Chapter 1 - Cis-Regulatory Elements and Epigenetic Changes …biology.hunter.cuny.edu/molecularbio/Class Materials... · 2011-02-08 · H genes which are then joined by NHEJ or other

CHAPTER 1

Advances in Immunology,ISSN 0065-2776, DOI: 10.1

* The Howard Hughes Meand Department of Genetic{ University of Vienna, Dr-

Cis-Regulatory Elements andEpigenetic Changes ControlGenomic Rearrangementsof the IgH Locus

Thomas Perlot*,† and Frederick W. Alt*

Contents 1. Introduction 2

Volu016/

dicals, HKarl

me 99 # 2008S0065-2776(08)00601-9 All righ

Institute, The Children’s Hospital, Immune Disease Institute,arvard Medical School, Boston, Massachusetts, USA-Lueger-Ring1, Vienna, Austria

Elsts

2. T
he Immunoglobulin Heavy Chain Locus 3
3. V
(D)J Recombination During B-Cell Development 4
4. C
lass Switch Recombination and
Somatic Hypermutation
7
5. Ig
H Rearrangements and Allelic Exclusion 8
6. A
ccessibility Control 10
6.1.
G ermline transcripts 10
6.2.
S patial organization and nuclear positioning
of the IgH locus
12
6.3.
C hromatin modifications 13
7. Ig
H Locus Control Through Cis-Regulatory Elements 16
7.1.
P romoter of DQ52 16
7.2.
V H promoters 17
7.3.
In tronic enhancer 18
7.4.
3 0 IgH regulatory region and I promoters 20
7.5.
A dditional potential regulatory elements 21
7.6.
In terplay between cis-regulatory elements 22
8. C
onclusions 23
Ackn
owledgments 23
Refe
rences 24
evier Inc.reserved.

1

2 Thomas Perlot and Frederick W. Alt

Abstract Immunoglobulin variable region exons are assembled from discon-

tinuous variable (V), diversity (D), and joining (J) segments by

the process of V(D)J recombination. V(D)J rearrangements of the

immunoglobulin heavy chain (IgH) locus are tightly controlled in a

tissue-specific, ordered and allele-specific manner by regulating

accessibility of V, D, and J segments to the recombination activating

gene proteins which are the specific components of the V(D)J

recombinase. In this review we discuss recent advances and estab-

lished models brought forward to explain the mechanisms underly-

ing accessibility control of V(D)J recombination, including research

on germline transcripts, spatial organization, and chromatin modifi-

cations of the immunoglobulin heavy chain (IgH) locus. Furthermore,

we review the functions of well-described and potential new cis-

regulatory elements with regard to processes such as V(D)J recombi-

nation, allelic exclusion, and IgH class switch recombination.

1. INTRODUCTION

An individual clone of mature B-cells expresses immunoglobulin (Ig)molecules as an antigen receptor. The typical subunit of an Ig moleculeconsists of two identical heavy chains (HC) and two identical light chains(LC). The N-terminal region of these chains contains the highly variableantigen binding site; whereas the C-terminal part is called constant region(C region). The C region of the IgH chain (CH) determines the effectorfunctions of antibodies, which are the secreted form of Ig molecules.

Immunoglobulin (Ig) and T-cell receptor (TCR) variable region exonsare assembled from large arrays of V (variable), D (diversity), and J( joining) gene segments during the development, respectively, of B andT lymphocytes. Once a functional immunoglobulin chain is expressed,allelic exclusion operates through a feedback mechanism to prevent fur-ther rearrangements of Ig heavy (IgH) and Ig light (IgL) chain genes.V(D)J recombination is mediated by a common recombinase complexthat includes the recombination-activating gene products RAG1 andRAG2, which harbor endonuclease activity that introduces DNAdouble-strand breaks (DSBs) at V, D, and J segments. The V(D)J reactionis completed by the ubiquitously expressed nonhomologous end-joining(NHEJ) factors that join the broken V, D, and J segments together. Still, Igloci are only fully assembled in B lineage cells and TCR loci are onlyassembled in T lineage cells. Within a lineage, different loci are rear-ranged in a specific order. For example, IgH locus variable region exonsare assembled before those of Ig light chains (IgL), and within the IgHlocus D to JH recombination precedes VH to DJH recombination. Given

Genomic Rearrangements of the IgH Locus 3

such locus-specific regulation and a common V(D)J recombinase, accessi-bility of the different loci to the common V(D)J recombinase must under-lie the cell-type and stage-dependent assembly of the different IgH andTCR gene families ( Jung et al., 2006).

Activation of mature B-cells can alter their IgH loci through a separateform of genomic rearrangement which is termed IgH class switch recom-bination (CSR). CSR allows B-cells to express IgH chains with differentconstant regions which can change the effector functions of antibodieswithout altering variable region specificity. CSR is initiated by activation-induced cytosine deaminase (AID), the activity of which ultimately leadsto DSBs in regions upstream of CH genes which are then joined by NHEJor other end-joining pathways to complete the CSR reaction (Chaudhuriet al., 2007).

Ig and TCR loci contain a number of cis-regulatory elements whichregulate V(D)J rearrangements, IgH CSR, and Ig gene expression at vari-ous levels. In this chapter, we will focus on the impact of cis-regulatoryelements on genetic and epigenetic regulation of recombination eventswithin the IgH locus.

2. THE IMMUNOGLOBULIN HEAVY CHAIN LOCUS

The murine IgH locus is a complex genomic region, spanning about 3 Mbclose to the telomere of the long arm on chromosome 12. The IgH locuscomprises arrays of V, D, and J segments upstream of several constantregion exons (Fig. 1.1A). Different mouse strains carry varying numbersof VH and D elements. Some 150 VH segments are distributed over �2.5Mb in the 50 part of the IgH locus and are classified in 16 VH gene familiesdefined by sequence similarities ( Johnston et al., 2006). These VH genefamilies are partially interspersed with one another but, depending onposition, can be divided into proximal (30 part of the VH cluster, close toIgH–D region, for example, VH7183), intermediate (e.g., VHS107), anddistal (50 part of the VH cluster, distant from IgH–D region, for example,VHJ558) families. 30 of the VH elements, separated by �90 kb, lie 10–15 Dsegments (Retter et al., 2007; Ye, 2004) followed by 4 JH elements. Becauseof the uniform transcriptional orientation of V, D, and J segments, V(D)Jrecombination events at the IgH locus result in deletion of the interveningsequence. The 30 part of the IgH locus contains a series of sets of differentconstant (C) region exons Cm, Cd, Cg3, Cg1, Cg2b, Cg2a, Ce, Ca, which willbe referred to as ‘‘CH genes’’ (Fig. 1.1B).

A large number of cis-regulatory elements were identified throughoutthe IgH locus. The intronic enhancer, Em, is located in the intron betweenJH4 and the CH exons (Fig. 1.1); the 30 IgH regulatory region (IgH 30RR)consists of several DNase hypersensitive sites and is located at the very

VHJ558 VHS107 VH7183 DH JH CH

5�RR?

A

VD RR? IgH 3�RRPDQ52 Em

VHDJH Em Cm Cd Cg 3 Cg 1 Cg 2b Cg 2a Ce Ca IgH 3�RR

HS

3A

HS

1,2

HS

3BH

S4

HS

5H

S6

HS

7

AAAAAAImP

BIg 1P

FIGURE 1.1 Schematic depiction of the murine IgH locus. (A) VH, DH, JH gene segments

and CH exons are shown as rectangles, known and potential regulatory elements as ovals.

The VH families VHJ558, VHS107, and VH7183 are depicted as examples for distal,

intermediate, and proximal VH families, respectively. The cis-regulatory elements PDQ52

(promoter of DQ52), Em (intronic enhancer), and IgH 30RR (IgH 30 regulatory region)

are depicted. The potential regulatory elements 50RR (50 regulatory region) and VD RR

(VH–DH intergenic regulatory region) are depicted with a question mark. Drawing not to

scale. (B) The 30 part of the IgH locus. An assembled VHDJH exon is shown as a white

rectangle, CH genes as squares, Em and individual DNaseI hypersensitive sites within the

IgH 30RR are depicted as black ovals, switch regions as white circles. I promoters are

located upstream of every switch region (Chaudhuri et al., 2007; Lennon and Perry, 1985;

Lutzker and Alt, 1988), only m and g1 I promoters (ImP, Ig1P) are depicted. Transcripts fromI promoters get spliced and polyadenylated. Switch regions also get transcribed in the

antisense orientation (Apel et al., 1992; Julius et al., 1988, Morrison et al., 1998; Perlot

et al., 2008). Concomitant transcription from ImP and, for example, Ig1P can target AID to

m and g1 switch regions and thereby initiate CSR to Cg1.


30 end of the IgH locus (Fig. 1.1). Transcriptional promoters are presentupstream of every VH segment (Fig. 1.2B), upstream of DH segments(Fig. 1.2A and C), and upstream of CH genes (Fig. 1.2A). In addition,antisense transcripts from less well-defined promoters were describedin the VH, DH, JH regions, and upstream of CH genes. Section 7 of thischapter contains a detailed discussion of these IgH cis-regulatoryelements.

3. V(D)J RECOMBINATION DURING B-CELL DEVELOPMENT

The IgH locus V(D)J exon is assembled at the pro-B-cell stage leadingto the production of m IgH heavy chains via splicing of the VHDJH exononto the adjacent Cm constant region exons. Functional mHC and surro-gate Ig light chain proteins form a complex that is expressed on thesurface of pre-B-cells and is known as the pre-B-cell receptor (pre-BCR)

CH

AAAAAA

DH

A

PDQ52 DQ52 JH1 JH2 JH3 JH4 Emm0

Im

DH-JH antisense germline transcripts

AAA AAA AAA

L VH L VH L VH

VH antisense germline transcripts

VH sense germline transcriptsB

VHP VHP VHP

or

CH

AAAAAADH

C

PDH DJH JH4 Em

DH antisense germline transcript

DmIm

CH

AAAAAAVHP JH4VHDJH Em

L

D

VH LSm

mHC-mRNA

Im

VHDJH antisense transcript Sm antisense transcript

FIGURE 1.2 Transcripts within the IgH locus. VH, DH, JH gene segments and CH exons are

shown as rectangles, enhancer and promoter elements as ovals. 12 bp and 23 bp RSSs are

depicted as black and white triangles, respectively. Drawings not to scale. (A) The IgH

locus in germline configuration is transcribed from the promoter of DQ52 (PDQ52) to

produce the m0 transcript (Alessandrini and Desiderio, 1991), and from within the Emenhancer to generate the Im transcript (Lennon and Perry, 1985; Su and Kadesch, 1990),

both of which are getting spliced and polyadenylated (Kottmann et al., 1994, Su and

Kadesch, 1990). DH and JH elements are transcribed in the antisense orientation (Bolland

et al., 2007; Chakraborty et al., 2007), suggested start sites (dashed arrows) are located

around PDQ52 (Chakraborty et al., 2007) and Em (Bolland et al., 2007). Sites of transcrip-

tional termination of DH–JH antisense germline transcripts are unknown. (B) Unrear-

ranged VH segments are transcribed in the sense orientation from the individual VHpromoters (VHP) (Yancopoulos and Alt, 1985). The intron between the leader (L) and the

VH exon (VH) is spliced out, and the VH sense germline transcript gets polyadenylated

(Yancopoulos and Alt, 1985). The VH segments and VH intergenic regions can also get

transcribed in the antisense orientation (Bolland et al., 2004). Start and termination sites

of VH antisense germline transcripts are unknown. Therefore, individual antisense tran-

scripts could comprise one VH segment and its adjacent regions or multiple VH segments

including intergenic regions, shown as short and long solid arrows, respectively. (C) Upon

D to JH recombination, the assembled DJH exon gets transcribed from the DH promoter

(PDH) and spliced to the Cm exons to generate the Dm transcript (Alessandrini and



(Cobb et al., 2006). Signaling through the pre-BCR induces proliferation,signals cessation of further VH to DJH rearrangements at the IgH locus(i.e., allelic exclusion, see below), and promotes the onset of IgL variableregion exon (VLJL) assembly. Thus, expression of the pre-BCR representsan important checkpoint at the pro- to pre-B-cell transition (Martenssonet al., 2007). Subsequently, Igk and Igl LC variable regions are assembledduring the pre-B-cell stage. Expression of a functional Igk or Igl LC alongwith mHC forms a complete Ig molecule which is expressed on the cellsurface of the resulting immature B-cells (Gorman and Alt, 1998). Imma-ture B-cells migrate to the periphery where mature naıve B-cells can beactivated and undergo further modification of their IgH locus includingIgH CSR and somatic hypermutation (SHM) (see below).

