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Regulatory Elements in the Promoter of a Murine TCRD V Gene Segment^{1 ,2}

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

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

	Abstract

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TCRD V segments rearrange in an ordered fashion during human and murine thymic development. Recombination requires the accessibility of substrate gene segments, and transcriptional enhancers and promoters have been shown to regulate the accessible chromatin configuration. We therefore investigated the regulation of TCRD V rearrangements by characterizing the promoter of the first TCRD V segment to be rearranged, DV101S1, under the influence of its own enhancer. Sequences required for full promoter activity were identified by transient transfections of normal and mutated promoters into a human lymphoma, and necessary elements fall between -86 and +66 nt, relative to the major transcription start site. They include a cAMP responsive element (CRE) at -62, an Ets site at -39, a TATA box at -26, the major transcriptional start site sequence (-8 to -5 and -2 to +11), and a downstream sequence (+12 to +33). Gel shift analyses and in vitro DNase I footprinting showed that nuclear proteins bind to the functionally relevant CRE, Ets, +1 to +10 sequence, and the +17 to +21 sequence. Nuclear proteins also bind to an E box at -52, and GATA-3 binds to a GATA motif at -5, as shown by Ab ablation-supershift experiments, but mutations that abrogated protein binding to these sites failed to affect DV101S1 promoter activity. We conclude that not all protein-binding sites within the DV101S1 minimal promoter are important for enhancer driven TCRD gene transcription. Further, the possibility remains that the GATA and E box sites function in enhancer independent DV101S1 germline transcription.

	Introduction

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The and ß T cell development in the thymus requires the assembly of corresponding TCR gene variable exons from V, D (in the case of TCR D and B genes only), and J gene segments through the V(D)J joining recombination process (1, 2, 3, 4). Once assembled, TCR genes are transcribed from promoters associated with the rearranged V gene segments (5). This transcription requires the activity of enhancer (Enh)⁴ elements located adjacent to the C exons (5). Subsequently, splicing of the primary RNA transcripts occurs and the mature mRNA species give rise to functional TCR polypeptides if there is a proper reading frame.

The four TCR genes are located at three distinct chromosomal loci. The TCRD locus is included within the TCRA locus such that rearrangement of the A TCR chain gene segments is accompanied by deletion of the D TCR gene (6). In addition, there is some overlap in V gene segment usage in TCR A and D chains, although the V gene segments used in A and D chains are largely distinct (7). Assembly of the four TCR gene variable exons is highly regulated and temporally ordered. TCR genes are rearranged only in lymphoid precursors; then, complete assembly of the genes occurs only in T lymphocyte progenitors (8, 9). Further, the TCR G, D, and B variable exons assemble earlier during T-cell differentiation than the TCRA variable exons (3, 10), and the assembly of TCRB variable exons occurs sequentially. D-to-J rearrangements precede V-to-D rearrangements in the TCRB locus (8). Finally, within the TCR G and D loci, the V gene segments rearrange in a distinct order during ontogeny. In the mouse, GV1S1, GV2S1A1, and DV101S1 predominate in rearrangements in the fetus, whereas GV3S1A1 and other TCRD V gene segments, especially DV105S1, predominate in the adult (7, 11, 12, 13).

A single enzymatic system, the V(D)J recombinase, catalyzes all V(D)J rearrangement events in T and B cells, and two of the recombination factors, RAG-1 and RAG-2, are primarily lymphoid specific (14). Thus, restricted expression of the RAG genes may be the reason why V(D)J recombination occurs only in lymphoid precursors. However, differential rearrangement of Ag receptor genes within cells with recombinase activity requires an alternative explanation. The recombination mechanism involves recognition of recombination signal sequences, and these are conserved. Consequently, Yancopoulos and Alt (15) proposed that regulation was achieved through substrate accessibility. They postulated that chromatin is normally in a state refractory to V(D)J recombination and that regulated changes in the accessibility of the chromatin to the V(D)J recombinase enzyme system occur (15). Support for this "accessibility model" has been provided largely through indirect studies that utilize transcription as a measure of locus accessibility. Rearrangement has been shown to correlate with prior transcription of the corresponding unrearranged genes (12, 16), and cessation of rearrangement has been shown to correlate with the disappearance of corresponding sterile transcripts (12, 17). More recently, it has been shown, in an in vitro system, that cleavage of particular recombination signal sequences within chromatin depends on the source of the chromatin, in terms of both cell type and developmental stage, confirming the role of locus accessibility in targeting gene rearrangement (18).

