of April 5, 2017.This information is current as

Gene Segment TCRD V Regulatory Elements in the Promoter of a Murine

Laura J. Kienker, Maya R. Ghosh and Philip W. Tucker

http://www.jimmunol.org/content/161/2/7911998; 161:791-804; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/161/2/791.full#ref-list-1

, 54 of which you can access for free at: cites 99 articlesThis article

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

Regulatory Elements in the Promoter of a Murine TCRD VGene Segment1,2

Laura J. Kienker,* Maya R. Ghosh,† and Philip W. Tucker 3†

TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires theaccessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessiblechromatin configuration. We therefore investigated the regulation ofTCRD V rearrangements by characterizing the promoter ofthe first TCRD V segment to be rearranged,DV101S1, under the influence of its own enhancer. Sequences required for fullpromoter activity were identified by transient transfections of normal and mutated promoters into a humangd lymphoma, andnecessary elements fall between286 and 166 nt, relative to the major transcription start site. They include a cAMP responsiveelement (CRE) at262, an Ets site at239, a TATA box at 226, the major transcriptional start site sequence (28 to 25 and 22to 111), and a downstream sequence (112 to 133). Gel shift analyses and in vitro DNase I footprinting showed that nuclearproteins bind to the functionally relevant CRE, Ets,11 to 110 sequence, and the117 to121 sequence. Nuclear proteins also bindto an E box at 252, and GATA-3 binds to a GATA motif at 25, as shown by Ab ablation-supershift experiments, but mutationsthat abrogated protein binding to these sites failed to affectDV101S1promoter activity. We conclude that not all protein-bindingsites within the DV101S1minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibilityremains that the GATA and E box sites function in enhancer independentDV101S1germline transcription. The Journal ofImmunology,1998, 161: 791–804.

T he gd andab T cell development in the thymus requiresthe assembly of correspondingTCRgene variable exonsfrom V, D (in the case ofTCR DandB genes only), and

J gene segments through theV(D)J joining recombination process(1–4). Once assembled,TCRgenes are transcribed from promotersassociated with the rearrangedV gene segments (5). This transcrip-tion requires the activity of enhancer (Enh)4 elements located ad-jacent to theC exons (5). Subsequently, splicing of the primaryRNA transcripts occurs and the mature mRNA species give rise tofunctional TCR polypeptides if there is a proper reading frame.

The fourTCRgenes are located at three distinct chromosomalloci. TheTCRDlocus is included within theTCRAlocus such thatrearrangement of theA TCR chain gene segments is accompaniedby deletion of theD TCR gene (6). In addition, there is someoverlap inV gene segment usage in TCRA andD chains, althoughtheV gene segments used inA andD chains are largely distinct (7).Assembly of the fourTCRgene variable exons is highly regulatedand temporally ordered.TCR genes are rearranged only in lym-phoid precursors; then, complete assembly of the genes occurs

only in T lymphocyte progenitors (8, 9). Further, theTCR G, D,andB variable exons assemble earlier during T-cell differentiationthan theTCRAvariable exons (3, 10), and the assembly ofTCRBvariable exons occurs sequentially.D-to-J rearrangements precedeV-to-D rearrangements in theTCRBlocus (8). Finally, within theTCR Gand D loci, the V gene segments rearrange in a distinctorder during ontogeny. In the mouse,GV1S1,GV2S1A1, andDV101S1predominate in rearrangements in the fetus, whereasGV3S1A1and otherTCRD Vgene segments, especiallyDV105S1,predominate in the adult (7, 11–13).

A single enzymatic system, theV(D)J recombinase, catalyzes allV(D)J rearrangement events in T and B cells, and two of the re-combination factors, RAG-1 and RAG-2, are primarily lymphoidspecific (14). Thus, restricted expression of the RAG genes may bethe reason whyV(D)J recombination occurs only in lymphoid pre-cursors. However, differential rearrangement of Ag receptor geneswithin cells with recombinase activity requires an alternative ex-planation. The recombination mechanism involves recognition ofrecombination signal sequences, and these are conserved. Conse-quently, Yancopoulos and Alt (15) proposed that regulation wasachieved through substrate accessibility. They postulated thatchromatin is normally in a state refractory toV(D)J recombinationand that regulated changes in the accessibility of the chromatin totheV(D)J recombinase enzyme system occur (15). Support for this“accessibility model” has been provided largely through indirectstudies that utilize transcription as a measure of locus accessibility.Rearrangement has been shown to correlate with prior transcrip-tion of the corresponding unrearranged genes (12, 16), and cessa-tion of rearrangement has been shown to correlate with the disap-pearance of corresponding sterile transcripts (12, 17). Morerecently, it has been shown, in an in vitro system, that cleavage ofparticular recombination signal sequences within chromatin de-pends on the source of the chromatin, in terms of both cell type anddevelopmental stage, confirming the role of locus accessibility intargeting gene rearrangement (18).

*Harold C. Simmons Arthritis Research Center, Department of Internal Medicine,University of Texas Southwestern Medical Center, Dallas, TX 75235; and†Institutefor Cellular and Molecular Biology, University of Texas, Austin, TX 78712

Received for publication October 8, 1997. Accepted for publication March 17, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grant GM41497-02.L.J.K. was supported by Public Health Service Award 5 T32 AR07341-13 from theNational Institute of Arthritis and Musculoskeletal and Skin Diseases.2 The nucleotide sequence in this paper has been submitted to the GenBank database(accession number L41687).3 Address correspondence and reprint requests to Dr. P. W. Tucker, Institute forCellular and Molecular Biology, The University of Texas at Austin, ExperimentalScience Building, Room 534A, 24th and Speedway, Austin, TX 78712-1095. E-mailaddress: philtucker@mail.utexas.edu4 Abbreviations used in this paper: Enh, enhancer; CAT, chloramphenicol acetyltrans-ferase; EMSA, electrophoretic mobility shift assay; Inr, initiator; m, murine; CRE,cAMP responsive element; nt, nucleotide.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

Attention has now focused on determining thecis-acting ele-ments that govern the accessible chromatin configuration requiredfor V(D)J recombination. Studies of transgenic mice carrying re-combination substrates, as well as studies eliminating regulatoryelements from endogenous loci by homologous recombination,have shown that Ag receptor transcriptional controlling regions(promoters, Enhs, and silencers) are necessary for the regulation ofV(D)J recombination in developing lymphocytes (19). Thetrans-acting factors involved, and the mechanism(s) by which transcrip-tional controlling regions help to mediate the accessibility of chro-mosomal substrates to the recombinase, remain to be elucidated.Transcription itself may be unnecessary for making a locus “ac-cessible,” as instances of rearrangement in the absence of tran-scription have been reported (20–22). However, even if transcrip-tion is required for rearrangement, transcriptional controllingregions will also regulate other molecular events that are prereq-uisites for recombination because transcription initiation fromVandD gene segments has been observed to be insufficient for re-arrangement of these gene segments (23, 24). Such events mayinclude changes in chromatin structure, CpG demethylation,and/or recruitment of components of theV(D)J recombinase (14,19), andcis-acting elements, within the Ag receptor gene promoterand Enh regions, distinct from those regulating transcription maycontrol them (24, 25).

We are interested in determining the role ofTCRD Vpromotersequences in establishing the developmental program ofTCRD Vrearrangements (12, 13, 26–28). As an initial step in examining thepossibility that promoter sequences regulateTCRD Vaccessibility,we have identified the transcriptional regulatory elements in themurine DV101S1promoter.DV101S1was selected because it isthe firstTCRD Vsegment to be rearranged, and it is used exclu-sively in TCRD chains.

Materials and MethodsCell lines

33BTE-140.9 (GV2S1A1,DV101S1), 70BET104 (GV1S1, DV101S1),33BTE-125.5 (GV3S1A1, DV2S8), and 33BTE-67.1 (GV3S1A1,DV104S1) are murinegd T cell hybridomas (29, 30). JAC-3 (GV1S1,DV101S1) is an IL-2-independent dendritic epidermal T cell line made bystably transfecting the ID2 cell line (31) with a CMV-driven human IL-2vector. Molt-13 is a humangd T cell line (32, 33). Jurkat is a humanabexpressing mature T cell line. EL4 and AKR117 are murineab expressingT cell lines. 38B9 and PD31 are murine pre-B cell lines (34). M12.4 is amature BALB/c B cell lymphoma (mIg2 Ia2 FcR1) (35). All cell lineswere propagated in RPMI 1640 medium supplemented with 10% FCS, 2mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyru-vate, 57.2mM 2-ME, 100 U/ml penicillin-streptomycin, and 25 mMHEPES (pH 7.3).

DV101S1promoter region fragment

fAIR, a 14.6-kbMboI fragment containing theDV101S1gene segmentand leader exons in the vectorlJ1, isolated from a B10.A liver genomiclibrary, was obtained from Dr. Y. H. Chien (Stanford University, Stanford,CA). A 3.4-kb BamHI fragment containing 53 bp of theV gene segment,the leader exons, and upstream DNA was then subcloned into pGEM-7Zf(1) (Promega, Madison, WI), and the dideoxy chain terminationmethod (36) was used to sequence 1287 bp at theV gene segment end ofthe insert.

S1 nuclease protection analyses

Total cellular RNA was isolated from T cell hybridomas and cell lines bythe guanidinium/hot phenol method (37). S1 nuclease protection analysiswas performed by using a dsDNA probe as described (38). Briefly, 30mgof total RNA was resuspended in 50ml of hybridization buffer (80% deion-ized formamide, 0.4 M NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA)containing the 59-end-labeled 432-bpSstI-RsaI fragment probe (see Fig. 1).Following incubation at 80°C for 10 min, the hybridization reaction wasincubated at 52°C for 16 h. Three hundred microliters of buffer containing0.28 M NaCl, 50 mM NaOAc (pH 4.6), 4.5 mM ZnSO4, and 500 U/ml S1

was then added, the reactions were digested at 37°C for 1 h, and the sam-ples were subjected to electrophoresis on 6% polyacrylamide/8 M ureagels. Sequencing ladders used to determine the 59 end of S1 protectedbands were generated by the dideoxy chain termination method (36) byusing an oligonucleotide (59-ACTGAAGATGAATATACA-39) comple-mentary to 18 bp at the 39 end of the coding strand of theSstI-RsaI frag-ment. The size markers were 59 end-labeledMspI digests of pBR322.

Reporter gene plasmid constructions

pGEM7ZCATTCRDEnh(1) was the reporter gene plasmid used in all con-structions. To generate this plasmid, the bacterial chloramphenicol acetyl-transferase (CAT) gene and the SV40 small t intron and polyadenylationsite from a derivative of pRSVmCAT (39) were first subcloned into theApaI-XhoI sites of pGEM-7Zf(1), thus generating pGEM7ZCAT. The cos3 K5 cosmid clone, a derivative of cos 3 (6) containing the BALB/cTCRDC1andTCR Dchain Enh (TCRDenh), was then obtained from Dr.Y. H. Chien, and the 4.5-kbEcoRI fragment containing theTCRDEnh wasexcised and subcloned into pGEM-7Zf(1). From that plasmid, theTCRDEnh was isolated usingHaeIII, subcloned into theSmaI site of pGEM-7Zf(1), excised again withKpnI andClaI, and finally inserted into theKpnI-ClaI sites of pGEM7ZCAT to generate pGEM7ZCATTCRDEnh(1).