All V, D, and J segments are flanked by recombination signalsequences (RSSs) that consist of a conserved palindromic heptamer anda conserved AT-rich nonamer separated by a less conserved 12 bp or 23bp spacer (Sakano et al., 1980). The RAG1/2 endonuclease recognizes andbinds a pair of RSSs with different spacer lengths in the context of the12/23 rule (Early et al., 1980; Sakano et al., 1980), which allows for efficientV(D)J recombination only between gene segments flanked by 12 bp and23 bp RSSs (Fugmann et al., 2000). The 12/23 restriction provides somedirection for which Ig gene segments can be assembled. For example, IgHD segments are flanked with 12 bp RSSs on both sides; whereas VH andJH segments are flanked with 23 bp RSSs, thus preventing direct VH–JHjoining. In the TCRb locus, however, direct Vb to Jb joints would beallowed according to the 12/23 rule but are denied by ‘‘beyond 12/23’’restrictions (Bassing et al., 2000). Differential composition of RSSs imple-ment ‘‘beyond 12/23’’ restriction at the nicking and pairing step of V(D)Jrecombination (Drejer-Teel et al., 2007; Jung et al., 2003).

Desiderio, 1991; Reth and Alt, 1984), which in one reading frame encodes for a short mHCmolecule (Reth and Alt, 1984). DH antisense germline transcription is present throughout

the remaining unrearranged DH segments (Chakraborty et al., 2007). Suggested origin of

DH antisense germline transcripts is the region around the promoter of the recombined

DH segment (depicted as PDH) (Chakraborty et al., 2007), transcriptional termination sites

are unknown. (D) Upon VH to DJH recombination, the promoter of the rearranged VHsegment (depicted as VHP) drives expression of mRNA encoding for the mHC. In additionto Im sense transcription, the Sm switch region is also transcribed in the antisense

orientation (Perlot et al., 2008) from promoters residing within Sm (Apel et al., 1992;

Morrison et al., 1998), the transcriptional termination site of the Sm antisense transcript

is unknown. The assembled VHDJH exon and the adjacent JH region are transcribed in the

antisense orientation potentially from start sites within the JH cluster (dashed arrow)

(Perlot et al., 2008), the transcriptional termination site of the VHDJH antisense transcript

is unknown. Upstream unrearranged VH segments are transcriptionally silenced upon

assembly of a functional VHDJH exon (Bolland et al., 2004; Yancopoulos and Alt, 1985).


RAG cutting precisely between RSSs and variable region gene seg-ments results in the formation of blunt RSS ends, and the formation ofcoding ends (CE) of the V, D, or J segments as closed hairpins. Codingjoints (CJs) are formed through a joining reaction mediated by membersof the NHEJ repair pathway. In this reaction, Ku proteins bind the freeCEs and recruit DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which activates the endonuclease activity of Artemis to open thehairpins. Subsequently, ends are joined by the XRCC4/DNA ligaseIVcomplex (Rooney et al., 2004). In contrast, the blunt SEs are preciselyligated to each other by NHEJ.

Tight regulation of V(D)J recombination is imperative to ensure properlymphocyte development and genomic integrity. While V(D)J recombina-tion is of enormous advantage in order to efficiently combat infections,erroneous V(D)J recombination can have adverse consequences includingchromosomal translocations, which can contribute to neoplastic transfor-mation and the development of leukemias and lymphomas.

4. CLASS SWITCH RECOMBINATION ANDSOMATIC HYPERMUTATION

After a VHDJH variable exon is assembled upstream of the C region exons,a promoter 50 of the rearranged VH segment drives expression of m and dHCmolecules in mature B-cells. Upon antigen encounter and activation, aB-cell can switch to expression of downstream CH genes to generateantibodies with the same variable region specificity but a different CH

effector function by CSR. Repetitive switch (S) regions are locatedupstream of every CH gene except Cd. Introduction of DSBs in Sm and adownstream switch region can result in joining of the two switch regions,deletion of the intervening sequence, and consequently expression of adownstream CH gene with the same variable region exon. AID is abso-lutely required for CSR. It appears to function by deaminating cytosinesto uracils within the substrate S region DNA with the resulting mis-matches somehow being processed into DSBs by cooption of normalrepair pathways (Di Noia and Neuberger, 2007). As AID is a single-strand DNA-specific cytosine deaminase, its activity on duplex S regionDNA is targeted by transcription (Chaudhuri et al., 2007). In this context,switch regions can be transcribed from an I (intervening) promoterlocated upstream of each S region, which allows for AID targeting tospecific transcribed S regions (Fig. 1.2B). In addition to these sense germ-line transcripts, antisense transcripts were described in several S regions(Apel et al., 1992; Julius et al., 1988; Morrison et al., 1998; Perlot et al., 2008).AID initiated S region DSBs are joined by NHEJ or an alternative end-joining pathway to complete CSR.


AID is also required for SHM, a process during which the variableregion exon gets mutated at a relatively high frequency in activatedB-cells. SHM is initiated by transcription-dependent targeting of AID toassembled variable regions followed by error prone repair of the resultingmismatches (Di Noia and Neuberger, 2007). Through affinity maturation,B-cell clones producing higher affinity antibodies are selected and anefficient adaptive immune response is elicited.

5. IgH REARRANGEMENTS AND ALLELIC EXCLUSION

Expression of RAG1 and RAG2 is absolutely required for V(D)J recombi-nation. In the hematopoietic lineage, RAG activity can first be demon-strated in common lymphoid progenitor (CLP) cells, which are precursorcells that can develop into B-cells, T-cells, natural killer (NK) cells, anddendritic cells (DC) (Borghesi et al., 2004). Together with the detection ofD to JH rearrangements in non-B-cell lymphoid lineages (Borghesi et al.,2004; Born et al., 1988; Kurosawa et al., 1981), expression of RAG in CLPssuggests that the first IgH rearrangement step can occur, at least at lowlevel, in CLPs. Thus, the IgH D to JH recombination step is not absolutelyrestricted to the B-lineage, in contrast to VH to DJH rearrangements whichnormally occur only in B-cells. Efficient D to JH rearrangement on bothIgH alleles takes place after B lineage commitment in the pro-B-cell stage(Alt et al., 1984).

Once DJH segments are formed, one of the upstream VH elements canjoin to form a complete VHDJH exon. In the murine IgH locus, proximalVH segments are preferentially rearranged compared to distal VH ele-ments throughout ontogeny (Malynn et al., 1990; Yancopoulos et al., 1984).However, peripheral B-cells do not show this preference as selectionalters the B-cell repertoire (Yancopoulos et al., 1988). Both D to JH andVH to DJH recombination take place at the pro-B-cell stage, however, in anordered manner such that D to JH rearrangement nearly always occursbefore VH to DJH rearrangement (Alt et al., 1984). In this regard, VH to DJHrecombination is the step that is regulated in the context of allelic exclu-sion to ensure expression of only one functional HC.

Successful VH to DJH recombination and expression of a mHC from oneIgH allele prevents a second DJH allele from undergoing VH to DJHrearrangement ( Jung et al., 2006). Considering the junctional diversitygenerated during V(D)J recombination, only one out of three VHDJHexons will be in frame with the downstream Cm exons; whereas two outof three will be out of frame and therefore unable to express a functionalmHC (Mostoslavsky et al., 2004). The percentage of functional recombina-tion events is further decreased by usage of VH pseudogenes containingstop codons, frameshifts, defective splice sites, or lacking an ATG


translation start site, by stop codons in DH segments as well as throughselection against certain reading frames of DJH joins (Gu et al., 1991). As aresult, a substantial fraction of developing B-cells will not be able togenerate a functional mHC from either IgH allele and will undergo apo-ptosis (Rajewsky, 1996). If a nonfunctional VH to DJH rearrangementoccurs on the first allele, the second DJH allele can still undergo VH toDJH rearrangement (Alt et al., 1984).

Allelic exclusion of VH to DJH rearrangement is mediated by feedbackregulation; a functional mHC together with surrogate light chains areassembled to a pre-BCR, which signals the cessation of further VH toDJH rearrangements (Alt et al., 1984; Jung et al., 2006). In this regard,endogenous IgH rearrangements are largely inhibited by the expressionof a preassembled membrane-bound mHC transgene (Nussenzweig et al.,1988). Likewise, allelic exclusion was broken by targeted deletion ofthe mHC transmembrane exons (Kitamura and Rajewsky, 1992), by lackof a functional pre-BCR (Loffert et al., 1996), and by combined deletionof the downstream pre-BCR signaling molecules Syk and ZAP-70(Schweighoffer et al., 2003). The combined data from these studiesstrongly support a feedback-mediated mechanism for allelic exclusionthat is mediated by signaling through a functional mHC in the pre-BCRsignaling complex.

The complete chain of events that leads to cessation of VH to DJHrearrangements and implementation of allelic exclusion is still elusive.However, it was shown that the onset of allelic exclusion after successfulVH to DJH recombination is accompanied by the transient downregulationof RAG (Grawunder et al., 1995), decontraction of the IgH locus (Roldanet al., 2005), and loss of accessibility correlates such as VH germlinetranscripts and marks of active chromatin (see below). It has been esti-mated that only 1 in 10,000 wild-type B-lymphocytes actually escapeallelic exclusion and express a functional mHC from both IgH alleles(Barreto and Cumano, 2000). Feedback regulation can explain cessationof VH to DJH rearrangement but would be ineffective if both IgH alleleswould rearrange simultaneously. Therefore, it was suggested that theV(D)J recombination machinery targets one allele at a time (Alt et al.,1980). Supportive of this hypothesis was the observation that all Ig locias well as the TCRb locus undergo asynchronous replication(Mostoslavsky et al., 2001; Norio et al., 2005). At the allelically excludedIgk locus it is thought that asynchronous replication facilitates allele-specific chromatin changes (Mostoslavsky et al., 1998) that lead to theearly replicating allele rearranging first (Mostoslavsky et al., 2001).A similar mechanism for VH to DJH recombination, the allelicallyexcluded IgH rearrangement step, was speculated, but has not yet beendemonstrated. Thus, asynchronous replication could conceivably play arole in the initiation phase of allelic exclusion. However, it does not


provide an explanation for the maintenance of allelic exclusion duringsubsequent B-cell stages, which prevents further IgH rearrangements inthe presence of RAG, which must be affected by feedback mechanismsthat influence accessibility.

6. ACCESSIBILITY CONTROL

The accessibility hypothesis was proposed to explain how a single com-mon V(D)J recombinase can target the different Ig and TCR loci in alineage- and stage-specific manner (Yancopoulos and Alt, 1985). Forexample, Ig variable region exons are only fully assembled in B-cellswhile TCR variable region exons are only rearranged in T-cells. Similarly,IgH loci are rearranged during the pro-B-cell stage and not in pre-B-cellswhere IgL variable region assembly occurs. The accessibility hypothesiswas first proposed based on the finding that germline VH gene segmentsare transcribed in pro-B-cells but not in subsequent B-cell stages, withgermline VH transcription providing a potential correlate of accessibility(Yancopoulos and Alt, 1985). This hypothesis was proven by experimentsthat showed transfected TCR gene substrates could be rearranged by pro-B lines that do not rearrange endogenous TCR gene segments, firstdemonstrating a common V(D)J recombinase (Yancoupouls, 1986). Thisconclusion was confirmed and extended by other studies (Krangel, 2003;Stanhope-Baker et al., 1996). However, the precise mechanisms that medi-ate differential accessibility of Ig and TCR gene segments to V(D)J recom-bination are still not clear. Over the decades, several correlates ofaccessibility have been defined and a general picture is beginning toemerge as to how accessibility control might be regulated and implemen-ted. Among the known correlates of accessibility are germline transcripts,chromatin modifications, DNase hypersensitivity, spatial organization,and positioning of Ig and TCR loci in the interphase nucleus.