Attention has now focused on determining the cis-acting elements that govern the accessible chromatin configuration required for V(D)J recombination. Studies of transgenic mice carrying recombination substrates, as well as studies eliminating regulatory elements from endogenous loci by homologous recombination, have shown that Ag receptor transcriptional controlling regions (promoters, Enhs, and silencers) are necessary for the regulation of V(D)J recombination in developing lymphocytes (19). The trans-acting factors involved, and the mechanism(s) by which transcriptional controlling regions help to mediate the accessibility of chromosomal substrates to the recombinase, remain to be elucidated. Transcription itself may be unnecessary for making a locus "accessible," as instances of rearrangement in the absence of transcription have been reported (20, 21, 22). However, even if transcription is required for rearrangement, transcriptional controlling regions will also regulate other molecular events that are prerequisites for recombination because transcription initiation from V and D gene segments has been observed to be insufficient for rearrangement of these gene segments (23, 24). Such events may include changes in chromatin structure, CpG demethylation, and/or recruitment of components of the V(D)J recombinase (14, 19), and cis-acting elements, within the Ag receptor gene promoter and Enh regions, distinct from those regulating transcription may control them (24, 25).

We are interested in determining the role of TCRD V promoter sequences in establishing the developmental program of TCRD V rearrangements (12, 13, 26, 27, 28). As an initial step in examining the possibility that promoter sequences regulate TCRD V accessibility, we have identified the transcriptional regulatory elements in the murine DV101S1 promoter. DV101S1 was selected because it is the first TCRD V segment to be rearranged, and it is used exclusively in TCR D chains.

	Materials and Methods

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Cell lines

33BTE-140.9 (GV2S1A1, DV101S1), 70BET104 (GV1S1, DV101S1), 33BTE-125.5 (GV3S1A1, DV2S8), and 33BTE-67.1 (GV3S1A1, DV104S1) are murine T cell hybridomas (29, 30). JAC-3 (GV1S1, DV101S1) is an IL-2-independent dendritic epidermal T cell line made by stably transfecting the ID2 cell line (31) with a CMV-driven human IL-2 vector. Molt-13 is a human T cell line (32, 33). Jurkat is a human ß expressing mature T cell line. EL4 and AKR117 are murine ß expressing T cell lines. 38B9 and PD31 are murine pre-B cell lines (34). M12.4 is a mature BALB/c B cell lymphoma (mIg^- Ia^- FcR⁺) (35). All cell lines were propagated in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 57.2 µM 2-ME, 100 U/ml penicillin-streptomycin, and 25 mM HEPES (pH 7.3).

DV101S1 promoter region fragment

AIR, a 14.6-kb MboI fragment containing the DV101S1 gene segment and leader exons in the vector J1, isolated from a B10.A liver genomic library, was obtained from Dr. Y. H. Chien (Stanford University, Stanford, CA). A 3.4-kb BamHI fragment containing 53 bp of the V gene segment, the leader exons, and upstream DNA was then subcloned into pGEM-7Zf(+) (Promega, Madison, WI), and the dideoxy chain termination method (36) was used to sequence 1287 bp at the V gene segment end of the insert.

S1 nuclease protection analyses

Total cellular RNA was isolated from T cell hybridomas and cell lines by the guanidinium/hot phenol method (37). S1 nuclease protection analysis was performed by using a dsDNA probe as described (38). Briefly, 30 µg of total RNA was resuspended in 50 µl of hybridization buffer (80% deionized formamide, 0.4 M NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA) containing the 5'-end-labeled 432-bp SstI-RsaI fragment probe (see Fig. 1). Following incubation at 80°C for 10 min, the hybridization reaction was incubated at 52°C for 16 h. Three hundred microliters of buffer containing 0.28 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 samples were subjected to electrophoresis on 6% polyacrylamide/8 M urea gels. Sequencing ladders used to determine the 5' end of S1 protected bands were generated by the dideoxy chain termination method (36) by using an oligonucleotide (5'-ACTGAAGATGAATATACA-3') complementary to 18 bp at the 3' end of the coding strand of the SstI-RsaI fragment. The size markers were 5' end-labeled MspI digests of pBR322.