Nine 59 deletion constructs were made by inserting promoter fragmentsinto theXhoI-KpnI sites of pGEM7ZCATTCRDEnh(1). In eight cases, thepromoter fragments were first cloned into pSP72 (Promega) and then ex-cised withSalI andKpnI for insertion into pGEM7ZCATTCRDEnh(1).The strategies for cloning promoter fragments into pSP72 were as follows:1) 22100 to1368, the 3.4-kbBamHI DV101S1promoter fragment de-scribed above was subcloned into theBamHI site of pSP72, generatingpSP72AIRPHAGE3.4(2); 2)2791 to 1368, pSP72AIRPHAGE3.4(2)was digested withSmaI andBalI to remove the internal;2.2-kb fragmentand then religated to generate pSP72BalI-BamHIAIRPHAGE3.4(2); 3)2708 to 1368, a 1076-bpDraI-BamHI fragment of pSP72BalI-BamHI-AIRPHAGE3.4(2) was subcloned intoSmaI-BamHI sites of pSP72; 4)2531 to1368, a 899-bpScaI-BamHI fragment of pSP72BalI-BamHIAIR-PHAGE3.4(2) was subcloned intoSmaI-BamHI sites of pSP72; 5)2393to 1368, a 760-bpBsphI-BamHI fragment of pSP72BalI-BamHIAIR-PHAGE3.4(2) was blunted and subcloned into theSmaI site of pSP72; and6) 286 to 1368, plasmid 4.1.6 containing the 668-bpSacI-BamHI frag-ment of the DV101S1promoter cloned into theSacI-BamHI sites ofpGEM-3 (Promega) was obtained from Dr. Y. H. Chien. TheSacI-BamHIfragment was excised from pGEM-3 withBamHI andEcoRI, the endsblunted,EcoRI linkers attached, and then the fragment was cloned into theEcoRI site of pGEM-7Zf(1), generating pGEM7Z59Vd1(2) andpGEM7Z59Vd1(1). A 460-bp HincII-EcoRI fragment of pGEM7Z59Vd1(1) was blunted and subcloned into theSmaI site of pSP72 gen-erating pSP72HincII-BamHIpGEM7Z59Vd1(1)(1). 7) 233 to 1368, a432-bp MaeIII-PstI fragment from pSP72HincII-BamHIpGEM7Z59Vd1(1)(1) was blunted and subcloned intoPstI-SmaI sites of pSP72;8) 14 to 1368, a 396-bpHinfI-PstI fragment from pSP72HincII-BamHIpGEM7Z59Vd1(1)(1) was blunted and subcloned intoPstI-SmaI sites ofpSP72. For the final 59 deletion construct,2300 to 1368, the 668-bpSstI-BamHI DV101S1 promoter fragment was excised frompGEM7Z59Vd1(1) with XhoI andKpnI and inserted into theXhoI-KpnIsites of pGEM7ZCATTCRDEnh(1).

Three 39deletion constructs were also made by inserting promoter frag-ments into theXhoI-KpnI sites of pGEM7ZCATTCRDEnh(1). In twocases, the 39 deleted promoter fragments were again cloned into pSP72 firstand then excised withXhoI andKpnI (286 to166) orSalI andKpnI (286to 16) for insertion into pGEM7ZCATTCRDEnh(1). For fragment286 to166, a 152-bp HincII-FokI fragment was excised frompGEM7Z59Vd1(1), the ends blunted, and then cloned into thePvuII site ofpSP72. For fragment286 to 16, a 110-bpEcoRI-HinfI fragment wasexcised from pSP72HincII-BamHIpGEM7Z59Vd1(1)(1), the endsblunted, and then cloned into theSmaI site of pSP72. The remaining pro-moter 39 deletion,286 to 191, was amplified from pGEM7Z59Vd1(2)using the PCR (40) and oligonucleotides incorporatingSalI or KpnI re-striction sites: DV101S1(286) 59-TGCC(GGTACC)AACATGCTACCAGTCCATGACCTCACTAGC-39, and 191 59-AGGT(GTCGAC)AGTTTCACTGAGGATGAGTTTGTA-39.

Eight plasmid constructions were made containing mutations in the286to 1368DV101S1promoter fragment. Constructions containing mutationsin the cAMP responsive element (CRE) or E box elements were producedby oligonucleotide-mediated site-directed mutagenesis using single-stranded phagemid DNA as described (41). The sequences of the two syn-thetic oligonucleotides used with inserted mutations underlined are muta-tion 9A, 59-AGAGACATGTGGCAAGCTAGCTGGTGATTGGACTGGTAGCATGTT-39, and mutation 11A, 59-TTAGTCACTTCCTCAG

792 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

TACGACCTGCGCTAGCTAGTGAGGTCATGGA-39. The six remainingmutantDV101S1286 to 1368 promoter constructions were produced byoverlap extension using the PCR (42) followed by cloning of the reactionproducts into theXhoI-KpnI sites of pGEM7ZCATTCRDEnh(1). Four oli-gonucleotide primers were used to generate each mutant PCR product: twounique oligonucleotide primers for the insertional mutagenesis and two com-mon oligonucleotide primers for the boundaries of the final PCR product.The two common oligonucleotides are:DV101S1(286) and 59-AGGT(GTCGAC)TCTAGAGGATCCCCAATTCCGGATCCCCA-39. The sequences ofthe pairs of unique oligomers, with inserted mutations underlined, are: muta-tion 1, 59-TGGTTTGATATTAGTCAACCGGATGTTACCGAATGTGGCAAGCTAGTGA-39 and 59-TGACTAATATCAAACCACCTTTACAG-39;mutation 2, 59-CCTCTGTCTCTGTCTCCACTGCAGGACGCTAAGTTGGTAAAGGTGGTTTGATATTAG-39 and 59-GGAGACAGAGACAGAGGTGAGGCT-39;mutation3,59-TGGGCTGCCCTGCTGAGTTGGCTAATGAAGCTGCCTCTAGTGAGAATCCTTATCTG-39 and 59-CTCAGCAGGGCAGCCCACCTT-39; mutation 7, 59-TGCTGTAAAGGTGGTTTGGCGAGAGTCACTTCCTCAGAGAGACA-39 and 59-CAAACCACCTTTACAGCAGATAAGGA-39;mutation14,59-CTCCGTGAGAATCCTTAAGTGCTGTAAAGGTGGTTTGAT-39 and 59-TAAGGATTCTCACGGAGACAGAGACAGAGGTGAGGCTC-39; and mutation 15, 59-CTTATCTGCTGTAAAGGCAGTTTGATATTAGTCACTTCC-39and59-CCTTTACAGCAGATAAGGATTCTCACGGAGACA-39.

Sequences of all amplified and mutatedDV101S1promoter fragmentswere verified by double-stranded dideoxy-DNA sequence analysis.

Transfections

Molt-13 cells (203 106) in 0.8 ml RPMI 1640 medium were transfectedwith 10 mg of pRSVLuciferase reference plasmid (43) and a 3-fold molarexcess of test plasmid by electroporation using a gene pulser (Bio-Rad,Richmond, CA) set at 960mFD and 280 V. Cells were harvested 48 h later,resuspended in 0.1 ml of 0.25 M Tris-HCl (pH 7.5) per 33 106 live cells,and extracts were prepared by three cycles of freeze-thaw lysis followed bycentrifugation. DNA used in transfections was prepared by CsCl densitycentrifugation and quantitated by visualization in a gel. Constructs weretransfected in singlet or duplicate within an experiment and each experi-ment with a series of constructs was repeated a minimum of three times.

Luciferase and CAT assays

Luciferase activity in 15ml of cell extract was determined as described(43). Briefly, the 15ml cell extract sample was diluted to 50ml with 0.25M Tris-HCl (pH 7.5) and then added to 250ml of 43.2 mM glycylglycine(pH 7.8) containing 7.4 mM ATP, 1 mM DTT, 22 mM MgSO4, 400mg/mlBSA, and 2.4 mM EDTA in a test tube. The tube was placed in a lumi-nometer and the reaction was initiated by the injection of 100ml of 0.54mM D-luciferin (Sigma, St. Louis, MO). Peak light emission was recorded.CAT assays (44) were conducted with volumes of cell extracts that yieldedthe same amount of luciferase activity. A 4- to 21-h incubation period wasused to assay cell extracts for acetylation of [14C]chloramphenicol. Acety-lated chloramphenicol was then separated from nonacetylated chloram-phenicol by TLC, and the amount of radioactivity in each form was de-termined using a Betascope (Betagen, Waltham, MA).

Electrophoretic mobility shift assay (EMSA) and DNase Ifootprinting

The binding reaction was conducted in a total volume of 20ml containing20 mM HEPES (pH 7.3), 20% (v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA,0.5 mM DTT, 0.25 mM PMSF, 0.01% Nonidet P-40, 6mg BSA, 2 mgpoly(dI-dC), 5 to 12mg nuclear extract protein, and 0.5–5 ng32P-labeledfragment. The assay mixture was incubated for 15 min at 30°C, loaded ontoa 4% polyacrylamide gel containing 0.045 M Tris-borate, 1.0 mM EDTA,and 2% glycerol, and electrophoresed at room temperature. For experi-ments involving inhibition of complex formation, cold fragments or oli-gonucleotides were added to the binding reaction and incubated for 5 minbefore the addition of labeled fragment. For EMSAs performed with PEA3and murine (m) GATA-3 protein produced by in vitro translation, NonidetP-40 and BSA were excluded from the binding reactions, only 100 to 400ng poly(dI-dC) was used, and reactions were incubated at either 30°C or37°C for 15 min before loading them on polyacrylamide gels and runningas above. Ab treatment of the fragment 99 EMSA was conducted withmonoclonal mouse anti-GATA-3 IgG1 Ab (HG3–31; Santa Cruz Biotech-nology, Santa Cruz, CA) and mouse IgG1 (Southern Biotechnology Asso-ciates, Birmingham, AL). The reaction procedure consisted of a 15 min30°C preincubation of 12mg of Molt-13 nuclear extract and 100, 200, or400 ng of Ab in reaction buffer, followed by a 15 min 30°C incubation with

radiolabeled fragment 99. Reaction mixtures were run on polyacrylamidegels as above.

The DNase I protection (footprint) analysis was performed as described(45); however, the binding reaction was set up as above. The sequencingladder used as a marker was generated by the G1 A reaction of theMaxam-Gilbert sequencing method (46).

