone and R A G ~ vanants is relatively stable, unlike most other ce11 lines in which RAG expression can v a q tremendously between subclones [124].

One particular concem with respect to the stability of RAG expression in certain lines is the fact that it decreases over time. This is possibly due to the fact that high, persistent levels of RAG have toxic effect on the ce11 and therefore clones continuing to express RAG are selected against during in vitro

propagation [ l3 11. Since the isolation of the R A G ~ variant C3-A 11N four years ago, no noticeable reductions in RAG expression have been observed to date. While, the RAG' variant C3-A1 IN is particularly stable since it cannot undergo any further remangement, the stability of the R A G ~

phenotype of the parental population during the clona1 expansion of this population is more of a concem since it undergoes ongoing rearrangements resulting in the production of R A G ~ ~ variants. However, the

rate at which these R A G ~ ~ clones are generated has been detennined to be very low (1.3 X 10- Skel~generation). Furthemore, previous studies in Our lab in which a series of parental subclones have been allowed to undergo 20 ce11 divisions (Le - 14 days in culture) all displayed the same comparable, low levels of RAG expression as the original parental clone [142]. Therefore, it appears that as long as

parental clones are propagated for limited durations before RNA is exuacted and are occasionally subcloned by limiting dilution, the RAG~O phenotype should persist. This relative stability of RAG expression observed in OC1 LY8 over time and between variants is important because it affords the reproducibility required for studying RAG-related differentiaily expressed genes.

In order to isolate genes CO-expressing with the RAG genes in OC1 LY8 variants, the methodology developed by Liang and Pardee for detecting genes which are differentially expressed between different ce11 lines (or between differing conditions within the same ce11 line) could be employed 1218, 2 191. The strategy of this method, caiied differential display, is to ampli@ partial cDNA sequences from subsets of RNAs by RT-PCR, and then to display these short amplicons on sequencing gels for comparison. The design of the primers in this technique are important; the 3' primers confer C

partial specificity, and are complementary to the mRNA polyA tail + 2 additional 5' bases, whereas the 5' primers are random 10-mers. Reportedly, the combination of 20 10-mers and 4, 3' primers (the

penultimate nucleotide being degenerate) is sufficient to cover a large fraction of the 10-15, 000 estimated expressed genes within a given ce11 while at the same time displaying an ideal amount (50- 100) of these species per primer pair [220]. Therefore, differential display could be performed on OCI LYS-C3P ( R A G ~ ) and C3-A11N ( R A G ~ ) total RNA. Specifically, afier the RNA from these variants is treated with DNAse in order to eliminate genomic DNA contamination, it can be reverse transcribed using the four possible 3' oligodT primers, and amplified using the various combinations of the random 10-mers. In order to eliminate the amount of false positives from cDNA amplification, samples could be amplified and run on sequencing gels for comparison in duplicate or tripkate. To fùrther confirm that an amplicon is tmly differentially expressed between OC1 LYS-C3P and C3-A1 IN, reproducible bands unique to one variant could be excised, purified, reamplified, and then used as probes in Northern analysis, (using the same total RNA in the Northern blot as the batch used for differential display). This reconfirmation is particularly important as a screening step since it appears in most cases that only 10- 15% of differentially expressed bands can be reconfirmed, the others either representing false negatives or false positives [22 1,2221.

Once a number of di fferentially expressed amplicons are isolated using various primer combinations, these genes could then be funher characterized. The first step in this characterization process would be to assess if the differential expression observed in OC1 LY8 can be generalized to other systems. This could be done by preparing RNA from a panel of human B cell lines and lymphoid tissue (primary and secondary) known to express differing amounw of RAG based on differentiation stage. Afier assessing RAG expression in these samples, differentially expressed cDNA fragments could then be employed as probes to assess whether RAG expression correlates with the presence or absence of the differentially expressed gene of interest. Those differentially expressed genes with

highly-correlating patterns in this screening step could then be employai as probes to isolate hill-length cDNAs from a cDNA Library derived fiom the original OC1 LY8 variant in which the differentially expressed gene was initially isolated. These cDNAs could then be sequenced and analyzed in sequence search programs. This type of investigation rnay yield useful clues as to what the gene rnay encode and rnay therefore be important in deciding what funcrional assays to perfom.