6.1. Germline transcripts

Germline transcription is the production of transcripts from V, D, or Jsegments and adjacent regions before they undergo rearrangement(Fig. 1.2). Sense germline transcripts starting from promoters upstreamof V, D, and J segments have been described in all Ig and TCR loci(Hesslein and Schatz, 2001), and their stage-specific expression patternsstrongly correlate with accessibility of these transcribed elements (e.g.,Yancopoulos and Alt, 1985). The precise role of sense germline transcriptsis still not understood and has been debated (Krangel, 2003). Recentstudies support the notion that active transcription mediates chromatinchanges that render the transcribed regions accessible to the recombinase


(Sen and Oltz, 2006). However, it has been debated whether germlinetranscripts are the cause or the effect of these chromatin changes, andneither possibility has been unequivocally proven or disproved. On onehand, the levels of germline transcripts exhibit a positive correlation withrearrangement efficiency (Sun and Storb, 2001), which could suggest thatthe process of transcription itself could promote RAG targeting. How-ever, others have shown that the correlation between individual rearran-gements and germline transcription is not absolute (Angelin-Duclos andCalame, 1998; Sikes et al., 2002).

The IgH locus in germline configuration is transcribed from the pro-moter of DQ52 (PDQ52), the 30 most DH segment, toward Cm, therebyproducing the so-called m0 transcript (Fig. 1.2A). After D to JH rearrange-ment, the recombined DJH element is transcribed (Fig. 1.2C) (Alessandriniand Desiderio, 1991; Reth and Alt, 1984); and at the same time, unrear-ranged VH segments are transcribed from their promoters (Fig. 1.2B).Germline VH transcription appears to be silenced upon a productiverearrangement (Corcoran, 2005; Yancopoulos et al., 1985).

More recently, antisense transcripts have been found to occur through-out the VH cluster (Fig. 1.2B) (Bolland et al., 2004), in the DH region(Fig. 1.2A and C) (Bolland et al., 2007; Chakraborty et al., 2007), and inthe JH region (Fig. 1.2A) (Bolland et al., 2007; Perlot et al., 2008). VH

antisense transcripts appear to be biallelic, and it has been argued thatsuch transcripts are large and span several VH segments and the adjacentintergenic regions; but formal proof of their initiation sites is still lacking.VH antisense transcription was shown to initiate during D to JH recombi-nation, and to be rapidly downregulated after VH to DJH recombination(Bolland et al., 2004). DH antisense transcripts were detected in RAG-deficient pro-B-cells as well as on the D–JH rearranged allele of B-celllines with a functionally assembled IgH gene (Chakraborty et al., 2007).DH antisense transcripts have been suggested to originate from the 30 mostDH (Chakraborty et al., 2007) or from the JH region (Bolland et al., 2007).

The functional significance of antisense transcription in the context ofV(D)J recombination has not been fully elucidated. It has been postulatedthat antisense transcription promotes an active chromatin state renderingthe locus more accessible (Bolland et al., 2004), based on the observedcorrelation between antisense VH germline transcription and active VH toDJH recombination (Bolland et al., 2004). Similar conclusions were reachedbased on the observation of reduced antisense DH transcripts and reducedD to JH rearrangements in mice lacking the intronic enhancer, Em (Afsharet al., 2006; Bolland et al., 2007). Conversely, others have raised the possi-bility that antisense transcripts, at least in the DSP DH segments, couldpair with low levels of postulated germline sense transcripts and elicitRNA interference-mediated transcriptional gene silencing (Chakrabortyet al., 2007; Koralov et al., 2008). It should be noted that true germline sense


transcripts have not been identified as yet in the germline DH segments,but their level may be as low as those originally identified in the S. pombecentromeric repeats and may only be revealed in an RNAi-deficientbackground (Volpe et al., 2002).

6.2. Spatial organization and nuclear positioningof the IgH locus

The spatial organization of the Ig and TCR loci was analyzed by three-dimensional fluorescence in situ hybridization (3D FISH), in whichnuclear organization remains preserved. Several groups showed thatbefore undergoing rearrangement, the IgH locus moves from its defaultposition at the nuclear periphery to a more central compartment (Fuxaet al., 2004; Kosak et al., 2002), going along with the observation that thenuclear periphery has a repressive effect on transcription (Andrulis et al.,1998; Baxter et al., 2002; Reddy et al., 2008) and, therefore, might keep theIgH locus in an inaccessible state. These observations are consistent withthe peripheral location of the IgH locus in thymocytes which have onlylow levels of D to JH and no VH to DJH rearrangements (Fuxa et al., 2004;Kosak et al., 2002; Kurosawa et al., 1981). The centrally located IgH locus inpro-B-cells can undergo D to JH rearrangement; however, for rearrange-ments of the distant VH elements, long-range contraction and looping ofthe IgH locus ( Jhunjhunwala et al., 2008) seems to be crucial as lack of IgHlocus contraction in Pax5-deficient pro-B-cells does not allow for rearran-gements of intermediate and distal VH families (Fuxa et al., 2004; Sayeghet al., 2005). After successful rearrangement, the IgH locus decontractsand, thereby, has been proposed to impede further VH to DJH rearrange-ments by increasing the distance between VH elements and the DJH region(Roldan et al., 2005). Therefore, it seems that one aspect of VH to DJHrecombination accessibility might be influenced by spatial arrangement ofthe IgH locus within the nucleus.

In B lineage stages subsequent to the pro-B stage, one IgH allele ispositioned in close proximity to centromeric heterochromatin (Roldanet al., 2005; Skok et al., 2001). This finding was interpreted as the mono-allelic silencing of the nonproductive IgH allele, because transcriptionallysilent genes have been shown to associate with centromeric heterochro-matin (Brown et al., 1997). However, the VH cluster gets silenced on bothalleles in the context of germline transcription, and considering the factthat rearrangements from both IgH alleles, productive and nonproductivein either DJH or VHDJH configuration, are expressed in all B-cell stagesthat were examined (Daly et al., 2007; Fukita et al., 1998; Ono and Nose,2007), monoallelic silencing might be either a short-time transient phe-nomenon or recruitment to centromeric heterochromatin might haveother implications in the process of allelic exclusion.


Studies of interactions between IgH and Igk alleles demonstratedcoordinated patterns of action of Ig loci during B-cell development. Inter-actions between IgH and Igk, predominantly in pre-B-cells, were demon-strated to reposition the interacting IgH allele to centromericheterochromatin and induce IgH locus decontraction (Hewitt et al.,2008). Therefore, this interchromosomal interaction could play a role inIgH allelic exclusion and the transition from accessible IgH alleles toaccessible Igk alleles.

6.3. Chromatin modifications

Eukaryotic DNA is packaged into nucleosomes in which genomic DNA iswrapped around histone octamers. The N-terminal ends of histones,called histone tails, can be marked by diverse modifications (e.g., acetyla-tion, methylation, phosphorylation, ubiquitination, and others). The ‘‘his-tone code’’ ( Jenuwein and Allis, 2001) translates patterns of histonemodifications into repression or activation of chromatin. An extensiveeffort has been made to investigate the effects of histone modificationsand also various other chromatin attributes such as DNA methylation,DNase sensitivity, and nucleosome remodeling on accessibility of Ig andTCR loci with hopes of shedding light on the epigenetic regulation ofV(D)J recombination.

Posttranslational modifications of N-terminal histone tails can affectgenome regulation in several ways. Histone modifications can directlyaffect chromatin structure, for example, through a change in charge. Inthis context, histone acetylation can loosen the association between DNAand the histone core or can alter higher order chromatin packaging.Alternatively chromatin modifications can disrupt or provide bindingsites for chromatin remodeling complexes or other effector molecules.Prominent examples of such specialized binding domains are bromodo-mains specifically binding acetylated lysines, and chromodomains bind-ing to dimethylated lysine 9 on histone 3 (Kouzarides and Berger, 2007).

Many studies showed that marks of active chromatin correlate withV(D)J rearrangements. For example, acetylated lysine 9 on histone 3(H3K9ac), hyperacetylated histone 4, and dimethylated lysine 4 on his-tone 3 (H3K4me2) are active chromatin marks (Kouzarides and Berger,2007). They are present in the D–JH region peaking around the 50 mostD segment, DFL16.1, and over the JH elements (Chakraborty et al., 2007;Morshead et al., 2003) in early pro-B-cells that are poised to undergo D toJH rearrangements. However, they are almost absent in thymocytes(Chakraborty et al., 2007). Following D to JH recombination, the proximalVH elements become hyperacetylated and, thereafter, in a manner that isdependent on IL-7R signaling and on its downstream effector STAT5(signal transducer and activator of transcription 5), the distal VH segments


become hyperacetylated (Bertolino et al., 2005; Chowdhury and Sen,2001). Acetylation patterns seem to be narrowly confined to the VH

segment, its promoter, and RSS ( Johnson et al., 2003).Histone hyperacetylation is lost after productive VH to DJH recombi-

nation, thereby contributing to rendering the VH cluster inaccessible inpre-B-cells (Chowdhury and Sen, 2003). Notably, an engineered locus thatactively recruits an H3K9 methyltransferase shows downregulation ofgermline transcripts and impaired V(D)J recombination (Osipovichet al., 2004). H3K9me2 is absent in the D–JH region of pro-B-cells andpresent in thymocytes (Chakraborty et al., 2007), and removal ofH3K9me2 from the VH region before VH to DJH recombination is depen-dent on Pax5 ( Johnson et al., 2004), a transcription factor essential forB-cell commitment (Busslinger, 2004). In agreement with this data, loss ofPax5 leads to an inability to rearrange distal VH gene families (Hessleinet al., 2003; Nutt et al., 1997).

The antagonistic Polycomb (PcG) and Trithorax (trxG) groups of pro-tein complexes establish and propagate a silenced or active chromatinstate, respectively (Ringrose and Paro, 2004). Curiously, targeted deletionof the PcG protein Ezh2, an H3K27 methyltransferase, inhibits rearrange-ments of the distal VHJ558 family without affecting germline transcription(Su et al., 2003). H3K27 methylation was reported to be a mark of inactivechromatin (Kouzarides and Berger, 2007); therefore, it remains to bedetermined whether the results observed in the Ezh2 knockout are director indirect effects.

Recent studies reported that the PhD finger domain of RAG2 specifi-cally binds to trimethylated H3K4 (Liu et al., 2007; Matthews et al., 2007), ahistone modification associated with transcriptional start regions(Pokholok et al., 2005) also shown to be present in accessible IgH regions(Liu et al., 2007). Mutation of the conserved tryptophan residue W453within the PhD finger domain of RAG2 abrogates RAG2 binding toH3K4me3 and impairs V(D)J recombination of chromosomal and extra-chromosomal substrates (Liu et al., 2007; Matthews et al., 2007). However,removal of the entire RAG2 noncore region, including the PhD domain,only leads to a partial impairment of V(D)J recombination (Liang et al.,2002). These seemingly contradicting data have been suggested to reflectthe presence of an inhibitory function within the noncore region ofRAG-2, which is relieved upon binding to H3K4me3, or can be circum-vented by deleting the entire noncore region (Liu et al., 2007;Matthews et al.,2007). These recent studies provide the first direct link between epigeneticcontrol of V(D)J rearrangement and RAG recombinase accessibility.

Chromatin remodeling complexes can change the composition, struc-ture, or position of nucleosomes within chromatin. These changes arenoncovalent and are dependent on ATP hydrolysis (Martens andWinston, 2003). The SWI/SNF chromatin remodeling complex contains


a bromodomain that allows it to efficiently bind acetylated chromatin andmobilize nucleosomes or change nucleosome structure (Martens andWinston, 2003). In this regard, it was shown that unmodified or evenhyperacetylated nucleosomes located directly on RSSs are inhibitory toRAG cleavage in vitro (Golding et al., 1999) and that addition of SWI/SNFimproved substrate cleavage (Kwon et al., 2000). RSSs strongly attractnucleosomes and, thus, may implement some aspect of accessibility con-trol (Baumann et al., 2003). Moreover, nucleosome positioning appearspivotal for V(D)J recombination in vivo (Cherry and Baltimore, 1999).Further supporting the importance of SWI/SNF complexes in V(D)Jrecombination, BRG1 (the ATPase subunit of SWI/SNF) was found toassociate at Ig and TCR loci within hyperacetylated chromatin regions(Morshead et al., 2003). Functional targeting of BRG1 to a TCRbminilocuslacking the essential Db promoter rescued V(D)J recombination of thissubstrate (Osipovich et al., 2007), substantiating the role of SWI/SNFcomplexes in V(D)J recombination and suggesting a role for transcrip-tional promoters in recruitment of chromatin remodeling complexes.