View larger version (52K): [in this window] [in a new window]   FIGURE 1. DV101S1-5' region nucleotide sequence and localization of six transcriptional start sites. A, Nucleotide sequence from -919 to +368 (numbering relative to the major transcriptional start site). The DV101S1 gene segment (V) and leader sequence (L), determined by comparison with published DV101S1 cDNA sequences (54, 55), are boxed. The solid arrow indicates the major mapped transcriptional start sites, and the open triangles the other 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 second strongest. Restriction enzymes used to generate promoter deletions for reporter gene plasmid constructions are indicated, as well as the RsaI site end-labeled on the S1 nuclease protection probe (*). B, S1 nuclease mapping of the transcription start site. RNA from three cell populations expressing DV101S1 transcripts (70BET104, 33BTE140.9, JAC-3, lanes 4–6) and from three negative controls (EL4, 33BTE67.1, 33BTE125.5, lanes 1–3) were annealed to the 5' end-labeled 432-bp SstI-RsaI fragment indicated in A. S1 nuclease protected fragments (arrows) and full-length probe (P) are indicated. (M), radiolabeled MspI digested pBR322, sizes in nt; (P), input probe; (G), (A), (T), (C), sequencing ladder generated using an oligonucleotide complimentary to 18 bp at the 3' end of the coding strand of the SstI-RsaI fragment.

pGEM7ZCATTCRDEnh(+) was the reporter gene plasmid used in all constructions. To generate this plasmid, the bacterial chloramphenicol acetyltransferase (CAT) gene and the SV40 small t intron and polyadenylation site from a derivative of pRSVmCAT (39) were first subcloned into the ApaI-XhoI sites of pGEM-7Zf(+), thus generating pGEM7ZCAT. The cos 3 K5 cosmid clone, a derivative of cos 3 (6) containing the BALB/c TCRDC1 and TCR D chain Enh (TCRD enh), was then obtained from Dr. Y. H. Chien, and the 4.5-kb EcoRI fragment containing the TCRD Enh was excised and subcloned into pGEM-7Zf(+). From that plasmid, the TCRD Enh was isolated using HaeIII, subcloned into the SmaI site of pGEM-7Zf(+), excised again with KpnI and ClaI, and finally inserted into the KpnI-ClaI sites of pGEM7ZCAT to generate pGEM7ZCATTCRDEnh(+).

Nine 5' deletion constructs were made by inserting promoter fragments into the XhoI-KpnI sites of pGEM7ZCATTCRDEnh(+). In eight cases, the promoter fragments were first cloned into pSP72 (Promega) and then excised with SalI and KpnI for insertion into pGEM7ZCATTCRDEnh(+). The strategies for cloning promoter fragments into pSP72 were as follows: 1) -2100 to +368, the 3.4-kb BamHI DV101S1 promoter fragment described above was subcloned into the BamHI site of pSP72, generating pSP72AIRPHAGE3.4(-); 2) -791 to +368, pSP72AIRPHAGE3.4(-) was digested with SmaI and BalI to remove the internal 2.2-kb fragment and then religated to generate pSP72BalI-BamHIAIRPHAGE3.4(-); 3) -708 to +368, a 1076-bp DraI-BamHI fragment of pSP72BalI-BamHIAIRPHAGE3.4(-) was subcloned into SmaI-BamHI sites of pSP72; 4) -531 to +368, a 899-bp ScaI-BamHI fragment of pSP72BalI-BamHIAIRPHAGE3.4(-) was subcloned into SmaI-BamHI sites of pSP72; 5) -393 to +368, a 760-bp BsphI-BamHI fragment of pSP72BalI-BamHIAIRPHAGE3.4(-) was blunted and subcloned into the SmaI site of pSP72; and 6) -86 to +368, plasmid 4.1.6 containing the 668-bp SacI-BamHI fragment of the DV101S1 promoter cloned into the SacI-BamHI sites of pGEM-3 (Promega) was obtained from Dr. Y. H. Chien. The SacI-BamHI fragment was excised from pGEM-3 with BamHI and EcoRI, the ends blunted, EcoRI linkers attached, and then the fragment was cloned into the EcoRI site of pGEM-7Zf(+), generating pGEM7Z5'V1(-) and pGEM7Z5'V1(+). A 460-bp HincII-EcoRI fragment of pGEM7Z5'V1(+) was blunted and subcloned into the SmaI site of pSP72 generating pSP72HincII-BamHIpGEM7Z5'V1(+)(+). 7) -33 to +368, a 432-bp MaeIII-PstI fragment from pSP72HincII-BamHIpGEM7Z5'V1(+)(+) was blunted and subcloned into PstI-SmaI sites of pSP72; 8) +4 to +368, a 396-bp HinfI-PstI fragment from pSP72HincII-BamHI pGEM7Z5'V1(+)(+) was blunted and subcloned into PstI-SmaI sites of pSP72. For the final 5' deletion construct, -300 to +368, the 668-bp SstI-BamHI DV101S1 promoter fragment was excised from pGEM7Z5'V1(+) with XhoI and KpnI and inserted into the XhoI-KpnI sites of pGEM7ZCATTCRDEnh(+).