Probes

Fragment 58 is the 113-bpSalI-EcoRI fragment or the 131-bpSalI-BglIIfragment from pSP72HincII-MaeIII(2) mini 7, a plasmid derived by blunt-ing the 76 bpEcoRI-MaeIII fragment from pSP72HincII-BamHIpGEM7Z59Vd1(1)(1) and ligating it into theSmaI site of pSP72. Fragment 99 isthe 144-bpSalI-EcoRI fragment from pSP72 MaeIII-FokIAirphage3.4(2)mini 2, a plasmid derived by first cloning the 216-bpMaeIII-XbaIDV101S1promoter fragment into theXbaI-SmaI sites of pSP72, then cut-ting out the 107-bpFokI-SacI fragment from that vector, blunting the ends,and ligating the fragment into theSmaI site of pSP72. TheHincII-HinfIprobe is the 147-bpSalI-EcoRI fragment from pSP72HincII-HinfI(2) mini36 derived by blunting the 110-bpEcoRI-HinfI fragment frompSP72HincII-BamHIpGEM7Z59Vd1(1)(1) and cloning it into theSmaIsite of pSP72. Mutated fragment 58 probes are 113-bpSalI-EcoRI frag-ments from plasmids pSP72HincII-MaeIII(2) with mutated CRE andpSP72HincII-MaeIII(1) with mutated E box. The mutatedHincII-HinfIprobe is the 147-bpSalI-EcoRI fragment from pSP72HincII-HinfI(2) withMut. 1. Mutated fragment 99 probes are 136-bpEcoRI-SalI fragments fromplasmids pSP72 MaeIII-FokI(2) with Mut. 2, 3, 7, and 14, respectively.These four plasmids were derived by blunting the 99-bpMaeIII-FokI frag-ments bearing mutations and ligating them into theSmaI site of pSP72. Foruse as probes, DNA fragments were labeled at their 59 ends and purifiedby PAGE.

Nuclear extracts and in vitro translated proteins

Nuclear extracts were prepared by the procedure of Dignam et al. (47). ForDNase I footprinting, Molt-13 nuclear extracts were purified by heparin-agarose chromatography. Extract was loaded onto a column and the col-umn was washed with a buffer containing 20 mM HEPES (pH 7.9), 20%(v/v) glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF,and 1mg/ml leupeptin. Bound proteins were eluted with the above buffercontaining 0.4 M KCl.

The PEA3 and mGATA-3 protein were generated by in vitro transcrib-ing mRNA from plasmids pGEM-7PEA3 and p34 mc5b8, respectively,and in vitro translating the protein using the TNT-coupled reticulocytelysate systems (Promega) according to the manufacturer’s instructions.Plasmid pGEM-7PEA3 contains the 1.5-kb murine PEA3 cDNA insertedin the HindIII-SacI sites of pGEM-7Zf and was a gift from Dr. J. Hassell(McMaster University, Hamilton, Ontario, Canada) (48). Plasmid p34mc5b8 contains the;2.1 kb mGATA-3 cDNA inserted in theEcoRI siteof pGEM-7Zf(1) and was a gift from Dr. R. Sen (Brandeis University,Waltham, MA) (49).

Oligonucleotides

ds oligonucleotides used for EMSA inhibition experiments were pre-pared by annealing equal amounts of unlabeled complementary oligo-nucleotides followed by purification on 15% polyacrylamide gels. Theycontained the following sequences and their complementary strands:CRE, 59-GATCCTGGGGGCGCCTCCTTGGCTGACGTCAGAGAGAGAGTTAACG-39, from the rat somatostatin promoter (50); GATAglobin, 59-GATCTCCGGCAACTGATAAGGATTCCCTG-39, from themousea1-globin promoter (51); GATA-Vd1, 59-TAGGATAGCCCTGAGATAACGCGAATATTCTC-39; Ets, 59-AATATTGAGCTCGGAGAGCGGAAGCGCGCGAACTCGAG-39, from the Moloney murinesarcoma virus long terminal repeat (52); AP1, 59-GATCCCCCGGATGAGTCATAGCTTATCGATACCG-39; E box, 59-GATCAATATTGAGCTCGGATTGCCACATGTCTCGACGCGAACTCGAG-39; TCF-1,59-GATCCAGGGAATCCAATTCTCTGGGCTTGCCGGA-39; StartSite, 59-CTCGAGTTCGCGAGGATTCTCATCCGAGCTCAATATT39; and Octamer, 59-ATGAATATGCAAATCAGGTG-39, from theBCL1 VH gene promoter (53).

All oligonucleotides were made on an Applied Biosystems (Foster City,CA) model 380A oligonucleotide synthesizer.

793The Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

ResultsTranscription initiates at six sites upstream of the murineTCRDV101S1gene segment

To initiate analysis of theTCRDV101S1promoter, 1234 upstreambp were sequenced beginning from the 53rd residue of theDV101S1gene segment (Fig. 1A). S1 nuclease protection analysiswas then performed to define the RNA start site (Fig. 1B). A432-bpSstI-RsaI 59 end-labeled fragment was used to protect RNAfrom two DV101S1-expressing T cell hybridomas and aDV101S1expressing T cell line. Six protected bands (110, 116, 118, 133,138, and 141 bp) were consistently observed with each of theseRNA samples, indicating multiple transcription initiation sites(Fig. 1B, lanes 4–6). In contrast, RNA from three negative con-trols (aDV104S11 and aDV2S81 T cell hybridoma, and anab Tcell line) did not produce any protected bands (Fig. 1B, lanes 1–3).These findings were confirmed using RNase protection analysis ofa uniformly [32P]CTP-labeled RNA probe (data not shown). Thesix transcription initiation sites fall into two clusters of three (Fig.1A, Table I), with the strongest start site (number 3, solid arrow inFig. 1A) associated with one cluster and the second strongest startsite (number 5, open arrow with the asterisk above it in Fig. 1A)associated with the other. The strongest start site is located 91 bpfrom the translation initiation codon and was designated as11.

A significant TATA box (consensus: 59-STATAWAWRSSSSSS-39(S 5 C or G; W 5 A or T; R 5 A or G), similaritycalculated according to Bucher (56) and 79% considered the sig-

nificant cut-off value (56)) is not present within 177 bp upstreamof the translation initiation site. The two best TATA boxes in theregion, however, each happen to lie 18 to 28 bp in front of one ofthe clusters of transcription initiation sites (Table I).

Several potential initiator (Inr) sequences are present in the220 to166 region encompassing theDV101S1transcription start sites. Us-ing the Inr consensus derived through sequence comparison of variousstart site regions (59-KCABHYBY-39 (K 5 G or T; B 5 C, G, or T;H 5 A, C, or T; Y 5 C or T); significant similarity cut-off value581.4% (56)) there are nine potential Inr sequences in this region. Fourof these nine coincide with experimentally identified transcriptionalstart sites (Table I, start sites 1, 2, 5, and 6). The others are located at218,135,143,156, and159. Using a different Inr consensus, onederived by functional studies (59-YYANWYY-3 9 (57)) and requiringa 5/7 bp match, an additional seven potential Inrs can be found withinthis region. These elements are at217,215,214,212,12,16, and129. However, if the critical bases in the experimentally defined Inrconsensus and the location of the pyrimidines are considered (57, 58),then only the additional212 and12 potential Inrs are likely func-tional Inrs (57, 58).

Sequences between286 and166 contribute substantially toTCRDV101S1promoter activity in the presence of the TCRD Enh

To map the region containing the functional elements of theDV101S1promoter, the22100 to1368DV101S1fragment (num-bering relative to the transcription start site) was inserted into a

FIGURE 1. DV101S1-59region nucleotide sequence and localization of six transcriptional start sites.A, Nucleotide sequence from2919 to 1368(numbering relative to the major transcriptional start site). TheDV101S1gene segment (V) and leader sequence (L), determined by comparison withpublishedDV101S1cDNA sequences (54, 55), are boxed. The solid arrow indicates the major mapped transcriptional start sites, and the open triangles theother mapped transcriptional start sites. Start sites are numbered 1 to 6 for reference, and number 5 (open triangle with the asterisk above it) is the secondstrongest. Restriction enzymes used to generate promoter deletions for reporter gene plasmid constructions are indicated, as well as theRsaI site end-labeledon the S1 nuclease protection probe (*).B, S1 nuclease mapping of the transcription start site. RNA from three cell populations expressingDV101S1transcripts (70BET104, 33BTE140.9, JAC-3,lanes 4–6) and from three negative controls (EL4, 33BTE67.1, 33BTE125.5,lanes 1–3) were annealed tothe 59 end-labeled 432-bpSstI-RsaI fragment indicated inA. S1 nuclease protected fragments (arrows) and full-length probe (P) are indicated. (M),radiolabeledMspI digested pBR322, sizes in nt; (P), input probe; (G), (A), (T), (C), sequencing ladder generated using an oligonucleotide complimentaryto 18 bp at the 39 end of the coding strand of theSstI-RsaI fragment.

794 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

CAT reporter construct for transient transfection studies in humangd T cells (Molt-13). This vector did not yield detectable CATactivity (data not shown), so the homologous murine coreTCRDEnh (59) was added to the construct. Detectable CAT activity wasobtained with this vector (Fig. 2A), and a series of constructs wasthen made with deletions at the 59 end of theDV101S1fragmentutilizing restriction enzyme sites (Fig. 1A). These plasmids wereelectroporated with the reference plasmid, pRSVLuciferase, intoMolt-13 cells and CAT activity was determined in cell extractsprepared 48 h after DNA transfection (Fig. 2A). CAT activity wasused as a measure of the transcriptional activity of the constructs,but CAT activity may not accurately reflect true promoter activitybecause we did not quantitate correctly initiatedDV101S1tran-scripts. Furthermore, the CAT activity obtained will depend uponthe stability of the RNA and the efficiency of CAT translation. Thepresence of multiple translation initiation sites after theDV101S1transcriptional start site in the reporter gene constructs complicatesthe translation issue. The context of the CAT translation initiationsite (AUGCAT) is the strongest of the multiple translation initiationsites present in mRNA derived from theDV101S1promoter re-porter constructs (Fig. 2; Ref. 61); however it is the fourth AUGcodon from the 59 end of the mRNA and varies as to whether it isin the same or different reading frame as the first translation ini-tiation codon, AUGVd1, and to whether an in-frame terminatorcodon occurs upstream of it. Hence, it is best to score the con-structs in Fig. 2 only as yielding or prohibiting CAT activity andto compare variabilities in CAT activity only within constructs thathave the translational start sites similarly oriented (grouped to-gether in Fig. 2; Refs. 61 and 62).

Significant CAT activity was observed with all promoter trun-cations down to position286. With additional deletions, one ex-tending to233 nt of the RNA initiation site and one eliminatingthe major RNA start site and two surrounding minor start sites(14), CAT gene activity was essentially abolished. These resultslocate the 59 boundary of the minimal functionalDV101S1pro-moter at286 nt from the mRNA start site. Fluctuations in the CATactivity obtained with the22100,2791, 2708, and2531 trun-cations, having AUGVd1 and AUGCAT in the same reading frame,may indicate negative regulatory sequences in the regions2708 to2791 and2393 to 2531, and sequences that can abrogate theeffects of these regulatory regions within2791 to 22100 and2531 to2708, respectively.

To determine the 39 boundary of the minimal functionalDV101S1promoter, a series of constructs was made with deletions

at the 39 end of the286 to 1368 DV101S1fragment. Deletionswere made by using restriction enzyme sites or the PCR (Fig. 1A),and constructs were tested for CAT activity by transient transfec-tion in Molt-13 cells (Fig. 2B) as above. A deletion of 277 nt to just59 of the methionine translation initiation codon in the first leaderexon, 191, had no appreciable affect on CAT activity. Further,when an additional 25 nt were removed to166, the promoterfragment yielded activity equivalent to that obtained with the286to 1368 construct. However, with the furthest 39 truncation,16,no significant CAT activity was obtained. These results show thatsequences within16 and 166 are essential for expression ofDV101S1and define the 39endpoint of the minimal functionalDV101S1promoter as166 nt from the mRNA start site.