Another readout to assess chromatin structure is the DNase sensitivityassay. Less tightly packed chromatin- or nucleosome-free DNA is moresensitive to DNase or restriction enzyme digestion than heterochromatinregions. While cis-acting elements such as promoters and enhancers canbe devoid of nucleosomes and, therefore, are DNase hypersensitive,accessible chromatin of Ig and TCR loci shows general DNase sensitivity(Yancopoulos et al., 1986). In this context, the region between DQ52 andEm is DNase sensitive before D to JH rearrangement and JH RSSs showenhanced sensitivity and seem to be nucleosome free (Maes et al., 2006).The VH region becomes nuclease sensitive before VH to DJH rearrange-ment and reverts to a refractory state after successful VH to DJH recombi-nation (Chowdhury and Sen, 2003).

Cytosines in mammalian DNA can be methylated in CpG dinucleo-tides. Generally, cytosine methylation corresponds to silenced genes(Stein et al., 1982; Vardimon et al., 1982) or silent regions throughout thegenome; whereas promoter regions of expressed genes are found in anunmethylated state. Cytosine methylation can act by inhibiting regulatoryproteins from binding to DNA (Watt and Molloy, 1988), or by recruitingmethyl-CpG binding proteins which in turn can interact with HDACs toenforce a silent chromatin state ( Jaenisch and Bird, 2003). In this regard,methylated V(D)J recombination substrates are refractory to active rear-rangement (Cherry and Baltimore, 1999; Hsieh and Lieber, 1992); inparticular, methylated RSSs can abolish RAG cleavage and V(D)J recom-bination (Whitehurst et al., 2000). Demethylation alone, however, is notsufficient to initiate V(D)J recombination (Cherry et al., 2000). The DH–JHcluster gets demethylated before the onset of D to JH recombination (Maeset al., 2001; Storb and Arp, 1983) characteristic of an accessible state. In this


context, the JCk region gets monoallelically demethylated and thisdemethylated allele undergoes rearrangement first (Mostoslavsky et al.,1998). The second allele stays in a repressive environment and somehowcan get demethylated, if the rearrangement on the first Igk allele isnonproductive (Goldmit and Bergman, 2004). More extensive studies onDNA methylation of the IgH locus could potentially help to elucidateaspects of accessibility control within this locus.

7. IgH LOCUS CONTROL THROUGHCIS-REGULATORY ELEMENTS

A formidable number of cis-regulatory elements have been identifiedthroughout the IgH locus (Fig. 1.1). Enhancers are located in the JH–Cmintronic region and at the very 30 end of the locus. Promoters are found 50

of VH and D segments as well as 50 of most CH genes. Cis-elements in theIgH locus not only govern gene expression, but also play crucial roles inaccessibility control in all its above-mentioned aspects and also controlCSR. An extensive effort has been made to elucidate the many roles ofthese transcription elements. Ongoing research also aims at identifyingmissing regulatory elements and elucidating their role in IgH locuscontrol.

7.1. Promoter of DQ52

DQ52 is the 30 most D segment. Its promoter becomes active before D to JHrearrangement to generate the m0 transcript (Fig. 1.2A) (Alessandrini andDesiderio, 1991; Kottmann et al., 1994; Schlissel et al., 1991a). This tran-script runs all the way through the Cm exons, which get spliced to the JH1splice donor site (Schlissel et al., 1991b). The same promoter region alsogives rise to a low-level antisense transcript (Chakraborty et al., 2007).It has been suggested that the repetitive nature of the DH regionin combination with bidirectional transcription can elicit RNAinterference-mediated transcriptional gene silencing that would lead tothe observed inactive chromatin state of the DSP elements. However, asmentioned above, only antisense and no sense transcripts have beendetected thus far in the germline DH region (Chakraborty et al., 2007).

Every DH element upstream of DQ52 has a bidirectional promoterwhich, upon D to JH rearrangement, potentially through approximationto the Em enhancer, gets activated to generate an antisense transcriptand a sense transcript (Fig. 1.2C) (Alessandrini and Desiderio, 1991;Chakraborty et al., 2007). The sense transcript gets spliced in a way thatthe rearranged DJH segment is joined to the Cm exons. In one readingframe, this mRNA can encode for a shorter version of the mHC (Reth and


Alt, 1984), which can inhibit subsequent VH to DJH rearrangements(Gu et al., 1991; Loffert et al., 1996; Malynn et al., 2002). Targeted deletionof the DQ52 promoter, which has both promoter and enhancer activity(Kottmann et al., 1994), in mice had no major impact on D to JH rearrange-ment, other than a slight shift in JH usage (Afshar et al., 2006; Nitschkeet al., 2001). However, in these studies, m0-like transcripts were stillevident, suggesting that activity of the heterogeneous promoter of DQ52was not entirely abrogated. In a different study, the intronic Em enhancerwas replaced with a phosphoglycerate kinase promoter–neomycin resis-tance gene cassette (PGK-NeoR), which resulted in complete absence ofm0 transcripts and complete inhibition of D to JH rearrangement (Perlotet al., 2005). In this regard, targeted deletion of an analogous promoterelement in the TCRb locus, the promoter of Db1, led to diminished germ-line transcripts from this promoter and reduced Db1 rearrangements(Whitehurst et al., 1999), demonstrating an accessibility control functionfor this element in Db–Jb recombination.

7.2. VH promoters

Every VH element has its own promoter that initiates VH germline tran-scripts before VH to DJH rearrangement (Fig. 1.2B), as well as transcriptsof the assembled VHDJH exon after rearrangement (Fig. 1.2D). Most VH

promoters can generate a germline transcript, in which a leader exon getsspliced to a VH exon (Fig. 1.2B). The transcript gets polyadenylated andcontains an open reading frame (Yancopoulos and Alt, 1985); however, noVH protein or its function has thus far been demonstrated. The mostconserved element across VH promoters is the octamer ATGCAAAT(Parslow et al., 1984). This sequence element has been shown to be neces-sary for VH transcription (Mason et al., 1985), and it binds the ubiquitouslyexpressed Oct-1 and the B-cell-specific Oct-2, both POU family transcrip-tion factors. Most but not all VH promoters contain a TATA box, aninitiator (Inr) element (Buchanan et al., 1997), a heptamer, and a pyrimi-dine stretch (Eaton and Calame, 1987). Additionally, binding sites for anumber of mostly B-lineage-specific transcription factors and chromatinremodeling complexes have been identified in VH promoter regions( Johnston et al., 2006).

Germline transcripts from unrearranged VH promoters are generatedupon D to JH rearrangement in pro-B-cells, and downregulated aftercompleted VH to DJH recombination and assembly and expression of afunctional VHDJH exon (Bolland et al., 2004; Hardy et al., 1991). Thepromoter of a recombined VH element stays active throughout B-celldevelopment, and cell line experiments showed that the first upstreamunrearranged VH segment can also be continuously expressed at reducedlevels (Wang and Calame, 1985). Promoter activity of a functionally


rearranged VH element was shown to be partially dependent on the 30

regulatory region (Pinaud et al., 2001). Thus, VH promoters might fulfill adual role: to help confer accessibility to germline VH segments and todrive expression of the assembled heavy chain gene.

7.3. Intronic enhancer

The IgH intronic enhancer (Em) was the first cellular (as opposed to viral)eukaryotic enhancer element described (Alt et al., 1982; Banerji et al., 1983;Gillies et al., 1983). Em comprises a 220 bp enhancer core (cEm) and twoflanking matrix attachment regions (MARs). Targeted deletion of bothMARs shows that they are dispensable for efficient V(D)J recombinationwithin the IgH locus (Sakai et al., 1999). Deletion of Em in B-cells (Chenet al., 1993; Sakai et al., 1999; Serwe and Sablitzky, 1993) and in the germ-line of mice (Afshar et al., 2006; Perlot et al., 2005) led to reduced D to JHrearrangement and severely impaired VH to DJH rearrangement. Theresidual V(D)J recombination activity in the IgH locus implies that activa-tion of IgH rearrangements may also involve one or more additionalenhancer type elements. One candidate for such a compensating elementis the promoter/enhancer region PDQ52, which was speculated to pro-mote D to JH recombination (Alessandrini and Desiderio, 1991). However,deletion of PDQ52 along with Em did not show increased impairmentabove that seen with deletion of Em alone (Afshar et al., 2006). However,since the deletion of PDQ52 appeared to be incomplete, this elementcannot yet be ruled out as having redundant functions with Em in confer-ring accessibility to the DH–JH region. Another candidate for cooperativefunction with Em is the 30 IgH regulatory region, but the double knockoutof Em and the IgH 30RR has not been generated. By analogy, deletion of theintronic Igk enhancer (iEk) reduces Vk to Jk rearrangements (Xu et al.,1996); whereas a double knockout of iEk and the 30Ek enhancercompletely blocks recombination of the Igk locus (Inlay et al., 2002). TheiEk and 30Ek in the Igk locus are the enhancer elements corresponding tothe position of Em and IgH 30RR in the IgH locus.

It has been puzzling why in Em knockout mice the VH to DJH step ismore severely impaired than the D to JH step, even though the Emenhancer has no obvious effect on germline transcripts of intermediateand distal VH families (Perlot et al., 2005). One explanation could besignificant underestimation of D to JH impairment in Em knockout mice.Initial very low levels of D to JH rearrangements could limit the crucialDJH substrates for subsequent VH to DJH rearrangements and, therefore,result in the observed strong reduction of VH to DJH recombination. Aftera productive rearrangement, feedback regulation inhibits further VH toDJH recombination, but does not block further D to JH rearrangements


(Reth et al., 1987). Therefore, D to JH recombination might ‘‘catch up’’ overthe course of B-cell development and mask a stronger impairment.

Notably, replacement of Em with a PGK-NeoR cassette (Chen et al.,1993; Perlot et al., 2005; Sakai et al., 1999) or introduction of PGK-NeoR

cassette just 50 of Em (Chen et al., 1993; Delpy et al., 2002) results in a muchmore severe impairment or a complete block of V(D)J recombination andconcomitant complete loss of m0 transcripts (Perlot et al., 2005). Thisphenomenon could be explained by a promoter competition/insulatingmechanism. In such a scenario, the PGK-NeoR gene and its promotermight compete with PDQ52 for activity from a downstream cis-elementsuch as the IgH 30RR, which is known to act over long distances (Pinaudet al., 2001). Similar promoter competition for the IgH 30RR has beenobserved between I promoters and the PGK-NeoR cassette introduced inthe CH region (see below). Alternatively, the PGK-NeoR cassette couldinduce local chromatin changes that impede m0 germline transcriptionand accessibility of D and JH segments.

Extensive studies revealed an array of binding sites for B-lineage-specific transcription factors and also for ubiquitously expressed proteinswithin the Em enhancer and the flanking MARs (Calame and Sen, 2004).The unique combination of these factors is likely to mediate the enhan-cer’s predominant activity in pro-B-cells (Inlay et al., 2006). In this context,replacement of iEk with Em leads to premature Igk rearrangement in pro-B-cells and absence of Igk rearrangements in pre-B-cells, the stage whenLC rearrangement normally takes place (Inlay et al., 2006), corroboratingthe pro-B-cell specificity of Em.