What are some of the potentially novel genes that rnay be detected by virtue of their CO-

expression with the RAG genes ? One category would be any trans-acting factor involved in the molecular control of RAG gene expression. These could inc1ude"upstream" signaling proteins, such as one of the PTK or other intermediates associated with the BCR signaiing cascade or a constitutively expressed transcription factor (s) , whose signal-independent presence or absence would be critical in regulating the protein-binding responsive elements of the RAG locus. To identiw a differentially expressed gene as a putative regulatory factor of RAG-1 and/or RAG-2 would involve looking for matches in the sequences of these differentially expressed cDNAs with known motifs in search programs. These would include SHI, SH2, and SH3 domains in signaling molecules and standard transcription factor motifs (such as helix-loop-helix, Ers, or leucine zipper domains). Candidate regulators of RAG-1 andor RAG-2 that match such motifs could then be functionaily characterized by using the full-length cDNAs in complementation assays. Essentially, the cDNAs of interest would be ligated into expression constructs and these constructs would then be transfected into the variant fiom which the cDNA was not isolated Mm. M G - 1 and RAG-2 expression before and after addition of this factor, along with appropriate control vector constructs, could then be assessed for reversion to the phenotype of the other variant. Funhermore, if the isolated gene represents a transcription factor, gel- shift assays could be perfonned to test for binding of such factors to RAG regulatory regions. The feasibility of this assay will naturally depend on whether the RAG-I anaor RAG-2 promoters and enhancers have been identified. In this context, there are currently three groups (including Our laboratory) working on the identification and characterization of the RAG-1 promoter region.

Other categories of novel factors that rnay CO-express with RAG-1 and RAG-2 in OC1 LY8 include other components of the recombinase system or other B-ceU specific genes. These factors could be CO-expressed because they are CO-regulated with the RAG genes by a common regulatory factor. Ln this context, it is therefore possible that the Rch-1 protein, which has been found to associate with RAG- 1 at the protein level [23,24], rnay also be CO-regulated at the level of RNA regulation. Furthemore, the recent report that RAG rnay have a similar function as the scid factor in fesolving coding joints leads one to speculate that the DNA-PK complex, or components of it, rnay also co-express with RAG-1 and RAG-2. More interesting, however, would be the identification of novel recombinase factors using this

method. Alternatively, since it has not yet been shown that the RAG genes play an indirect, rather than a

direct role in V@)J recombination, the RAG genes themselves may be regulating the expression of such coexpressed recombinase component(s). This would be particularly interesting since it would mle out the possibility of the products of the RAG locus as direct recombinase components. This possibility could be tested by complementation analysis in which RAG expression vectors would be transfected inrc? the variant in which the differentially expressed putative recombinase component has not been isolated fiom, and expression levels of the differentially expressed gene would then be assessed.

OC1 LY8 C3P and C3-A 1 LN would be particularly well suited for idenwing regulatory factors because the constitutive differences in RAG-1 and RAG-2 expression between these two variants appear to be de novo synthesis dependent (Fig. 3.9), thereby making it iikely that such regulatory factors would be detected at the level of differential RNA expression. However, one difficulty with using this technique specificaily for the purpose of finding transcriptional regulators of RAG expression is that transcription factor messages may belong to rare message classes, and rnay thus be difficult to detect unless the total RNA is somehow depleted of abundant common vanscripts that may consume reagents in the PCR reaction. Funhermore, it would be a relatively labor-intensive, indirect way of finding of a transcription factor which in any event may mm out to be one of the already known B-celi specific DNA binding proteins (such as Ig transcription factors). Thus, considering al1 the possible categories of mRNAs that could be differentially expressed between these variants, while the chance of finding a specific regulatory factor is quite low, the chance of finding some novel genes that coexpress with the

RAG genes is considerably higher.

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