Molt-13 nuclear factor(s) specifically bind to the minimalDV101S1promoter region

To identify proteins regulating the activity of the minimalTCRDV101S1promoter, EMSAs were performed using two32P-labeled fragments that comprise all of the286 to 166 DV101S1promoter region (Fig. 3A). These fragments are the 58-bpHincII-MaeIII fragment, representing286 to229, and the 99-bpMaeIII-FokI fragment representing233 to 166. Numerous retardedDNA-protein bands were observed in Molt-13 nuclear extractswith fragment 58 (Fig. 3B, lane 7). To establish the specificity ofthe DNA-protein interactions, unlabeled fragment 58 (Fig. 3B,lanes 2–6) and an irrelevant fragment (Fig. 3B, lanes 8–12), wereused as inhibitors in the binding assay. Four specific retarded pro-tein-DNA bands were observed (arrows in Fig. 3B).

Figure 3Ddemonstrates that nuclear extract from Molt-13 cellsalso contains factor(s) which specifically bind to fragment 99. Fourof the migration retarded species observed with Molt-13 nuclearextract and fragment 99 (Fig. 3D,lane 8) could be specificallyinhibited by unlabeled fragment 99 (Fig. 3D, lanes 2–7), whereasthe irrelevant fragment (Fig. 3D, lanes 9–14) did not inhibit for-mation of these species.

EMSAs were also performed with the two probes using nuclearextracts from other cell lines including TCR-gd 70BET104 and33BTE125.5, TCR-ab EL4 and AKR117, pre-B 38B9 and PD31,and mature B M12.4. The results (summarized in Fig. 3,C andE)demonstrated that the probe 99 nuclear protein binding species 4was T cell specific (Fig. 3E).

Table I. Potential TATA boxes and Inr sites within theDV101S1promoter

TATA Boxes Initiator Sites

Location (bp)a Sequenceb

Similarityto the

consensusc

%

ExperimentallydeterminedDV101S1

start site no. Locationa (bp) Sequenceb

Similarity to theconsensusc

(%)

Distance fromTATA boxto start site

(bp)

Consensus STATAWAWRSSSSSSd Consensus KCABHYBYe 30f

228 TAATATCAAACCACC 70.4 1 210 ACAGCAGA 83.5 182 27 GCAGATAA 82.8 213 22 TAAGGATT 48.9 26

27 GCAGATAAGGATTCT 74.0 4 114 AGACAGAG 51.3 205 116 ACAGAGAC 81.6 226 122 ACAGAGGT 85.1 28

a Numbering relative to the major transcription initiation site.b Sequence on the nontemplate strand from 59 to 39. B 5 C, G, or T; H5 A, C, or T; K 5 G or T; R 5 A or G; S 5 C or G; W 5 A or T; Y 5 C or T.c Similarity to the consensus was calculated according to Bucher (56); a similarity greater than 79% (TATA box) or 81.4% (Inr) is considered significant.d Consensus (Ref. 56): center of TATA box is underlined.e Consensus (Ref. 56): start sites are underlined.f The distance from the underlined T in the TATA box consensus to the start site is 306 2 bp in 60% of the entries in the Eukaryotic Promoter Database.

795The Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

The CRE, Ets, E Box, GATA, and two elements in the 59untranslated leader bind Molt-13 nuclear factor(s)

To identify the binding sites of the proteins interacting with theDV101S1minimal promoter, this region was searched initially forconsensus recognition sequences of transcription factors involvedin gene regulation during T-cell development (63, 64) and later forconsensus recognition sequences listed in the National Center forBiotechnology Information transcription factor database (Fig. 4D;Ref. 68). Duplex oligonucleotides containing most of the observedtranscription factor binding motifs were then used as competitorsin EMSAs. A CRE, two E boxes, and an Ets site were observed infragment 58 at262,252,247, and239 nt from the mRNA startpoint, respectively (Fig. 4D). Duplex oligonucleotides containingthese consensus sequences were used as inhibitors in EMSAs with32P-labeled fragment 58 and Molt-13 nuclear extract (Fig. 4,A andB). An oligonucleotide containing a CRE site from the rat soma-tostatin promoter (50) inhibited the formation of migration re-tarded species 1 and 4 with this probe in a titratable manner (Fig.4A, lanes 2–7), whereas formation of species 2, 3, and 4 wasinhibited in a titratable manner by an oligonucleotide containingthe region256 to 243 of theDV101S1promoter (E box oligo;

Fig. 4B,lanes 2–7). An oligonucleotide containing an Ets site fromthe Moloney murine sarcoma virus long terminal repeat (52) didnot inhibit formation of any of the migration retarded species (Fig.4A, lanes 9–14) despite the presence of the consensus Ets bindingsite in the fragment. Two irrelevant duplex oligomers, one con-taining a GATA site from the mousea1-globin promoter (GATA-globin oligo; Ref. 66) and the other containing an AP1 site, alsohad no inhibitory capacity (data not shown).

Fragment 99 contains two AP1 sites at232 and226 nt from themRNA start point, the viral Enh core motif at215, a GATA el-ement at25, an Sp1 site at166, the 2 putative TATA boxes, andthe 16 potential Inr sequences discussed earlier. The GATA-globinoligo inhibited the formation of migration retarded species 4 whenit was added to the binding assay of Molt-13 nuclear extract and32P-labeled fragment 99 (Fig. 4C, lanes 2–7). Because the GATA-globin oligo is identical to fragment 99 at 6 bp immediately 39 ofthe GATA site, a duplex oligonucleotide containing only theDV101S1GATA site (GATA-Vd1 oligo) and a duplex oligonu-cleotide containing the 10-bp region immediately 39 of the GATAsite (11 to 110; Start site oligo), were used as competitors inEMSAs. Interference of formation of species 4 was again seen

FIGURE 2. Deletional analysis of the;2.5-kbDV101S1promoter fragment reveals that 152 bp is sufficient to generate high levels of promoter activity.VariousDV101S1promoter region fragments were fused to the CAT reporter gene and tested for CAT activity in the presence of the homologousTCRDEnh by transient transfection into Molt-13 cells. Transfection efficiencies were controlled by transfecting pRSVLuciferase along with the test plasmid. Thepromoterless plasmid, pGEM7ZCATTCRDEnh(1), served as a negative control and pRSVmCAT as a positive control in each experiment. Within eachexperiment, extract volumes were normalized for luciferase activity before analysis of CAT activity. The percent acetylation with the negative control wasthen subtracted from measurements of test constructs before their CAT activities were evaluated relative to that of the full-length parent construct.A, Resultswith the 22100 to1368 promoter fragment progressively deleted at the 59 end. Four experiments are presented, with the data expressed as the percentof CAT activity displayed by the22100 to1368 promoter reporter construct. pGEM7ZCATTCRDEnh(1) produced 1.0, 0.9, 1.2, and 0.2% acetylation,and the22100 to1368 construct produced 8.7, 3.3, 11.5, and 3.5% acetylation in experiments 1 through 4, respectively. Mean percentages of CAT activityare listed in the last data column.B, Results with the286 to 1368 DV101S1promoter fragment progressively deleted at the 39end. Three experimentsare presented with the data expressed as the percent of CAT activity displayed by the286 to 1368 promoter reporter construct.pGEM7ZCATTCRDEnh(1) produced 0.2, 0.2, and 0.2% acetylation, and the286 to1368 construct produced 35.0, 22.4, and 22.3% acetylation in thesethree experiments, respectively. Mean percentages of CAT activity are listed in the last data column. The orientation of translational start sites in mRNApresumed to be derived from the reporter gene constructs is diagramed in the right hand side of the tables inA andB. Constructs yielding identical mRNAare blocked together. Parentheses around an AUG indicate that the codon is out of frame with the 59-proximal AUGDV101S1(designated AUGVd1). STOPindicates a terminator codon in frame with AUGVd1. Base pairs between translation initiation codons or between translation initiation and terminationcodons are indicated. The sequences of the four translation initiation sites found in mRNA from the reporter gene constructs in order of their predictedstrength (60) and with base pairs homologous to the consensus sequence underlined are: AUGCAT, CTCCACAAC(ATG)G; AUGVd1, AGTGAAACT(ATG)C; ATG2, GCTTTGGAG(ATG)T; and ATG3, CCTCTTTGG(ATG)T.

796 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

with the GATA-Vd1 oligo, but all migration retarded species wereobserved with the Start site oligonucleotide (data not shown), con-firming that a GATA binding factor interacts with fragment 99. Anoligonucleotide containing an AP1 site did not interfere with theformation of any of the four migration-retarded species (data notshown). Also, two irrelevant duplex oligonucleotides, one contain-ing a potential TCF-1 site (59-YCTYTKWW-39; Ref. 63) from theDV101S1intronic region1191 to1201 (Fig. 4C, lanes 9–14) andthe other an Ets site (data not shown), did not interfere with theformation of any of the 4 migration retarded species.

To further examine nuclear protein binding sites in the minimalDV101S1promoter region, fragments 58 and 99 were subjected toDNase I protection studies using Molt-13 nuclear extract. Bothfragments were labeled on either strand at the 59 end and fourprotected regions were identified (Fig. 5). All four protected re-gions were detected on only one of the DNA strands, and three hadat least one DNase I hypersensitive site at their boundary. The firstprotected region overlaps the 39end of the E box motif, protectingnt 248 to 242 (Fig. 5A). Another protected region spans the Etssite, protecting nt239 to 233 (Fig. 5A). The third protected re-gion overlaps the major transcription initiation site and two poten-tial Inr elements, protecting nt11 to 110 (Fig. 5B). Finally, thelast protected region is downstream of the major transcription ini-tiation site and overlaps one potential Inr, protecting nt117 to121 (Fig. 5C).

The GATA-3 transcription factor binds to the GATA element

The demonstration that a T cell-specific nuclear factor binds theGATA element at 25 prompted us to examine whether theGATA-3 transcription factor binds this element. GATA-3 is one ofa family of six transcription factors that bind the GATA consensusrecognition sequence (69–71). It is the predominant member ex-pressed in T cells and has been shown previously to be importantin regulation ofTCR A,B, andD genes as well as the CD8a gene(49, 72–76). GATA-3 binding to theTCRDV101S1promoter sitewas first examined by comparing the mobility of the species 4complex generated with fragment 99 and Molt-13 nuclear extractwith that of the complex generated with this probe and mGATA-3.Nonradioactive mGATA-3 was produced by in vitro translationand production was confirmed by performing parallel reactions inthe presence of [35S]methionine and analyzing reaction productson a SDS-polyacrylamide gel (data not shown). In vitro-translatedproducts yielded a GATA-3 protein of;48 kDa when the sensemRNA was synthesized from p34 mc5b8 (49) with the T7 poly-merase, but no radioactively labeled protein was made when theantisense mRNA was synthesized with the SP6 polymerase (datanot shown). The other control, PEA3, an irrelevant transcriptionfactor belonging to the Ets gene family (48), yielded a major spe-cies of ;68 kDa from the sense mRNA as expected (data notshown). The in vitro-translated mGATA-3/fragment 99 nucleopro-tein complex (Fig. 6A, lanes 4and9) comigrates with specific T

FIGURE 3. Factor(s) in Molt-13 nuclear extractsspecifically bind to the minimalDV101S1promoter.A, Restriction endonuclease map of the minimalDV101S1promoter. Relevant enzymes are noted aswell as the fragments used as probes. Fragments arereferred to here and in subsequent figures by theirlength in base pairs.B, EMSA analysis of fragment58. 32P-labeled fragment 58 was incubated without(lane 1) or with (lanes 2–12) Molt-13 nuclear ex-tract. A 5-, 10-, 25-, 50-, and 100-fold molar excessof unlabeled fragment 58 and the irrelevant 265-bpBalI-EcoRI CAT coding region fragment was addedto the binding assay inlanes 6–2and8–12, respec-tively. Migration of the arbitrarily numbered four re-tarded species is indicated on the left.C, Summary ofthe fragment 58 nuclear protein-binding activities de-tected in different lymphoid cell nuclear extracts.D,EMSA using 32P-labeled fragment 99 and Molt-13nuclear extract (lanes 2–14); lane 1 received no ex-tract. A 1-, 5-, 10-, 25-, 50-, and 100-fold molar ex-cess of unlabeled fragment 99 and the irrelevant265-bpBalI-EcoRI CAT coding region fragment wasadded to the binding assay inlanes 7–2and 9–14,respectively. Migration of the arbitrarily numberedfour retarded species is indicated on the left.E, Sum-mary of the fragment 99 nuclear protein-binding ac-tivities detected in different lymphoid cell nuclearextracts.