Em was suggested to play a role in regulating antisense transcriptsthrough the JH and DH region (Afshar et al., 2006; Bolland et al., 2007), andadditionally a promoter region within Em was identified that gives rise tothe Im transcript (Lennon and Perry, 1985; Su and Kadesch, 1990). Startingat Em, this transcript extends through the m switch region and Cm. Tran-scription of switch regions was shown to be necessary for CSR, probablyfor targeting AID, and in this regard, deletion of Em leads to reduced Imtranscript levels and reduced CSR (Bottaro et al., 1998; Perlot et al., 2005).Deletion of Em has no obvious effect on somatic hypermutation of VHDJHexons in mature B-cells (Perlot et al., 2005). An open question is how theactivity of AID is specifically targeted to regions within Ig loci. It wasspeculated that cis-regulatory elements could determine this specificity,but neither cEm nor the IgH 30RR, alone (Morvan et al., 2003), seems tohave a crucial role in targeting SHM to the IgH locus VHDJH segments.While an absolute requirement of cEm or the IgH 30RR for SHM can beexcluded, there is a possibility that smaller defects of SHM in thesemutants are masked by selection processes during affinity maturation.Also, a combined function of cEm and the IgH 30RR in promoting ortargeting SHM is another possibility that needs to be tested.


7.4. 30 IgH regulatory region and I promoters

I promoters are located upstream of all switch regions (Chaudhuri et al.,2007; Lennon and Perry, 1985; Lutzker and Alt, 1988). Transcripts initiat-ing from I promoters are processed in such a way that an I (intervening)exon, located immediately downstream of the I promoter, is spliced tothe associated CH exons. In this process, the intronic region including theS region is spliced out and the transcript gets polyadenylated. However,these transcripts appear ‘‘sterile,’’ as they do not contain an open readingframe and could not be shown to encode for a protein (Chaudhuri et al.,2007). Active transcription from I promoters is necessary for CSR as onlytranscribed S regions can become AID targets during CSR. In this context,deletion of I promoters abrogates efficient CSR to the associated CH genes;while replacement of I promoters with a constitutively active promoterdirects CSR to the associated CH gene (Manis et al., 2002). Transcriptionfrom different I promoters prior to CSR can be induced upon stimulationwith different activators or cytokines. Corresponding surface receptorsfor these molecules and their associated downstream signaling pathwaysaffect different combinations of activating or repressive response ele-ments within I promoter regions, which leads to CSR to different IgHisotypes under different stimulation conditions (Stavnezer, 2000). Most Ipromoters do not appear to act in isolation as efficient transcription fromthem also requires the IgH 30RR (Pinaud et al., 2001) and physical interac-tion between the IgH 30RR and specific I promoters has been implicated(Wuerffel et al., 2007).

The IgH 30RR is located downstream of Ca at the very 30 end of the IgHlocus (Fig. 1.2B). This regulatory region consists of a number of DNaseIhypersensitive sites scattered over �35 kb (Dariavach et al., 1991; Garrettet al., 2005; Lieberson et al., 1991; Matthias and Baltimore, 1993; Petterssonet al., 1990); up until now, none of them was shown to play a role in V(D)Jrecombination but more studies are needed (Khamlichi et al., 2000; Pinaudet al., 2001). The most striking function, control of IgH CSR, has beenassigned to HS3b, HS4 within the IgH 30RR. Targeted deletions in micerevealed severely reducedCSR tomost IgH isotypes and reduced germlinetranscription from I promoters through the corresponding S regions(Pinaud et al., 2001), a process required for CSR (Jung et al., 1993; Zhanget al., 1993). Deletion of the more 50 DNaseI hypersensitive sites within theIgH 30RR HS3a and HS1,2 had no effect on CSR (Manis et al., 1998);however, replacement ofHS3a orHS1,2with a PGK-NeoR cassette resultedin a similar defect as in the HS3b, HS4 deletion (Cogne et al., 1994, Maniset al., 1998). The latter observations suggested a potential promoter compe-tition/insulation between I promoters and the PGK-NeoR cassette forsignals from within the IgH 30RR. This hypothesis was strengthened byinsertion of a PGK-NeoR cassette at the Ig2b promoter or the Ce gene,


respectively. In both cases, germline transcription and class switching toCH genes 30 of the inserted PGK-NeoR cassette was unaffected; whilegermline transcription and class switching to CH genes 50 of the insertedPGK-NeoR cassette was impaired (Seidl et al., 1999). These results suggestthat the inserted PGK-NeoR cassette can interfere with the long-rangecontrol effect of IgH 30RR on CSR in a position-dependent manner.

The IgH 30RR is necessary for efficient expression of the rearrangedHC from the promoter upstream of the assembled VHDJH exon (Pinaudet al., 2001), whereas the much more proximal Em enhancer is not requiredfor HC expression (Perlot et al., 2005). Because it can influence expressionof rearranged VHDJH segments, the IgH 30 RR can function over a distanceof at least 200 kb. Such long-range activity may be important for activatingoncogenes translocated into the upstream portions of the CH locus inlymphomas. Not all of the seven described hypersensitivity sites in thespacious 30 regulatory region have been knocked out yet, therefore, otherpotential functions still remain to be discovered. Apart from the above-mentioned effects on germline transcription, CSR, and IgH expression, ithas been speculated that parts of the 30 regulatory region might have arole in long-range chromatin organization. Finally, activity of the Ig1promoter does not appear to be dependent on the IgH 30RR; suggestingthat it carries sufficient regulatory elements itself or that there are otherlong-range IgH locus elements that function in CSR to be defined.

7.5. Additional potential regulatory elements

Several laboratories suggested that the IgH locus can be associated withthe nuclear periphery via its 50 region (Kosak et al., 2002; Yang et al., 2005).The 50 end of the IgH locus does not get deleted in the course of V(D)Jrecombination and as such is an attractive location for amissing regulatoryelement that controls processes such as accessibility control of the distalVH genes, positioning of the IgH locus, or feedback regulation. In fact,�30kb upstream of the most distal VH element an array of DNaseI hypersensi-tive sites has been identified (Pawlitzky et al., 2006). One of these sites,HS1,was reported to be pro-B-cell specific and potentially contain bindingsites for the transcription factors PU.1, Pax5, and E2A. However, prelimi-nary knockout experiments, in which HS1 was deleted, showed no effecton the IgH locus, as targeted alleles could still undergo efficient V(D)Jrecombination including all VH gene families. Furthermore, allelic exclu-sion was unaffected (Perlot, Pawlitzky, Brodeur, and Alt, unpublisheddata). Other potential functions of these sites, including acting as a bound-ary area as was suggested by DNA modifications confined to one side of50IgH hypersensitive sites (Reddy et al., 2008), are still being tested.

Another area that was speculated to harbor a regulatory element is the�90 kb region between the VH and the DH clusters. This region could


contain an element that ensures the ordered rearrangement of the D to JHand VH to DJH steps, such as a boundary element that influences activa-tion of separate IgH locus domains. Moreover, the VH to DH intergenicregion is deleted on a productively rearranged allele but remains in placeon a DJH rearranged allele, suggesting an element might reside in thisregion that is responsible for shutting down the incompletely rearrangedallele in the context of allelic exclusion. Potential support for such anelement came from placement of a VH segment into the DH region,which resulted in breaking of lineage specificity, ordered rearrangement,and allelic exclusion of the introduced VH segment (Bates et al., 2007).Preliminary studies in which this intergenic region has been deleted haveprovided direct support for the notion that this region contains elementsimportant for regulation of lineage specificity of VH to DJH rearrangement(Giallourakis, Franklin, and Alt, unpublished data).

7.6. Interplay between cis-regulatory elements

The transition from an inactive to an active chromatin state of the IgHlocus is in part governed by Em. The intronic enhancer plays an importantrole in placing active chromatin marks throughout the DH–JH region(Chakraborty, Perlot, Subrahmanyam, Alt, and Sen, unpublished data).In addition, Em promotes transcripts from PDQ52 and supports formationof the DNaseI hypersensitive site at this promoter element (Perlot et al.,2005; Chakraborty, Perlot, Subrahmanyam, Alt, and Sen, unpublisheddata). These data argue in favor of a direct interaction between Em andPDQ52 reminiscent of a corresponding promoter/enhancer holocomplexdescribed in the TCRb locus (Oestreich et al., 2006). The fact that Em is notabsolutely required for stimulation of PDQ52 suggests partial compensa-tion by another cis-element. It was speculated that the IgH 30RR can takeover this role of interaction with PDQ52 and of activating the DH–JHregion, because its ability to function over a long range to activate I regionpromoters and to influence expression of the promoter of a rearrangedVHDJH segment (Pinaud et al., 2001).

No single cis-regulatory element has thus far been identified that isresponsible for the bulk of VH germline transcription. It seems to be likelythat VH promoters can be activated in trans by B-lineage and stage-specific factors. VH antisense transcripts might be a prerequisite of VH

sense germline transcripts by initializing an active chromatin state andaccessibility of the VH locus (Bolland et al., 2004). Start sites of thesetranscripts still remain elusive, which exacerbates the manipulation ofsuch transcripts and a direct proof for this hypothesis.

After rearrangement of a complete VHDJH exon, the assembledVHDJH–Cm gene is transcribed from the VH promoter 50 of the rearrangedVH. Transcription from the rearranged VH promoter is mainly supported


by the IgH 30RR (Pinaud et al., 2001) suggesting direct interaction of thetwo cis-elements. In this regard, the ability of the IgH 30RR to form a directcomplex with a region around Em and downstream I promoters wasdemonstrated during CSR (Wuerffel et al., 2007). However, cEm does notappear to be involved in that interaction, since deletion of that elementdoes not affect complex formation (Wuerffel et al., 2007). As mentionedabove, the interaction of the IgH 30RR with I region promoters likelyunderlies the cooperation of these two elements in I region transcriptionand regulation of IgH CSR.

8. CONCLUSIONS

Antigen receptor genes are assembled by the process of V(D)J recombina-tion in a developmentally controlled manner. Differential accessibility atIg and TCR loci is regulated at least in part by cooperative action of cis-regulatory elements. Tremendous progress has been made in identifyingand elucidating the multiple layers of control of the IgH locus duringV(D)J recombination; however, many important questions remain unan-swered and new questions are emerging. Among these are processesinvolved in ordered IgH rearrangements, asynchronous VH to DJH rear-rangements, enforcement of feedback regulation, the precise relevanceand impact of chromosome positioning and movements, the role of anti-sense transcription throughout the IgH locus, and chromatin modifica-tions. Likewise, a remarkable amount of progress has been made inelucidating the role of cis-acting elements in the regulation of IgH CSR,but again there are still many unanswered questions including preciselyhow these elements function to specifically target AID to S regions andthe precise mechanisms by which the IgH 30RR and I region promoterselements cooperate in response to external stimuli to specifically activateCSR to particular CH genes. To fully understand the genetic and epige-netic regulation of the IgH locus, all involved cis-regulatory elementsand trans acting factors need to be identified and analyzed. More workalso will need to be done to understand how these factors influenceregulation at the level of chromatin structure and spatial organization.Understanding the mechanisms governing the IgH locus, a model systemfor gene expression and epigenetic regulation will also advance ourunderstanding of various other unsolved biological problems.

ACKNOWLEDGMENTS

We thank Cosmas Giallourakis and John Manis for critically reviewing the manuscript andfor discussions. T. P. received a Boehringer Ingelheim Fonds PhD scholarship. This workwassupported by National Institutes of Health Grants PO1CA092625-05 and 2PO1AI031541-15(to F.W.A.). F.W.A. is an investigator of the Howard Hughes Medical Institute.


REFERENCES

Afshar, R., Pierce, S., Bolland, D. J., Corcoran, A., and Oltz, E. M. (2006). Regulation of IgHgene assembly: Role of the intronic enhancer and 50△y52 region in targeting DHJHrecombination. J. Immunol. 176(4), 2439–2447.

Alessandrini, A., and Desiderio, S. V. (1991). Coordination of immunoglobulin DJHtranscription and D-to-JH rearrangement by promoter-enhancer approximation.Mol. Cell. Biol. 11(4), 2096–2107.

Alt, F. W., Enea, V., Bothwell, A. L., and Baltimore, D. (1980). Activity of multiple light chaingenes in murine myeloma cells producing a single, functional light chain. Cell 21(1), 1–12.

Alt, F. W., Rosenberg, N., Casanova, R. J., Thomas, E., and Baltimore, D. (1982). Immuno-globulin heavy-chain expression and class switching in a murine leukaemia cell line.Nature 296(5855), 325–331.

Alt, F. W., Yancopoulos, G. D., Blackwell, T. K., Wood, C., Thomas, E., Boss, M., Coffman, R.,Rosenberg, N., Tonegawa, S., and Baltimore, D. (1984). Ordered rearrangement of immu-noglobulin heavy chain variable region segments. EMBO J. 3(6), 1209–1219.

Andrulis, E. D., Neiman, A. M., Zappulla, D. C., and Sternglanz, R. (1998). Perinuclearlocalization of chromatin facilitates transcriptional silencing. Nature 394(6693), 592–595.

Angelin-Duclos, C., and Calame, K. (1998). Evidence that immunoglobulin VH-DJ recombi-nation does not require germ line transcription of the recombining variable genesegment. Mol. Cell. Biol. 18(11), 6253–6264.

Apel, T. W., Mautner, J., Polack, A., Bornkamm, G. W., and Eick, D. (1992). Two antisensepromoters in the immunoglobulin mu-switch region drive expression of c-myc in theBurkitt’s lymphoma cell line BL67. Oncogene 7(7), 1267–1271.

Banerji, J., Olson, L., and Schaffner, W. (1983). A lymphocyte-specific cellular enhancer islocated downstream of the joining region in immunoglobulin heavy chain genes. Cell33(3), 729–740.

Barreto, V., and Cumano, A. (2000). Frequency and characterization of phenotypic Ig heavychain allelically included IgM-expressing B cells in mice. J. Immunol. 164(2), 893–899.

Bassing, C. H., Alt, F. W., Hughes, M. M., D’Auteuil, M., Wehrly, T. D., Woodman, B. B.,Gartner, F., White, J. M., Davidson, L., and Sleckman, B. P. (2000). Recombination signalsequences restrict chromosomal V(D)J recombination beyond the 12/23 rule. Nature 405

(6786), 583–586.Bates, J. G., Cado, D., Nolla, H., and Schlissel, M. S. (2007). Chromosomal position of a VH

gene segment determines its activation and inactivation as a substrate for V(D)J recombi-nation. J. Exp. Med. 204(13), 3247–3256.

Baumann, M., Mamais, A., McBlane, F., Xiao, H., and Boyes, J. (2003). Regulation of V(D)Jrecombination by nucleosome positioning at recombination signal sequences. EMBO J. 22

(19), 5197–5207.Baxter, J., Merkenschlager, M., and Fisher, A. G. (2002). Nuclear organisation and gene

expression. Curr. Opin. Cell Biol. 14(3), 372–376.Bertolino, E., Reddy, K., Medina, K. L., Parganas, E., Ihle, J., and Singh, H. (2005). Regulation

of interleukin 7-dependent immunoglobulin heavy-chain variable gene rearrangementsby transcription factor STAT5. Nat. Immunol. 6(8), 836–843.

Bolland, D. J., Wood, A. L., Johnston, C. M., Bunting, S. F., Morgan, G., Chakalova, L.,Fraser, P. J., and Corcoran, A. E. (2004). Antisense intergenic transcription in V(D)Jrecombination. Nat. Immunol. 5(6), 630–637.

Bolland, D. J., Wood, A. L., Afshar, R., Featherstone, K., Oltz, E. M., and Corcoran, A. E.(2007). Antisense intergenic transcription precedes Igh D-to-J recombination and iscontrolled by the intronic enhancer Emu. Mol. Cell. Biol. 27(15), 5523–5533.

Borghesi, L., Hsu, L. Y., Miller, J. P., Anderson, M., Herzenberg, L., Herzenberg, L.,Schlissel, M. S., Allman, D., and Gerstein, R. M. (2004). B lineage-specific regulation of


V(D)J recombinase activity is established in common lymphoid progenitors. J. Exp. Med.

199(4), 491–502.Born, W., White, J., Kappler, J., and Marrack, P. (1988). Rearrangement of IgH genes in

normal thymocyte development. J. Immunol. 140(9), 3228–3232.Bottaro, A., Young, F., Chen, J., Serwe, M., Sablitzky, F., and Alt, F. W. (1998). Deletion of the

IgH intronic enhancer and associated matrix-attachment regions decreases, but does notabolish, class switching at the mu locus. Int. Immunol. 10(6), 799–806.

Brown, K. E., Guest, S. S., Smale, S. T., Hahm, K., Merkenschlager, M., and Fisher, A. G.(1997). Association of transcriptionally silent genes with Ikaros complexes at centromericheterochromatin. Cell 91(6), 845–854.

Buchanan, K. L., Smith, E. A., Dou, S., Corcoran, L. M., and Webb, C. F. (1997). Family-specific differences in transcription efficiency of Ig heavy chain promoters. J. Immunol.

159(3), 1247–1254.Busslinger, M. (2004). Transcriptional control of early B cell development. Annu. Rev. Immu-

nol. 22, 55–79.Calame, K., and Sen, R. (2004). Transcription of immunoglobulin genes. In ‘‘Molecular

Biology of B Cells’’ (T. Honjo, F. W. Alt, and M. S. Neuberger, Eds.), pp. 83–100. ElsevierAcademic Press, London.

Chakraborty, T., Chowdhury, D., Keyes, A., Jani, A., Subrahmanyam, R., Ivanova, I., andSen, R. (2007). Repeat organization and epigenetic regulation of the DH-Cmu domain ofthe immunoglobulin heavy-chain gene locus. Mol. Cell 27(5), 842–850.

Chaudhuri, J., Basu, U., Zarrin, A., Yan, C., Franco, S., Perlot, T., Vuong, B., Wang, J.,Phan, R. T., Datta, A., Manis, J., and Alt, F. W. (2007). Evolution of the immunoglobulinheavy chain class switch recombination mechanism. Adv. Immunol. 94, 157–214.

Chen, J., Young, F., Bottaro, A., Stewart, V., Smith, R. K., and Alt, F. W. (1993). Mutations ofthe intronic IgH enhancer and its flanking sequences differentially affect accessibility ofthe JH locus. EMBO J. 12(12), 4635–4645.

Cherry, S. R., and Baltimore, D. (1999). Chromatin remodeling directly activates V(D)Jrecombination. Proc. Natl. Acad. Sci. USA 96(19), 10788–10793.

Cherry, S. R., Beard, C., Jaenisch, R., and Baltimore, D. (2000). V(D)J recombination is notactivated by demethylation of the kappa locus. Proc. Natl. Acad. Sci. USA 97(15),8467–8472.

Chowdhury, D., and Sen, R. (2001). Stepwise activation of the immunoglobulin mu heavychain gene locus. EMBO J. 20(22), 6394–6403.

Chowdhury, D., and Sen, R. (2003). Transient IL-7/IL-7R signaling provides amechanism forfeedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity 18

(2), 229–241.Cobb, R. M., Oestreich, K. J., Osipovich, O. A., and Oltz, E. M. (2006). Accessibility control of

V(D)J recombination. Adv. Immunol. 91, 45–109.Cogne, M., Lansford, R., Bottaro, A., Zhang, J., Gorman, J., Young, F., Cheng, H. L., and

Alt, F. W. (1994). A class switch control region at the 30 end of the immunoglobulin heavychain locus. Cell 77(5), 737–747.

Corcoran, A. E. (2005). Immunoglobulin locus silencing and allelic exclusion. Semin.Immunol. 17(2), 141–154.

Daly, J., Licence, S., Nanou, A., Morgan, G., and Martensson, I. L. (2007). Transcription ofproductive and nonproductive VDJ-recombined alleles after IgH allelic exclusion. EMBOJ. 26(19), 4273–4282.

Dariavach, P., Williams, G. T., Campbell, K., Pettersson, S., and Neuberger, M. S. (1991). Themouse IgH 30-enhancer. Eur. J. Immunol. 21(6), 1499–1504.

Delpy, L., Decourt, C., Le Bert, M., and Cogne, M. (2002). B cell development arrest uponinsertion of a neo gene between JH and Emu: Promoter competition results in transcrip-tional silencing of germline JH and complete VDJ rearrangements. J. Immunol. 169(12),6875–6882.


Di Noia, J. M., and Neuberger, M. S. (2007). Molecular mechanisms of antibody somatichypermutation. Annu. Rev. Biochem. 76, 1–22.

Drejer-Teel, A. H., Fugmann, S. D., and Schatz, D. G. (2007). The beyond 12/23 restriction isimposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination.Mol. Cell. Biol. 27(18), 6288–6299.

Early, P., Huang, H., Davis, M., Calame, K., and Hood, L. (1980). An immunoglobulin heavychain variable region gene is generated from three segments of DNA: VH, D and JH. Cell19(4), 981–992.

Eaton, S., and Calame, K. (1987). Multiple DNA sequence elements are necessary for thefunction of an immunoglobulin heavy chain promoter. Proc. Natl. Acad. Sci. USA 84(21),7634–7638.

Fugmann, S. D., Lee, A. I., Shockett, P. E., Villey, I. J., and Schatz, D. G. (2000). The RAGproteins and V(D)J recombination: Complexes, ends, and transposition. Annu. Rev.Immunol. 18, 495–527.

Fukita, Y., Jacobs, H., and Rajewsky, K. (1998). Somatic hypermutation in the heavy chainlocus correlates with transcription. Immunity 9(1), 105–114.

Fuxa, M., Skok, J., Souabni, A., Salvagiotto, G., Roldan, E., and Busslinger, M. (2004). Pax5induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18(4), 411–422.

Garrett, F. E., Emelyanov, A. V., Sepulveda,M. A., Flanagan, P., Volpi, S., Li, F., Loukinov, D.,Eckhardt, L. A., Lobanenkov, V. V., and Birshtein, B. K. (2005). Chromatin architecturenear a potential 30 end of the igh locus involves modular regulation of histone modifica-tions during B-cell development and in vivo occupancy at CTCF sites.Mol. Cell. Biol. 25(4),1511–1525.

Gillies, S. D., Morrison, S. L., Oi, V. T., and Tonegawa, S. (1983). A tissue-specific transcrip-tion enhancer element is located in the major intron of a rearranged immunoglobulinheavy chain gene. Cell 33(3), 717–728.

Golding, A., Chandler, S., Ballestar, E., Wolffe, A. P., and Schlissel, M. S. (1999). Nucleosomestructure completely inhibits in vitro cleavage by the V(D)J recombinase. EMBO J. 18(13),3712–3723.

Goldmit, M., and Bergman, Y. (2004). Monoallelic gene expression: A repertoire of recurrentthemes. Immunol. Rev. 200, 197–214.

Gorman, J. R., and Alt, F. W. (1998). Regulation of immunoglobulin light chain isotypeexpression. Adv. Immunol. 69, 113–181.

Grawunder, U., Leu, T. M., Schatz, D. G., Werner, A., Rolink, A. G., Melchers, F., andWinkler, T. H. (1995). Down-regulation of RAG1 and RAG2 gene expression in preBcells after functional immunoglobulin heavy chain rearrangement. Immunity 3(5),601–608.

Gu, H., Kitamura, D., and Rajewsky, K. (1991). B cell development regulated by generearrangement: Arrest of maturation by membrane-bound D mu protein and selectionof DH element reading frames. Cell 65(1), 47–54.

Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D., and Hayakawa, K. (1991). Resolu-tion and characterization of pro-B and pre-pro-B cell stages in normal mouse bonemarrow. J. Exp. Med. 173(5), 1213–1225.

Hesslein, D. G., and Schatz, D. G. (2001). Factors and forces controlling V(D)J recombination.Adv. Immunol. 78, 169–232.

Hesslein, D. G., Pflugh, D. L., Chowdhury, D., Bothwell, A. L., Sen, R., and Schatz, D. G.(2003). Pax5 is required for recombination of transcribed, acetylated, 50 IgH V genesegments. Genes Dev. 17(1), 37–42.

Hewitt, S. L., Farmer, D., Marszalek, K., Cadera, E., Liang, H. E., Xu, Y., Schlissel, M. S., andSkok, J. A. (2008). Association between the Igk and Igh immunoglobulin loci mediated by


the 30 Igk enhancer induces ‘decontraction’ of the Igh locus in pre-B cells. Nat. Immunol.