797The Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

cell nuclear extract/fragment 99 complex species 4 that is inhibit-able with the duplex GATA-globin oligonucleotide (Fig. 6A, lanes7 and11–13) supporting the notion that it is GATA-3 in the T cellnuclear extracts that binds this site. Additional support derivesfrom the observation that mutation of the GATA site in fragment99 (Mut. 2) disrupts formation of Molt-13 nuclear extract/DNAcomplex species 4 and significantly decreases binding of in vitrotranslated mGATA-3 to this promoter region fragment (Fig. 6A,lanes 8and10, respectively).

Ab ablation-supershift experiments using an anti-GATA-3 mAbproved that GATA-3 is present in species 4 formed between frag-ment 99 and proteins in Molt-13 nuclear extract (Fig. 6B). Species4 was ablated with anti-GATA-3 mAb (Fig. 6B, lanes 3–5) but notwith the control Ab (Fig. 6B,lanes 6–8), establishing thatGATA-3 specifically binds to the GATA site at25 within theTCRDV101S1promoter.

The CRE, Ets site, 59 TATA box, and nucleotides in the region28 to 133 are essential for maximalDV101S1promoterfunction in the presence of theTCRD Enh

The functional significance ofcis-elements in theDV101S1pro-moter identified by either computer search, oligonucleotide inhi-bition of gel retarded species, or DNase I protection studies wasassessed by site-directed mutagenesis. Fig. 7Ashows the mutationsintroduced into elements in the reporter construct containing286to 1368 of theDV101S1promoter and the coreTCRDEnh. Theeffects of the mutations on CAT activity and on protein binding areshown in Figs. 7B and 8, respectively. Transient transfection ex-periments of mutated constructs into Molt-13 cells showed thatmutation of the CRE element (Mut. 9A) reduced CAT activity to40% of the level obtained with the wild-type sequence. Mutationof the Ets site and 8 upstream nucleotides, including 1 nt within the

FIGURE 4. Synthetic oligonucleotides containing CRE, E Box, or GATA elements inhibit interaction of Molt-13 nuclear protein with radiolabeledDV101S1promoter fragments.A andB, 32P-labeled fragment 58 was incubated without (A andB, lane 1) or with (A, lanes 2–14;B, lanes 2–8) Molt-13nuclear extract. Binding was competed with a 1-, 5-, 10-, 25-, 50-, and 100-fold molar excess of a CRE containing ds oligonucleotide (A, lanes 7–2,respectively), an Ets containing ds oligonucleotide (A, lanes 9–14), or an E box containing ds oligonucleotide (B, lanes 7–2). C, 32P-labeled fragment 99was incubated without (lane 1) or with (lanes 2–14) Molt-13 nuclear extract. Binding was competed with a 1-, 5-, 10-, 25-, 50- and 100-fold molar excessof the ds GATA-globin oligonucleotide (lanes7–2, respectively) or an irrelevant ds oligonucleotide containing a TCF-1 site (lanes 9–14). D, Schematicof the minimal functionalDV101S1promoter with computer-identified transcription factor consensus recognition sequences boxed. Potential TATA boxesare underlined (see Table I). AP1, 59-TGASTMA-39 (M 5 A or C) (65); CRE, 59-TGANNTCA-39 (64); E Box, 59-CANNTG-39 (N 5 any nucleotide) (64);Ets, 59-SMGGAWGY-39(65); GATA, 59-WGATAR-39 (66); Sp1, 59-KRGGCKRRK-39 (65); and viral Enh core, 59-GTGGWWWG-39(67).

798 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

E box element (Mut. 1) also decreased CAT activity, but only to83% of the level obtained with the wild-type construct. Resultswith the E box mutation (Mut. 11A) demonstrated that this elementis not important for the functionality of the promoter. Thus, theCRE binding site is an essential upstreamDV101S1promoter el-ement and the Ets binding site is necessary for full transcriptionalactivity. Replacing 5 nt at positions227 to223 (Mut. 7) reducedCAT activity by more than 90%, confirming the importance of allor some of these nucleotides inTCRDgene transcription. Nucle-otides227 to223 fall within the 59 putative TATA box, and thereplacement nucleotides reduce the similarity to the TATA con-sensus from 70.4% to 37.9%. Thus, this TATA box may be im-portant forDV101S1promoter activity, but nt227 and226 alsofall within an AP1 element; without an independent mutation it isunclear which element(s) the227 to223 replacement mutation isaffecting. However, under the conditions of our binding assay, noAP1-inhibitable nuclear factors bound to theDV101S1minimalpromoter. Thus, the likely element affected by mutation 7 is theTATA box and the inability of the putative 39 TATA at 298 bp,relative to the translation initiation site, to compensate indicatesthat the 39TATA-like sequence does not direct transcription ini-tiation from DV101S1start site numbers 4 to 6. Nucleotides216and 217 (Mut. 15) were replaced to test the importance of theDV101S1viral Enh core site. These particular base substitutionshave previously been used to demonstrate the importance of a viralEnh core site for activity of theTCRG Enh (77) and in theDV101S1promoter; they do not significantly alter the overlapping59 TATA box (similarity to the consensus was lowered by,1%).Results with mutation 15 demonstrated that the viral Enh core isnot important forDV101S1promoter activity. In contrast, nucle-otides within the region28 to 111 are critical forDV101S1geneexpression since substituting 17 of these 19 nt surrounding themajor transcription initiation site (Mut. 2) reduced CAT activity by

.70%. Mutation 2 disrupts six potential Inr sequences and theGATA consensus sequence and to independently evaluate the im-portance of the GATA site, nt24 and23 were substituted withinthe DV101S1minimal promoter (Mut. 14). The second and thirdnucleotides of the GATA consensus site were altered since chang-ing these nucleotides within otherTCRgene GATA sites revealedthe functional significance of these elements (72, 73). Further, thereplacement nucleotides only serve to increase the similarity of thetwo overlapping Inr sequences to the consensus Inr sequence(;1.2%). Mutation 14 did not alterDV101S1promoter activity,implying that this GATA site is not necessary forDV101S1pro-moter function. Finally, nucleotides within the region112 to133,downstream of the major transcription start site (Mut. 3), are es-sential for DV101S1gene expression in vivo as substitution ofthese 22 bases reduced CAT activity by 89%. Nucleotides withinthe 59untranslated region could affectTCRDtranscriptional activ-ity, but the lower CAT activity levels could also reflect a reductionin the transfected cells of translation efficiency and/or mRNAstability.

Binding of factor(s) present in Molt-13 nuclear extract tomutatedDV101S1promoter region fragments

To ensure that the lack of an effect of a mutation in a proteinbinding site onDV101S1promoter activity is due to the unimpor-tance of that element rather than to the failure of that mutation todisrupt protein binding, EMSAs were performed with Molt-13 nu-clear extracts and32P-labeledDV101S1promoter region fragmentscontaining individual mutations (Fig. 8). Mutation 11A (E box),mutation 14 (GATA), and mutation 15 (viral Enh core) were theonly mutations that failed to affectDV101S1promoter activity.The E box mutation did disrupt formation of migration retardedspecies 1, 3, and 4 normally observed with Molt-13 nuclear extractand fragment 58 (Fig. 8A, lane 6). Migration retarded species 2, 3,

FIGURE 5. DNase I protection analyses of themigration retarded species observed with Molt-13extract and fragments 58 and 99 from theDV101S1promoter.A, Fragment 58 59end-labeled to estab-lish coding strand contacts.B, Fragment 99 59end-labeled to establish noncoding strand contacts.C,Fragment 99 59end-labeled to establish codingstrand contacts.Lanes 1and5 in A andB and lane3 in C represent the G1A-specific modification/cleavage reaction conducted on the respective frag-ments for orientation.Lanes 2and4 in A andB, andlane 2 in C represent the pattern of DNase I cleav-age of the unbound DNA fragment (digested in thepresence of nuclear proteins). Partial sequence ofthe fragments are given. *, hypersensitivity sites; •,complete protection.

799The Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

and 4 are the Molt-13/fragment 58 nucleoprotein complexes in-hibitable with a ds E box containing oligonucleotide (Fig. 4B).Likewise, the GATA mutation disrupted the Molt-13 nuclear ex-tract/fragment 99 complex that is inhibitable with a ds GATA con-taining oligonucleotide, species 4 (Fig. 8C, lane 9). Thus, our pre-vious conclusion that the E box and the GATA site areunimportant forDV101S1gene expression in Molt-13 cells is rea-sonable. However, it is not possible to definitively rule out thefunctional significance of the viral Enh core inDV101S1promoteractivity as the binding of nuclear factors to either the wild-type ormutatedDV101S1viral Enh core was not examined in this study.

Of the five mutations that affected promoter activity, three dis-rupted binding of nuclear proteins toDV101S1promoter frag-ments. Mutation 2 (the28 to 111 region) completely disruptedformation of migration retarded species 4 and partially disruptedformation of migration retarded species 1 (Fig. 8C, lane 8) nor-mally observed with wild-type fragment 99 and Molt-13 nuclearextract (Fig. 8C, lane 6). Mutation 3 (the112 to133 region) alsopartially disrupted formation of migration retarded species 1 (Fig.8C, lane 10). The Ets site mutation (Mut. 1) was examined usingthe 93-bpHincII-HinfI fragment (see Fig. 1) since the mutationdestroys theMaeIII restriction site. This fragment routinely yieldsfive specific migration retarded species (data not shown, arrows inFig. 8B, lane 3), but when the Ets site was mutated only retardedspecies 5 was generated (Fig. 8B, lane 4).

The last two mutations that significantly reduced promoter ac-tivity, mutations 7 (TATA box/AP1) and 9A (CRE), unexpectedlydid not alter binding of nuclear proteins toDV101S1promoterfragments (Fig. 8C, lane 7, and Fig. 8A, lane 5, respectively). This

is actually reasonable in the case of the TATA box/AP1 mutationbecause no AP1 inhibitable migration retarded species were ob-served with wild-type fragment 99, and it has been impossible todetect TFIID by gel retardation with crude extracts (78). However,the results with the CRE mutation (Mut. 9A) are more difficult toexplain and may imply that complex interactions occur betweenfactors that bind to the multiple regulatory elements in theDV101S1promoter.