9(4), 396–404.Hsieh, C. L., and Lieber, M. R. (1992). CpG methylated minichromosomes become inaccessi-

ble for V(D)J recombination after undergoing replication. EMBO J. 11(1), 315–325.Inlay, M., Alt, F. W., Baltimore, D., and Xu, Y. (2002). Essential roles of the kappa light chain

intronic enhancer and 30 enhancer in kappa rearrangement and demethylation.Nat. Immunol. 3(5), 463–468.

Inlay, M. A., Lin, T., Gao, H. H., and Xu, Y. (2006). Critical roles of the immunoglobulinintronic enhancers in maintaining the sequential rearrangement of IgH and Igk loci.J. Exp. Med. 203(7), 1721–1732.

Jaenisch, R., and Bird, A. (2003). Epigenetic regulation of gene expression: How the genomeintegrates intrinsic and environmental signals. Nat. Genet. 33(Suppl), 245–254.

Jenuwein, T., and Allis, C. D. (2001). Translating the histone code. Science 293(5532),1074–1080.

Jhunjhunwala, S., van Zelm, M. C., Peak, M. M., Cutchin, S., Riblet, R., van Dongen, J. J.,Grosveld, F. G., Knoch, T. A., and Murre, C. (2008). The 3D structure of the immunoglob-ulin heavy-chain locus: Implications for long-range genomic interactions. Cell 133(2),265–279.

Johnson, K., Angelin-Duclos, C., Park, S., and Calame, K. L. (2003). Changes in histoneacetylation are associated with differences in accessibility of V(H) gene segments toV-DJ recombination during B-cell ontogeny and development. Mol. Cell. Biol. 23(7),2438–2450.

Johnson, K., Pflugh, D. L., Yu, D., Hesslein, D. G., Lin, K. I., Bothwell, A. L., Thomas-Tikhonenko, A., Schatz, D. G., and Calame, K. (2004). B cell-specific loss of histone 3 lysine9 methylation in the V(H) locus depends on Pax5. Nat. Immunol. 5(8), 853–861.

Johnston, C. M., Wood, A. L., Bolland, D. J., and Corcoran, A. E. (2006). Complete sequenceassembly and characterization of the C57BL/6mouse Ig heavy chain V region. J. Immunol.

176(7), 4221–4234.Julius, M. A., Street, A. J., Fahrlander, P. D., Yang, J. Q., Eisenman, R. N., and Marcu, K. B.

(1988). Translocated c-myc genes produce chimeric transcripts containing antisensesequences of the immunoglobulin heavy chain locus in mouse plasmacytomas. Oncogene

2(5), 469–476.Jung, S., Rajewsky, K., and Radbruch, A. (1993). Shutdown of class switch recombination by

deletion of a switch region control element. Science 259(5097), 984–987.Jung, D., Bassing, C. H., Fugmann, S. D., Cheng, H. L., Schatz, D. G., and Alt, F. W. (2003).

Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJrecombination in nonlymphoid cells. Immunity 18(1), 65–74.

Jung, D., Giallourakis, C., Mostoslavsky, R., and Alt, F. W. (2006). Mechanism and control ofV(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 24,541–570.

Khamlichi, A. A., Pinaud, E., Decourt, C., Chauveau, C., and Cogne, M. (2000). The 30 IgHregulatory region: A complex structure in a search for a function. Adv. Immunol. 75,

317–345.Kitamura, D., and Rajewsky, K. (1992). Targeted disruption of mu chain membrane exon

causes loss of heavy-chain allelic exclusion. Nature 356(6365), 154–156.Koralov, S. B., Muljo, S. A., Galler, G. R., Krek, A., Chakraborty, T., Kanellopoulou, C.,

Jensen, K., Cobb, B. S., Merkenschlager, M., Rajewsky, N., and Rajewsky, K. (2008).Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage.Cell 132(5), 860–874.

Kosak, S. T., Skok, J. A., Medina, K. L., Riblet, R., Le Beau, M. M., Fisher, A. G., and Singh, H.(2002). Subnuclear compartmentalization of immunoglobulin loci during lymphocytedevelopment. Science 296(5565), 158–162.


Kottmann, A. H., Zevnik, B., Welte, M., Nielsen, P. J., and Kohler, G. (1994). A secondpromoter and enhancer element within the immunoglobulin heavy chain locus. Eur. J.Immunol. 24(4), 817–821.

Kouzarides, T., and Berger, S. L. (2007). Chromatin modifications and their mechanism ofaction. In ‘‘Epigenetics’’ (C. D. Allis, T. Jenuwein, D. Reinberg, and M. L. Caparros, Eds.),pp. 191–209. Cold Spring Harbor Academic Press, Cold Spring Harbor, NY.

Krangel, M. S. (2003). Gene segment selection in V(D)J recombination: Accessibility andbeyond. Nat. Immunol. 4(7), 624–630.

Kurosawa, Y., von Boehmer, H., Haas, W., Sakano, H., Trauneker, A., and Tonegawa, S.(1981). Identification of D segments of immunoglobulin heavy-chain genes and theirrearrangement in T lymphocytes. Nature 290(5807), 565–570.

Kwon, J., Morshead, K. B., Guyon, J. R., Kingston, R. E., and Oettinger, M. A. (2000). Histoneacetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage ofnucleosomal DNA. Mol. Cell 6(5), 1037–1048.

Lennon, G. G., and Perry, R. P. (1985). C mu-containing transcripts initiate heterogeneouslywithin the IgH enhancer region and contain a novel 50-nontranslatable exon. Nature 318

(6045), 475–478.Liang, H. E., Hsu, L. Y., Cado, D., Cowell, L. G., Kelsoe, G., and Schlissel, M. S. (2002). The

‘‘dispensable’’ portion of RAG2 is necessary for efficient V-to-DJ rearrangement during Band T cell development. Immunity 17(5), 639–651.

Lieberson, R., Giannini, S. L., Birshtein, B. K., and Eckhardt, L. A. (1991). An enhancer at the30 end of the mouse immunoglobulin heavy chain locus.Nucleic Acids Res. 19(4), 933–937.

Liu, Y., Subrahmanyam, R., Chakraborty, T., Sen, R., and Desiderio, S. (2007). A planthomeodomain in RAG-2 that binds hypermethylated lysine 4 of histone H3 is necessaryfor efficient antigen-receptor-gene rearrangement. Immunity 27(4), 561–571.

Loffert, D., Ehlich, A., Muller, W., and Rajewsky, K. (1996). Surrogate light chain expressionis required to establish immunoglobulin heavy chain allelic exclusion during early B celldevelopment. Immunity 4(2), 133–144.

Lutzker, S., and Alt, F. W. (1988). Structure and expression of germ line immunoglobulingamma 2b transcripts. Mol. Cell. Biol. 8(4), 1849–1852.

Maes, J., O’Neill, L. P., Cavelier, P., Turner, B. M., Rougeon, F., and Goodhardt, M. (2001).Chromatin remodeling at the Ig loci prior to V(D)J recombination. J. Immunol. 167(2),866–874.

Maes, J., Chappaz, S., Cavelier, P., O’Neill, L., Turner, B., Rougeon, F., and Goodhardt, M.(2006). Activation of V(D)J recombination at the IgH chain JH locus occurs within a6-kilobase chromatin domain and is associated with nucleosomal remodeling. J. Immunol.

176(9), 5409–5417.Malynn, B. A., Yancopoulos, G. D., Barth, J. E., Bona, C. A., and Alt, F. W. (1990). Biased

expression of JH-proximal VH genes occurs in the newly generated repertoire of neonataland adult mice. J. Exp. Med. 171(3), 843–859.

Malynn, B. A., Shaw, A. C., Young, F., Stewart, V., and Alt, F. W. (2002). Truncatedimmunoglobulin Dmu causes incomplete developmental progression of RAG-deficientpro-B cells. Mol. Immunol. 38(7), 547–556.

Manis, J. P., van der Stoep, N., Tian, M., Ferrini, R., Davidson, L., Bottaro, A., and Alt, F. W.(1998). Class switching in B cells lacking 30 immunoglobulin heavy chain enhancers.J. Exp. Med. 188(8), 1421–1431.

Manis, J. P., Tian, M., and Alt, F. W. (2002). Mechanism and control of class-switchrecombination. Trends Immunol. 23(1), 31–39.

Martens, J. A., and Winston, F. (2003). Recent advances in understanding chromatinremodeling by Swi/Snf complexes. Curr. Opin. Genet. Dev. 13(2), 136–142.

Martensson, I. L., Keenan, R. A., and Licence, S. (2007). The pre-B-cell receptor. Curr. Opin.

Immunol. 19(2), 137–142.


Mason, J. O., Williams, G. T., and Neuberger, M. S. (1985). Transcription cell type specificityis conferred by an immunoglobulin VH gene promoter that includes a functional consen-sus sequence. Cell 41(2), 479–487.

Matthews, A. G., Kuo, A. J., Ramon-Maiques, S., Han, S., Champagne, K. S., Ivanov, D.,Gallardo, M., Carney, D., Cheung, P., Ciccone, D. N., Walter, K. L., Utz, P. J., et al. (2007).RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination.Nature 450(7172), 1106–1110.

Matthias, P., and Baltimore, D. (1993). The immunoglobulin heavy chain locus containsanother B-cell-specific 30 enhancer close to the alpha constant region. Mol. Cell. Biol.13(3), 1547–1553.

Morrison, A. M., Jager, U., Chott, A., Schebesta, M., Haas, O. A., and Busslinger, M. (1998).Deregulated PAX-5 transcription from a translocated IgH promoter in marginal zonelymphoma. Blood 92(10), 3865–3878.

Morshead, K. B., Ciccone, D. N., Taverna, S. D., Allis, C. D., and Oettinger, M. A. (2003).Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1and flanked by peaks of histoneH3 dimethylated at lysine 4. Proc. Natl. Acad. Sci. USA 100

(20), 11577–11582.Morvan, C. L., Pinaud, E., Decourt, C., Cuvillier, A., and Cogne, M. (2003). The immuno-

globulin heavy-chain locus hs3b and hs4 30 enhancers are dispensable for VDJ assemblyand somatic hypermutation. Blood 102(4), 1421–1427.

Mostoslavsky, R., Singh, N., Kirillov, A., Pelanda, R., Cedar, H., Chess, A., and Bergman, Y.(1998). Kappa chainmonoallelic demethylation and the establishment of allelic exclusion.Genes Dev. 12(12), 1801–1811.

Mostoslavsky, R., Singh, N., Tenzen, T., Goldmit, M., Gabay, C., Elizur, S., Qi, P.,Reubinoff, B. E., Chess, A., Cedar, H., and Bergman, Y. (2001). Asynchronous replicationand allelic exclusion in the immune system. Nature 414(6860), 221–225.

Mostoslavsky, R., Alt, F. W., and Rajewsky, K. (2004). The lingering enigma of the allelicexclusion mechanism. Cell 118(5), 539–544.

Nitschke, L., Kestler, J., Tallone, T., Pelkonen, S., and Pelkonen, J. (2001). Deletion of theDQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJrecombination and altered D gene usage. J. Immunol. 166(4), 2540–2552.

Norio, P., Kosiyatrakul, S., Yang, Q., Guan, Z., Brown, N. M., Thomas, S., Riblet, R., andSchildkraut, C. L. (2005). Progressive activation of DNA replication initiation in largedomains of the immunoglobulin heavy chain locus during B cell development. Mol. Cell

20(4), 575–587.Nussenzweig, M. C., Shaw, A. C., Sinn, E., Campos-Torres, J., and Leder, P. (1988). Allelic

exclusion in transgenic mice carrying mutant human IgM genes. J. Exp. Med. 167(6),1969–1974.

Nutt, S. L., Urbanek, P., Rolink, A., and Busslinger, M. (1997). Essential functions of Pax5(BSAP) in pro-B cell development: Difference between fetal and adult B lymphopoiesisand reduced V-to-DJ recombination at the IgH locus. Genes Dev. 11(4), 476–491.

Oestreich,K. J., Cobb, R.M., Pierce, S., Chen, J., Ferrier, P., andOltz, E.M. (2006). Regulation ofTCRbeta gene assembly by a promoter/enhancer holocomplex. Immunity 24(4), 381–391.