DiscussionThis study has defined the 59DV101S1 cis-sequences necessaryand sufficient forD Enh dependent basal promoter activity to in-vestigate the regulation of orderedTCRD Vrearrangements duringontogeny. Several findings are inconsistent with a previous char-acterization of this promoter (79). First, although we confirmed thepresence of multipleDV101S1transcription start sites, the majorDV101S1initiation site was mapped 13 bp further downstreamthan where it was mapped previously (79). This leads to a TATAbox being properly positioned upstream from the major transcrip-tion start site and the importance of this element inDV101S1geneexpression was confirmed by mutational analyses. Therefore, theDV101S1promoter is properly identified as a TATA-box-contain-ing rather than a TATA-box-deficient promoter (79). Second, withregard to regulatory elements located upstream of theDV101S1start site, we also identified the CRE and Ets sites as functionallysignificant promoter elements. However, we did not find that theEts site is critical forDV101S1promoter activity. Mutation of theEts site reducedDV101S1promoter activity by 17% in the human

FIGURE 6. mGATA-3 binds to the GATA site in theDV101S1promoter region.A, EMSAs performed with32P-labeled wild-typeDV101S1promoterregion fragment 99 (W;lanes 1–5,7, 9, and11–15) or 32P-labeled fragment 99 bearing mutation 2 (m;lanes 6,8, and10) and unlabeled, in vitro translatedproteins from sense PEA3 RNA (lane 2), antisense mGATA-3 RNA (lane 3), sense mGATA-3 RNA (lanes 4,9, and10), or Molt-13 nuclear extract (lanes7, 8, and11–15). In lanes 1,5, and6, radiolabeled fragments were incubated without in vitro-translated protein or nuclear extract. Binding was competedwith a 50- and 100-fold molar excess of the double-stranded GATA-globin oligonucleotide (lanes 12and 13) or the Ets containing double-strandedoligonucleotide (lanes 14and15). Arrows indicate the specific migration retarded species seen with mGATA-3 protein and fragment 99 and complex 4previously seen with the Molt-13 nuclear extract/fragment 99 binding reaction.B, EMSAs with32P-labeledDV101S1promoter region fragment 99 andMolt-13 nuclear extract (lanes 2–8); binding reactions inlanes 1and9–14contained no extract. Nuclear extract was preincubated with 100, 200, or 400ng of the anti-GATA-3 mAb (lanes 3–5) or isotype-matched control Ab (lanes 6–8) for 15 min before the addition of radiolabeled fragment 99. The arrowindicates the specific complex 4 previously seen with the Molt-13/fragment 99 binding reaction.

800 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

gd T cell line, Molt-13. In contrast, Punturieri et al. (79) reportedthat mutation of the Ets site completely abolishedDV101S1pro-moter activity in the murineDV101S11 dendritic epidermal T cellhybridoma, T245/BW. Furthermore, we failed to demonstrate anyfunctional significance of the252 E box, mutation of which, Pun-turieri et al. (79) showed, reducedDV101S1promoter activity by40% in T245/BW cells. The reasons for these discrepancies remainunclear, but may be related to the different cells used for transfec-tion analyses (see below).

We observed that the 59 untranslated leader region and the nu-cleotides around the major transcription start site are involved inDV101S1gene expression. The importance of both of these re-gions was not noted previously (79), and their identification herewas allowed by the nature of our mutations. Transient transfectionanalyses of 39 deletion mutants initially revealed the necessity ofelements within the 66 nt immediately downstream of the majorRNA start site. Analyses of mutations replacing the sequences28to 111 (Mut. 2) and112 to 133 (Mut. 3) then confirmed theimportance of nucleotides within the first 33 bases downstream ofthe major CAP site for in vivoDV101S1gene expression. Thefunctional significance of the134 to166 region was not directlyaddressed by our mutational studies so we are unsure of the con-tribution of elements within this region (e.g., Sp1) toDV101S1gene expression. However, all of the reporter constructs used byPunturieri et al. (79) included only 37 nt of downstreamDV101S1

sequence, relative to our major transcription start site, so presum-ably, this region is unimportant for promoter activity. Several othereukaryotic genes have been found to have regulatory elementswithin their 59 untranslated leader regions including RAG-1 (80).Interestingly, the112 to125 region from theDV101S1promoteris highly homologous to the111 to 125 region from the RAG-1promoter (80). Thirteen of theDV101S1nucleotides match theRAG-1 nucleotides, if a 1 base gap is introduced. Regulatory el-ements within the 59untranslated leader region could operate ateither the DNA or RNA level, and affect either transcription (81–83) or posttranscriptional processes (84–86). DNase I protectionanalyses showed that nuclear factors bind to theDV101S1down-stream sequence. Protected regions were observed at11 to 110and 117 to 121. These binding motifs overlap potential Inr se-quences, but no obvious similarities with other known consensussequences were found. Mutational analyses demonstrated thatbinding of factor(s) to the11 to110 and117 to121 regions wasintegral for in vivo DV101S1gene expression. This observationsupports the notion that theDV101S1downstream sequence influ-ences transcriptional efficiency, but additional studies examiningsteady-state mRNA levels and mRNA stability are required tothoroughly address this issue.

The mutant substituting the wild-typeDV101S128 to 111 se-quence revealed the critical nature of nucleotides near the major

FIGURE 7. Mutational analysis identifies fivecis-regulatory elements important forDV101S1promoter activity.A, Schematic of the286 to 1368DV101S1promoter region with computer identified transcription factor consensus recognition sequences boxed, potential TATA boxes underlined, tran-scriptional start sites indicated, and introduced element mutations listed below the wild-type sequence. Base changes are noted, and a (2) indicates sequenceidentity. Mutations were introduced intocis-regulatory elements individually in the reporter gene construct containing286 to 1368 of theDV101S1promoter.B, Promoter activity of the mutated reporter gene constructs assessed by transient transfection into Molt-13 cells. Transfection efficiencies werecontrolled by transfecting pRSVLuciferase along with the test plasmid. The promoterless plasmid, pGEM7ZCATTCRDEnh(1), served as a negative controland pRSVmCAT as a positive control in each experiment. Within each experiment, extract volumes were normalized for luciferase activity before analysisof CAT activity. The percent acetylation with the negative control was then subtracted from measurements of test constructs before their CAT activity wasevaluated relative to the CAT activity of the wild-type286 to 1368 construct. Reported are the percentages of the wild-type286 to 1368 construct’sCAT activity obtained with the mutated constructs. Each construct was tested a minimum of three times, and the mean activity is listed in the last column.pGEM7ZCATTCRDEnh(1) produced 0.2, 0.6, 0.2, 0.3, 0.2, 1.1, 1.1, 1.3, 1.3, and 1.3% acetylation, and the286 to 1368 wild-type construct produced9.0, 24.5, 18.9, 22.6, 16.9, 23.9, 25.9, 25.0, 26.7, and 19.3% acetylation in experiments 1–10, respectively.

801The Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

transcription start site for in vivoDV101S1gene expression. Re-porter gene activity was reduced by;70% suggesting that the startsite region either acts as an Inr (58, 87) or contains a binding sitefor an essential activator (88). Inrs are basal control elements thatoverlap transcription start sites and serve to direct RNA polymer-ase II to a precise transcription start site in TATA-less promoters(89), and to increase promoter strength in TATA-containing pro-moters, if they are located;25 bp downstream of the TATA box(89–91). Both general transcription factors (92–94) and specificactivator proteins (Ref. 95, reviewed in Ref. 58) have been shownto recognize Inrs, but their mechanism of action remains unclear.Consistent with the idea that theDV101S1start site possessesfunctional Inr activity is the observation that the start site mutationdisrupts six potential Inr sequences, identified using the two avail-able loose consensus sequences (56, 57). However, before it can beconcluded that an Inr element functions as a basic regulator forTCRDV101S1promoter activity, it needs to be shown that theDV101S1 start site functions as a transcriptional positioningelement.

Currently, only theDV101S1andAV11S1gene promoters havebeen characterized from theTCR A/Dlocus (this paper; Refs. 79and 96). Upstream sequence information is available, however, foranotherTCRD V gene segment (DV105S1; Ref. 97), and threeTCRA Vgene segments (AV1S3,AV12S1, andAV3S1; Refs. 96,98, and 99) allowing a comparison of thetrans-acting factor bind-ing motifs within these promoter regions. Analysis of theAV11S1promoter identified a single element, the GT box, critical forAV11S1promoter activity (96), and this element is shared by twoof the three otherTCRA V segment promoters (AV1S3 andAV12S1), but not by either of the twoTCRD Vsegment promoters.Thus, the GT box may be a conserved element inTCRA Vpro-moters similar to the conserved decanucleotide found in 13 of 14characterized murineTCRB Vpromoters (100) and the conservedoctanucleotide, and its inversion, found in all Ig VH and VL pro-moters respectively (101). Neither the CRE or Ets site are con-servedTCRD Vpromoter elements as they are present 59 of AV1S3and AV11S1, and absent 59 of DV105S1. The viral Enh core se-quence is uniquely shared by the twoTCRD Vupstream sequences,but preliminary studies reported here suggest that this element may

be unimportant forDV101S1promoter activity. Thus, all of theTCRD Vpromoter elements may be heterogeneous.

An interesting observation from this study is that not all of theprotein-binding sites within the murineDV101S1minimal pro-moter are important for Enh-drivenTCRD gene transcription inMolt-13 cells. EMSA analyses showed that the GATA consensusat 25 is recognized by recombinant GATA-3 as well as GATA-3in Molt-13 nuclear extracts. However, a 2-base substitution in theGATA site that abolished GATA-3 binding had no effect onDV101S1promoter activity. Likewise, oligonucleotide competi-tion experiments demonstrated that ubiquitously expressed nuclearproteins bind theDV101S1252 E box, but a complete substitutionof the E box abrogating protein binding did not alterDV101S1promoter activity. It is conceivable that the GATA and E box sitesfunction at a stage in T cell development different from that rep-resented by Molt-13. For example, Molt-13 is well beyond thepredicted stage for Enh-independentDV101S1germline transcrip-tion. On the other hand, GATA-3 and E box binding proteins maybe exclusively involved in the developmentally regulated selectionof DV101S1for rearrangement in early mouse ontogeny sincecis-elements of theDV101S1promoter that are essential for targetinggene rearrangement may not be the same as those essential fortranscriptional activation (24, 25). The possibility that the GATAsite is required for theDV101S1promoter to mediate V(D)J re-combinational accessibility is particularly intriguing given that aGATA motif is only present upstream of theDV101S1segment outof the two TCRD and four TCRA Vsegment promoter regionscompared (see above), and GATA-3 is transcribed as early as day13/14 of gestation (42, 102), coincident with the onset ofTCRDV101S1rearrangements (11). Regardless, our original re-sults showing the lack of an effect of certain mutations onDV101S1promoter activity in Molt-13 cells should be confirmedwith a murineDV101S11 cell line.