Ono, M., and Nose, M. (2007). Persistent expression of an unproductive immunoglobulinheavy chain allele with DH-JH-gamma configuration in peripheral tissues. APMIS 115

(12), 1350–1356.Osipovich, O., Milley, R., Meade, A., Tachibana, M., Shinkai, Y., Krangel, M. S., and

Oltz, E. M. (2004). Targeted inhibition of V(D)J recombination by a histone methyltrans-ferase. Nat. Immunol. 5(3), 309–316.

Osipovich, O., Cobb, R. M., Oestreich, K. J., Pierce, S., Ferrier, P., and Oltz, E. M. (2007).Essential function for SWI-SNF chromatin-remodeling complexes in the promoter-directed assembly of Tcrb genes. Nat. Immunol. 8(8), 809–816.


Parslow, T. G., Blair, D. L., Murphy, W. J., and Granner, D. K. (1984). Structure of the 5 endsof immunoglobulin genes: A novel conserved sequence. Proc. Natl. Acad. Sci. USA 81(9),2650–2654.

Pawlitzky, I., Angeles, C. V., Siegel, A. M., Stanton, M. L., Riblet, R., and Brodeur, P. H.(2006). Identification of a candidate regulatory element within the 5 flanking region of themouse Igh locus defined by pro-B cell-specific hypersensitivity associated with bindingof PU.1, Pax5, and E2A. J. Immunol. 176(11), 6839–6851.

Perlot, T., Alt, F. W., Bassing, C. H., Suh, H., and Pinaud, E. (2005). Elucidation of IgHintronic enhancer functions via germ-line deletion. Proc. Natl. Acad. Sci. USA 102(40),14362–14367.

Perlot, T., Li, G., and Alt, F. W. (2008). Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions. Proc. Natl. Acad. Sci. USA 105(10), 3843–3848.

Pettersson, S., Cook, G. P., Bruggemann, M., Williams, G. T., and Neuberger, M. S. (1990).A second B cell-specific enhancer 30 of the immunoglobulin heavy-chain locus.Nature 344

(6262), 165–168.Pinaud, E., Khamlichi, A. A., Le Morvan, C., Drouet, M., Nalesso, V., Le Bert, M., and

Cogne, M. (2001). Localization of the 30 IgH locus elements that effect long-distanceregulation of class switch recombination. Immunity 15(2), 187–199.

Pokholok, D. K., Harbison, C. T., Levine, S., Cole, M., Hannett, N. M., Lee, T. I., Bell, G. W.,Walker, K., Rolfe, P. A., Herbolsheimer, E., Zeitlinger, J., Lewitter, F., et al. (2005).Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122(4),517–527.

Rajewsky, K. (1996). Clonal selection and learning in the antibody system. Nature 381(6585),751–758.

Reddy, K. L., Zullo, J. M., Bertolino, E., and Singh, H. (2008). Transcriptional repressionmediated by repositioning of genes to the nuclear lamina. Nature.

Reth, M. G., and Alt, F. W. (1984). Novel immunoglobulin heavy chains are produced fromDJH gene segment rearrangements in lymphoid cells. Nature 312(5993), 418–423.

Reth, M., Petrac, E., Wiese, P., Lobel, L., and Alt, F. W. (1987). Activation of V kappa generearrangement in pre-B cells follows the expression of membrane-bound immunoglobu-lin heavy chains. EMBO J. 6(11), 3299–3305.

Retter, I., Chevillard, C., Scharfe, M., Conrad, A., Hafner, M., Im, T. H., Ludewig, M.,Nordsiek, G., Severitt, S., Thies, S., Mauhar, A., Blocker, H., et al. (2007). Sequence andcharacterization of the Ig heavy chain constant and partial variable region of the mousestrain 129S1. J. Immunol. 179(4), 2419–2427.

Ringrose, L., and Paro, R. (2004). Epigenetic regulation of cellular memory by the Polycomband Trithorax group proteins. Annu. Rev. Genet. 38, 413–443.

Roldan, E., Fuxa, M., Chong, W., Martinez, D., Novatchkova, M., Busslinger, M., andSkok, J. A. (2005). Locus ‘decontraction’ and centromeric recruitment contribute to allelicexclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6(1), 31–41.

Rooney, S., Chaudhuri, J., and Alt, F. W. (2004). The role of the non-homologous end-joiningpathway in lymphocyte development. Immunol. Rev. 200, 115–131.

Sakai, E., Bottaro, A., Davidson, L., Sleckman, B. P., and Alt, F. W. (1999). Recombination andtranscription of the endogenous Ig heavy chain locus is effected by the Ig heavy chainintronic enhancer core region in the absence of the matrix attachment regions. Proc. Natl.

Acad. Sci. USA 96(4), 1526–1531.Sakano, H., Maki, R., Kurosawa, Y., Roeder, W., and Tonegawa, S. (1980). Two types of

somatic recombination are necessary for the generation of complete immunoglobulinheavy-chain genes. Nature 286(5774), 676–683.

Sayegh, C., Jhunjhunwala, S., Riblet, R., and Murre, C. (2005). Visualization of loopinginvolving the immunoglobulin heavy-chain locus in developing B cells. Genes Dev. 19

(3), 322–327.


Schlissel, M. S., Corcoran, L. M., and Baltimore, D. (1991a). Virus-transformed pre-B cellsshow ordered activation but not inactivation of immunoglobulin gene rearrangementand transcription. J. Exp. Med. 173(3), 711–720.

Schlissel, M., Voronova, A., and Baltimore, D. (1991b). Helix-loop-helix transcription factorE47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrange-ment in a pre-T-cell line. Genes Dev. 5(8), 1367–1376.

Schweighoffer, E., Vanes, L., Mathiot, A., Nakamura, T., and Tybulewicz, V. L. (2003).Unexpected requirement for ZAP-70 in pre-B cell development and allelic exclusion.Immunity 18(4), 523–533.

Seidl, K. J., Manis, J. P., Bottaro, A., Zhang, J., Davidson, L., Kisselgof, A., Oettgen, H., andAlt, F. W. (1999). Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus. Proc.Natl. Acad. Sci. USA 96(6), 3000–3005.

Sen, R., and Oltz, E. (2006). Genetic and epigenetic regulation of IgH gene assembly. Curr.Opin. Immunol. 18(3), 237–242.

Serwe, M., and Sablitzky, F. (1993). V(D)J recombination in B cells is impaired but notblocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.EMBO J. 12(6), 2321–2327.

Sikes, M. L., Meade, A., Tripathi, R., Krangel, M. S., and Oltz, E. M. (2002). Regulation of V(D)J recombination: A dominant role for promoter positioning in gene segment accessibility.Proc. Natl. Acad. Sci. USA 99(19), 12309–12314.

Skok, J. A., Brown, K. E., Azuara, V., Caparros, M. L., Baxter, J., Takacs, K., Dillon, N.,Gray, D., Perry, R. P., Merkenschlager, M., and Fisher, A. G. (2001). Nonequivalentnuclear location of immunoglobulin alleles in B lymphocytes.Nat. Immunol. 2(9), 848–854.

Stanhope-Baker, P., Hudson, K. M., Shaffer, A. L., Constantinescu, A., and Schlissel, M. S.(1996). Cell type-specific chromatin structure determines the targeting of V(D)J recombi-nase activity in vitro. Cell 85(6), 887–897.

Stavnezer, J. (2000). Molecular processes that regulate class switching. Curr. Top. Microbiol.Immunol. 245(2), 127–168.

Stein, R., Razin, A., and Cedar, H. (1982). In vitro methylation of the hamster adeninephosphoribosyltransferase gene inhibits its expression in mouse L cells. Proc. Natl.

Acad. Sci. USA 79(11), 3418–3422.Storb, U., and Arp, B. (1983). Methylation patterns of immunoglobulin genes in lymphoid

cells: Correlation of expression and differentiation with undermethylation. Proc. Natl.

Acad. Sci. USA 80(21), 6642–6646.Su, L. K., and Kadesch, T. (1990). The immunoglobulin heavy-chain enhancer functions as

the promoter for I mu sterile transcription. Mol. Cell. Biol. 10(6), 2619–2624.Su, I. H., Basavaraj, A., Krutchinsky, A. N., Hobert, O., Ullrich, A., Chait, B. T., and

Tarakhovsky, A. (2003). Ezh2 controls B cell development through histone H3 methyla-tion and Igh rearrangement. Nat. Immunol. 4(2), 124–131.

Sun, T., and Storb, U. (2001). Insertion of phosphoglycerine kinase (PGK)-neo 50 of Jlambda1dramatically enhances VJlambda1 rearrangement. J. Exp. Med. 193(6), 699–712.

Vardimon, L., Kressmann, A., Cedar, H., Maechler, M., and Doerfler, W. (1982). Expressionof a cloned adenovirus gene is inhibited by in vitromethylation. Proc. Natl. Acad. Sci. USA

79(4), 1073–1077.Volpe, T. A., Kidner, C., Hall, I. M., Teng, G., Grewal, S. I., and Martienssen, R. A. (2002).

Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.Science 297(5588), 1833–1837.

Wang, X. F., and Calame, K. (1985). The endogenous immunoglobulin heavy chain enhancercan activate tandem VH promoters separated by a large distance. Cell 43(3 Pt 2), 659–665.


Watt, F., and Molloy, P. L. (1988). Cytosine methylation prevents binding to DNA of a HeLacell transcription factor required for optimal expression of the adenovirus major latepromoter. Genes Dev. 2(9), 1136–1143.

Whitehurst, C. E., Chattopadhyay, S., and Chen, J. (1999). Control of V(D)J recombinationalaccessibility of the D beta 1 gene segment at the TCR beta locus by a germline promoter.Immunity 10(3), 313–322.

Whitehurst, C. E., Schlissel, M. S., and Chen, J. (2000). Deletion of germline promoter PD beta1 from the TCR beta locus causes hypermethylation that impairs D beta 1 recombinationby multiple mechanisms. Immunity 13(5), 703–714.

Wuerffel, R., Wang, L., Grigera, F., Manis, J., Selsing, E., Perlot, T., Alt, F. W., Cogne, M.,Pinaud, E., and Kenter, A. L. (2007). S-S synapsis during class switch recombination ispromoted by distantly located transcriptional elements and activation-induced deami-nase. Immunity 27(5), 711–722.

Xu, Y., Davidson, L., Alt, F. W., and Baltimore, D. (1996). Deletion of the Ig kappa light chainintronic enhancer/matrix attachment region impairs but does not abolish V kappa Jkappa rearrangement. Immunity 4(4), 377–385.

Yancopoulos, G. D., and Alt, F. W. (1985). Developmentally controlled and tissue-specificexpression of unrearranged VH gene segments. Cell 40(2), 271–281.

Yancopoulos, G. D., Desiderio, S. V., Paskind, M., Kearney, J. F., Baltimore, D., and Alt, F. W.(1984). Preferential utilization of the most JH-proximal VH gene segments in pre-B-celllines. Nature 311(5988), 727–733.

Yancopoulos, G. D., Blackwell, T. K., Suh, H., Hood, L., and Alt, F. W. (1986). Introduced Tcell receptor variable region gene segments recombine in pre-B cells: Evidence that B andT cells use a common recombinase. Cell 44(2), 251–259.

Yancopoulos, G. D., Malynn, B. A., and Alt, F. W. (1988). Developmentally regulated andstrain-specific expression of murine VH gene families. J. Exp. Med. 168(1), 417–435.

Yang, Q., Riblet, R., and Schildkraut, C. L. (2005). Sites that direct nuclear compartmentaliza-tion are near the 5 end of the mouse immunoglobulin heavy-chain locus.Mol. Cell. Biol. 25(14), 6021–6030.

Ye, J. (2004). The immunoglobulin IGHD gene locus in C57BL/6 mice. Immunogenetics 56(6),399–404.

Zhang, J., Bottaro, A., Li, S., Stewart, V., and Alt, F. W. (1993). A selective defect in IgG2bswitching as a result of targeted mutation of the I gamma 2b promoter and exon. EMBO J.

12(9), 3529–3537.

Documents

Chapter 1 - Cis-Regulatory Elements and Epigenetic Changes …biology.hunter.cuny.edu/molecularbio/Class Materials... · 2011-02-08 · H genes which are then joined by NHEJ or other