AcknowledgmentsWe thank Drs. Y. H. Chien, J. Hassell, and R. Sen for phage, cosmid, andplasmid DNA; Drs. R. Baer, W. Born, A. Clausell, J. Forman, and D. Yuanfor cell lines; Dr. K. Meek for providing laboratory space to complete a

FIGURE 8. Mutating cis-regulatory elementswithin theDV101S1promoter does not always alterMolt-13 nuclear protein binding. EMSAs performedwith Molt-13 nuclear extract and32P-labeled wild-type or mutatedDV101S1promoter region frag-ments.A, Wild-type and mutated fragment 58 asprobes.B, Wild-type and mutated 92-bpHincII-HinfI fragment (286 to 14) as probes. The 92-bpHincII-HinfI fragment (see Fig. 1) had to be usedsince mutation 1 destroys theMaeIII restriction site.C, Wild-type and mutated fragment 99 as probes.The presence (1) or absence (2) of Molt-13 nuclearextract and the radiolabeled fragments used in thebinding reactions are indicated above the lanes. Ar-rows and numbers, to the right in each panel, indi-cate the specific migration-retarded species observedwith the wild-type fragment.

802 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

portion of the project; and Dr. K. Arizumii for critically reading themanuscript.

References1. Mombaerts, P., J. Iacomini, R. S., Johnson, K. Herrup, S. Tonegawa, and

V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lym-phocytes.Cell 68:896.

2. Shinkai, Y., G. Rathbun, K.-P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn,J. Charron, M. Datta, F. Young, A. M. Stall, and F. W. Alt. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)Jrearrangement.Cell 68:855.

3. Fowlkes, B. J., and D. M. Pardoll. 1989. Molecular and cellular events of T celldevelopment.Adv. Immunol. 44:207.

4. Strominger, J. L. 1989. Developmental biology of T cell receptors.Science244:943.

5. Leiden, J. M. 1993. Transcriptional regulation of T cell receptor genes.Annu.Rev. Immunol. 11:539.

6. Chien, Y.-H., M. Iwashima, K. B. Kaplan, J. F. Elliott, and M. M. Davis. 1987.A new T-cell receptor gene located within thea locus and expressed early inT-cell differentiation.Nature 327:677.

7. Raulet, D. H. 1989. The structure, function, and molecular genetics of theg/dT cell receptor.Annu. Rev. Immunol. 7:175.

8. Kronenberg, M., G. Siu, L. E. Hood, and N. Shastri. 1986. The molecular ge-netics of the T-cell antigen receptor and T-cell antigen recognition.Annu. Rev.Immunol. 4:529.

9. Cook, W. D., and A. M. Balaton. 1987. T-cell receptor and immunoglobulingenes are rearranged together in abelson virus-transformed pre-B and pre-Tcells.Mol. Cell. Biol. 7:266.

10. Shortman, K., and L. Wu. 1996. Early T lymphocyte progenitors.Annu. Rev.Immunol. 14:29.

11. Chien, Y.-H., M. Iwashima, D. A. Wettstein, K. B. Kaplan, J. F. Elliot, W. Born,and M. Davis. 1987. T-cell receptord gene rearrangements in early thymocytes.Nature 330:722.

12. Goldman, J. P., D. M. Spencer, and D. H. Raulet. 1993. Ordered rearrangementof variable region genes of the T cell receptorg locus correlates with transcrip-tion of the unrearranged genes.J. Exp. Med. 177:729.

13. Raulet, D., D. Spencer, Y.-H. Hsiang, J. Goldman, M. Bix, N.-S. Liao,M. Zijlstra, R. Jaenisch, and I. Correa. 1991. Control ofgd T cell development.Immunol. Rev. 120:185.

14. Alt, F. W., E. M. Oltz, F. Young, J. Gorman, G. Taccioli, and J. Chen. 1992.VDJ recombination.Immunol. Today 13:306.

15. Yancopoulos, G. D., and F. W. Alt. 1985. Developmentally controlled and tis-sue-specific expression of unrearranged VH gene segments.Cell 40:271.

16. Fondell, J. D., and K. B. Marcu. 1992. Transcription of germ-line Va segmentscorrelates with ongoing T-cell receptora-chain rearrangement.Mol. Cell. Biol.12:1480.

17. Anderson, S. J., K. M. Abraham, T. Nakayama, A. Singer, and R. Perlmutter.1992. Inhibition of T-cell receptorb-chain gene rearrangement by overexpres-sion of the nonreceptor protein tyrosine kinase p56lck. EMBO J. 11:4877.

18. Stanhope-Baker, P., K. M. Hudson, A. L. Shaffer, A. Constantinescu, andM. S. Schlissel. 1996. Cell type-specific chromatin structure determines thetargeting of V(D)J recombinase activity in vitro.Cell 85:887.

19. Sleckman, B. P., J. R. Gorman, and F. W. Alt. 1996. Accessibility control ofantigen-receptor variable-region gene assembly: role ofcis-acting elements.Annu. Rev. Immunol. 14:459.

20. Kallenbach, S., C. Babinet, S. Pournin, P. Cavelier, M. Goodhardt, andF. Rougeon. 1993. The intronic immunoglobulink gene enhancer acts indepen-dently on rearrangement and on transcription.Eur. J. Immunol. 23:1917.

21. Lauster, R., C.-A. Reynaud, I.-L. Martensson, A. Peter, D. Bucchini, J. Jami,and J.-C. Weill. 1993. Promoter, enhancer and silencer elements regulate rear-rangement of an immunoglobulin transgene.EMBO J. 12:4615.

22. Alvarez, J. D., S. J. Anderson, and D. Y. Loh. 1995. V(D)J recombination andallelic exclusion of a TCR-b-chain minilocus occurs in the absence of a func-tional promoter.J. Immunol. 155:1191.

23. Okada, A., M. Mendelsohn, and F. Alt. 1994. Differential activation of tran-scription versus recombination of transgenic T cell receptorb variable regiongene segments in B and T lineage cells.J. Exp. Med. 180:261.

24. Fernex, C., M. Capone, and P. Ferrier. 1995. The V(D)J recombinational andtranscriptional activities of the immunoglobulin heavy-chain intronic enhancercan be mediated through distinct protein-binding sites in a transgenic substrate.Mol. Cell. Biol. 15:3217.

25. Jenuwein, T., W. C. Forrester, R.-G. Qiu, and R. Grosschedl. 1993. The immu-noglobulinm enhancer core establishes local factor access in nuclear chromatinindependent of transcriptional stimulation.Genes Dev. 7:2016.

26. Ferrier, P., B. Krippl, T. K. Blackwell, A. J. W. Furley, H. Suh, A. Winoto,W. D. Cook, L. Hood, F. Constantini, and F. W. Alt. 1990. Separate elementscontrol DJ and VDJ rearrangement in a transgenic recombination substrate.EMBO J. 9:117.

27. Lauzurica, P., and M. S. Krangel. 1994. Enhancer-dependent and -independentsteps in the rearrangement of a human T cell receptord transgene.J. Exp. Med.179:43.

28. Sunaga, S., K. Maki, Y. Komagata, J.-I. Miyazaki, and K. Ikuta. 1997. Devel-opmentally ordered V-J recombination in mouse T cell receptorg locus is notperturbed by targeted deletion of the Vg4 gene.J. Immunol. 158:4223.

29. Born, W., C. Miles, J. White, R. O’Brien, J. H. Freed, P. Marrack, J. Kappler,and R. T. Kubo. 1987. Peptide sequences of T-cell receptord andg chains areidentical to predicted X andg proteins.Nature 330:572.

30. O’Brien, R. L., M. P. Happ, A. Dallas, E. Palmer, R. Kubo, and W. K. Born.1989. Stimulation of a major subset of lymphocytes expressing T cell receptorgd by an antigen derived from mycobacterium tuberculosis.Cell 57:667.

31. Takashima, A., J. L. Nixon-Fulton, P. R. Bergstresser, and R. E. Tigelaar. 1988.Thy-11 dendritic epidermal cells in mice: precursor frequency analysis andcloning of concanavalin A-reactive cells.J. Invest. Dermatol. 90:671.

32. Hata, S., M. B. Brenner, and M. S. Krangel. 1987. Identification of putativehuman T cell receptord complementary DNA clones.Science 238:678.

33. Loh, E. Y., L. L. Lanier, C. W. Turck, D. R. Littman, M. M. Davis, Y.-H. Chien,and A. Weiss. 1987. Identification and sequence of a fourth human T cell an-tigen receptor chain.Nature 330:569.

34. Lewis, S., N. Rosenberg, F. Alt, and D. Baltimore. 1982. Continuingk-generearrangement in a cell line transformed by Abelson murine leukemia virus.Cell30:807.

35. Kim, K. J., C. Kanellopoulos-Langevin, R. M. Merwin, D. H. Sachs, andR. Asofsky. 1979. Establishment and characterization of BALB/c lymphomalines with B cell properties.J. Immunol. 122:549.

36. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chaintermination inhibitors.Proc. Natl. Acad. Sci. USA 74:5463.

37. Feramisco, J. R., J. E. Smart, K. Burridge, D. M. Helfman, and G. P. Thomas.1982. Co-existence of vinculin and a vinculin-like protein of higher molecularweight in smooth muscle.J. Biol. Chem. 257:11024.

38. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Mapping RNA with nucleaseS1. In Molecular Cloning: A Laboratory Manual. C. Nolan, ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, p. 7.58.

39. Gorman, C. M., G. T. Merlino, M. C. Willingham, I. Pastan, and B. H. Howard.1982. The Rous sarcoma virus long terminal repeat is a strong promoter whenintroduced into a variety of eukaryotic cells by DNA-mediated transfection.Proc. Natl. Acad. Sci. USA 79:6777.

40. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn,K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplificationof DNA with a thermostable DNA polymerase.Science 239:487.

41. Zoller, M. J., and M. Smith. 1982. Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for the production ofpoint mutations in any fragment of DNA.Nucleic Acids Res. 10:6487.

42. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989.Site-directed mutagenesis by overlap extension using the polymerase chain re-action.Gene 77:51.

43. De Wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani. 1987.Firefly luciferase gene: structure and expression in mammalian cells.Mol. Cell.Biol. 7:725.

44. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombinant genomeswhich express chloramphenicol acetyltransferase in mammalian cells.Mol. Cell.Biol. 2:1044.

45. Landolfi, N. F., J. D. Capra, and P. W. Tucker. 1987. Protein-nucleotide contactsin the immunoglobulin heavy-chain promoter region.Proc. Natl. Acad. Sci. USA84:3851.

46. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeled DNA with base-specific chemical cleavages.Methods Enzymol. 65:499.

47. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcriptioninitiation by RNA polymerase II in a soluble extract isolated from mammaliannuclei.Nucleic Acids Res. 11:1475.

48. Xin, H.-H, A. Cowie, P. Lachance, and J. A. Hassell. 1992. Molecular cloningand characterization of PEA3, a new member of the Ets oncogene family that isdifferentially expressed in mouse embryonic cells.Genes Dev. 6:481.

49. Ko, L. J., M. Yamamoto, M. W. Leonard, K. M. George, P. Ting, andJ. D. Engel. 1991. Murine and Human T-lymphocyte GATA-3 factors mediatetranscription through acis-regulatory element within the human T-cell receptord gene enhancer.Mol. Cell. Biol. 11:2778.

50. Montminy, M. R., K. A. Sevarino, J. A. Wagner, G. Mandel, andR. H. Goodman. 1986. Identification of a cyclic-AMP-responsive elementwithin the rat somatostatin gene.Proc. Natl. Acad. Sci. USA 83:6682.

51. Martin, D. I. K., and S. H. Orkin. 1990. Transcriptional activation and DNAbinding by the erythroid factor GF-1/NF-E1/Eryf 1.Genes Dev. 4:1886.

52. Gunther, C. V., J. A. Nye, R. S. Bryner, and B. J. Graves. 1990. Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptionalactivator sequence within the long terminal repeat of the Moloney murine sar-coma virus.Genes Dev. 4:667.

53. Landolfi, N. F., J. D. Capra, and P. W. Tucker. 1986. Interaction of cell-typespecific nuclear proteins with immunoglobulin VH promoter region sequences.Nature 323:548.

54. Asarnow, D. M., W. A. Kuziel, M. Bonyhadi, R. E. Tigelaar, P. W. Tucker, andJ. P. Allison. 1988. Limited diversity ofgd antigen receptor genes of Thy-11

dendritic epidermal cells.Cell 55:837.55. McConnell, T. J., W. M. Yohoyama, G. E. Kikuchi, G. P. Einhorn, G. Stingl,

E. M. Shevach, and J. E. Coligan. 1989.d-chains of dendritic epidermal T cellreceptors are diverse but pair withg-chains in a restricted manner.J. Immunol.142:2924.

56. Bucher, P. 1990. Weight matrix descriptions of four eukaryotic RNA polymer-ase II promoter elements derived from 502 unrelated promoter sequences.J. Mol. Biol. 212:563.

57. Javahery, R., A. Khachi, K. Lo, B. Zenzie-Gregory, and S. Smale. 1994. DNAsequence requirements for transcriptional initiator activity in mammalian cells.Mol. Cell. Biol. 14:116.

803The Journal of Immunology

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

58. Smale, S. 1994. Core promoter architecture for eukaryotic protein-coding genes. InTranscription Mechanisms and Regulation. R. C. Conaway and J. W. Conaway,eds. Raven Press, New York, NY p. 63.

59. Gill, L. L., D. Zaninetta, and K. Karjalainen. 1991. A transcriptional enhancerof the mouse T cell receptord gene locus.Eur. J. Immunol. 21:807.

60. Kozak, M. 1986. Point mutations define a sequence flanking the AUG initiatorcodon that modulates translation by eukaryotic ribosomes.Cell 44:283.

61. Kozak, M. 1989. The scanning model for translation: an update.J. Cell Biol.108:229.

62. Kozak, M. 1995. Adherence to the first AUG rule when a second AUG codonfollows closely upon the first.Proc. Natl. Acad. Sci. USA 92:2662.

63. Leiden, J. 1992. Transcriptional regulation during T-cell development: theaTCR gene as a molecular model.Immunol. Today 13:22.

64. Leiden, J. M., and C. B. Thompson. 1994. Transcriptional regulation of T-cellgenes during T-cell development.Curr. Opin. Immunol. 6:231.

65. Faisst, S., and S. Meyer. 1992. Compilation of vertebrate-encoded transcriptionfactors.Nucleic Acids Res. 20:3.

66. Evans, T., M. Reitman, and G. Felsenfeld. 1988. An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globingenes.Proc. Natl. Acad. Sci. USA 85:5976.

67. Weiher, H., M. Zonig, and P. Gruss. 1983. Multiple point mutations affectingthe simian virus 40 enhancer.Science 219:626.

68. Ghosh, D. 1992. TFD: the transcription factors database.Nucleic Acids Res.20:2091.

69. Laverriere, A. C., C. MacNeill, C. Mueller, R. E. Poelmann, J. B. E. Burch, andT. Evans. 1994. GATA-4/5/6, a subfamily of three transcription factors tran-scribed in developing heart and gut.J. Biol. Chem. 269:23177.

70. Orkin, S. H. 1992. GATA-binding transcription factors in hematopoietic cells.Blood 80:575.

71. Yamamoto, M., L. J. Ko, M. W. Leonard, H. Beug, S. H. Orkin, and J. D. Engel.1990. Activity and tissue-specific expression of the transcription factor NF-E1multigene family.Genes Dev. 4:1650.

72. Landry, D. B., J. D. Engel, and R. Sen. 1993. Functional GATA-3 binding siteswithin murine CD8a upstream regulatory sequences.J. Exp. Med. 178:941.

73. Henderson, A. J., S. McDougall, J. Leiden, and K. L. Calame. 1994. GATAelements are necessary for the activity and tissue specificity of the T-cell re-ceptorb-chain transcriptional enhancer.Mol. Cell. Biol. 14:4286.

74. Ho, I-C., P. Vorhees, N. Marin, B. K. Oakley, S-F. Tsai, S. H. Orkin, andJ. M. Leiden. 1991. Human GATA-3: a lineage-restricted transcription factorthat regulates the expression of the T cell receptora gene.EMBO J. 10:1187.

75. Joulin, V., D. Bories, J.-F. Eleouet, M.-C. Labastie, S. Chretien, M.-G. Mattei,and P.-H. Romeo. 1991. A T-cell specific TCR-d DNA binding protein is amember of the human GATA family.EMBO J. 10:1809.

76. Marine, J., and A. Winoto. 1991. The human enhancer-binding protein Gata3binds to several T-cell receptor regulatory elements.Proc. Natl. Acad. Sci. USA88:7284.

77. Hsiang, Y.-h., D. Spencer, S. Wang, N. Speck, and D. H. Raulet. 1993. The roleof viral enhancer “core” motif-related sequences in regulating T cell receptor-gand -d gene expression.J. Immunol. 150:3905–3916.

78. Sawadogo, M., and A. Sentenac. 1990. RNA polymerase B (II) and generaltranscription factors.Annu. Rev. Biochem. 59:711.

79. Punturieri, A., Y. Shirakata, C. Bovolenta, G. Kikuchi, and J. E. Coligan. 1993.Multiple cis-acting elements are required for proper transcription of the mouseVd1 T cell receptor promoter.J. Immunol. 150:139.

80. Brown, S. T., G. A. Miranda, Z. Galic, I. Z. Hartman, C. J. Lyon, andR. J. Aguilera. 1997. Regulation of the RAG-1 promoter by the NF-Y transcrip-tion factor.J. Immunol. 158:5071.

81. Amrolia, P. J., J. M. Cunningham, P. Ney, A. W. Nienhuis, and S. M. Jane.1995. Identification of two novel regulatory elements within the 59-untranslatedregion of the humanAg-globin gene.J. Biol. Chem. 270:12892.

82. Burke, T. W., and J. T. Kadonaga. 1996.DrosophilaTFIID binds to a conserveddownstream basal promoter element that is present in many TATA-box-defi-cient promoters.Genes Dev. 10:711.

83. Guzowski, J. F., J. Singh, and E. K. Wagner. 1994. Transcriptional activation ofthe herpes simplex virus type 1 UL38 promoter conferred by thecis-actingdownstream activation sequence is mediated by a cellular transcription factor.J. Virol. 68:7774.

84. Dix, D. J., P.-N. Lin, Y. Kimata, and E. C. Theil. 1992. The iron regulatoryregion of ferritin mRNA is also a positive control element for iron-independenttranslation.Biochemistry 31:2818.

85. Geballe, A. P., R. R. Spaete, and E. S. Mocarski. 1986. Acis-acting elementwithin the 59 leader of a cytomegalovirusb transcript determines kinetic class.Cell 46:865.

86. Prats, A.-C., S. Vagner, H. Prats, and F. Amalric. 1992.cis-Acting elementsinvolved in the alternative translation initiation process of human basic fibro-blast growth factor mRNA.Mol. Cell. Biol. 12:4796.

87. Kollmar, R., and P. J. Farnham. 1993. Site-specific initiation of transcription byRNA polymerase II.Proc. Soc. Exp. Biol. Med. 203:127.

88. Zenzie-Gregory, B., P. Sheridan, K. A. Jones, and S. T. Smale. 1993. HIV-1core promoter lacks a simple initiator element but contains a bipartite activatorat the transcription start site.J. Biol. Chem. 268:15823.

89. Smale, S. T., and D. Baltimore. 1989. The “initiator” as a transcription controlelement.Cell 57:103.

90. O’Shea-Greenfield, A., and S. T. Smale. 1992. Roles of TATA and Initiatorelements in determining the start site location and direction of RNA polymeraseII transcription.J. Biol. Chem. 267:1391.

91. Zenzie-Gregory, B., A. Khachi, I. P. Garraway, and S. T. Smale. 1993. Mech-anism of initiator-mediated transcription: evidence for a functional interactionbetween the TATA-binding protein and DNA in the absence of a specific rec-ognition sequence.Mol. Cell. Biol. 13:3841.

92. Kaufmann, J., and S. Smale. 1994. Direct recognition of initiator elements by acomponent of the transcription factor IID complex.Genes Dev. 8:821.

93. Purnell, B. A., P. A. Emanuel, and D. S. Gilmour. 1994. TFIID sequence rec-ognition of the initiator and sequences further downstream inDrosophilaclassII genes.Genes Dev. 8:830.

94. Verrijzer, C. P., J.-L. Chen, K. Yokomori, and R. Tijan. 1995. Binding of TAFsto core elements directs promoter selectivity by RNA polymerase II.Cell 81:1115.

95. Du, H., A. L. Roy, and R. G. Roeder. 1993. Human transcription factor USFstimulates transcription through the initiator-elements of the HIV-1 and theAd-ML promoters.EMBO J. 12:501.

96. Kingsley, C., and A. Winoto. 1992. Cloning of GT box-binding proteins: a novelSp1 multigene family regulating T-cell receptor gene expression.Mol. Cell.Biol. 12:4251.

97. Korman, A. J., J. Maruyama, and D. H. Raulet. 1989. Rearrangement by inver-sion of a T-cell receptord variable region gene located 39 of the d constantregion gene.Proc. Natl. Acad. Sci. USA 86:267.

98. Dembic, Z., W. Haas, S. Weiss, J. McCubrey, H. Kiefer, H. von Boehmer, andM. Steinmetz. 1986. Transfer of specificity by murinea andb T-cell receptorgenes.Nature 320:232.

99. Hayday, A. C., D. J. Diamond, G. Tanigawa, J. S. Heilig, V. Folsom, H. Saito,and S. Tonegawa. 1985. Unusual organization and diversity of T-cell receptora-chain genes.Nature 316:828.

100. Anderson, S. J., S. Miyake, and D. Y. Loh. 1989. Transcription from a murineT-cell receptor Vb promoter depends on a conserved decamer motif similar tothe cyclic AMP response element.Mol. Cell. Biol. 9:4835.

101. Staudt, L. M., and M. J. Lenardo. 1991. Immunoglobulin gene transcription.Annu. Rev. Immunol. 9:373.

102. Oosterwegel, M., J. Timmerman, J. Leiden, and J. Clevers. 1992. Expression ofGATA-3 during lymphocyte differentiation and mouse embryogenesis.Dev. Im-munol. 3:1.

804 MURINE TCRDV101S1PROMOTER CHARACTERIZATION

by guest on April 5, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from