Three 3' deletion constructs were also made by inserting promoter fragments into the XhoI-KpnI sites of pGEM7ZCATTCRDEnh(+). In two cases, the 3' deleted promoter fragments were again cloned into pSP72 first and then excised with XhoI and KpnI (-86 to +66) or SalI and KpnI (-86 to +6) for insertion into pGEM7ZCATTCRDEnh(+). For fragment -86 to +66, a 152-bp HincII-FokI fragment was excised from pGEM7Z5'V1(+), the ends blunted, and then cloned into the PvuII site of pSP72. For fragment -86 to +6, a 110-bp EcoRI-HinfI fragment was excised from pSP72HincII-BamHIpGEM7Z5'V1(+)(+), the ends blunted, and then cloned into the SmaI site of pSP72. The remaining promoter 3' deletion, -86 to +91, was amplified from pGEM7Z5'V1(-) using the PCR (40) and oligonucleotides incorporating SalI or KpnI restriction sites: DV101S1(-86) 5'-TGCC(GGTACC)AACATGCTACCAGTCCATGACCTCACTAGC-3', and +91 5'-AGGT(GTCGAC)AGTTTCACTGAGGATGAGTTTGTA-3'.

Eight plasmid constructions were made containing mutations in the -86 to +368 DV101S1 promoter fragment. Constructions containing mutations in the cAMP responsive element (CRE) or E box elements were produced by oligonucleotide-mediated site-directed mutagenesis using single-stranded phagemid DNA as described (41). The sequences of the two synthetic oligonucleotides used with inserted mutations underlined are mutation 9A, 5'-AGAGACATGTGGCAAGCTAGCTGGTGATTGGACTGGTAGCATGTT-3', and mutation 11A, 5'-TTAGTCACTTCCTCAGTACGACCTGCGCTAGCTAGTGAGGTCATGGA-3'. The six remaining mutant DV101S1 -86 to +368 promoter constructions were produced by overlap extension using the PCR (42) followed by cloning of the reaction products into the XhoI-KpnI sites of pGEM7ZCATTCRDEnh(+). Four oligonucleotide primers were used to generate each mutant PCR product: two unique oligonucleotide primers for the insertional mutagenesis and two common oligonucleotide primers for the boundaries of the final PCR product. The two common oligonucleotides are: DV101S1(-86) and 5'-AGGT(GTCGAC)TCTAGAGGATCCCCAATTCCGGATCCCCA-3'. The sequences of the pairs of unique oligomers, with inserted mutations underlined, are: mutation 1, 5'-TGGTTTGATATTAGTCAACCGGATGTTACCGAATGTGGCAAGCTAGTGA-3' and 5'-TGACTAATATCAAACCACCTTTACAG-3'; mutation 2, 5'-CCTCTGTCTCTGTCTCCACTGCAGGACGCTAAGTTGGTAAAGGTGGTTTGATATTAG-3' and 5'-GGAGACAGAGACAGAGGTGAGGCT-3'; mutation 3, 5'-TGGGCTGCCCTGCTGAGTTGGCTAATGAAGCTGCCTCTAGTGAGAATCCTTATCTG-3' and 5'-CTCAGCAGGGCAGCCCACCTT-3'; mutation 7, 5'-TGCTGTAAAGGTGGTTTGGCGAGAGTCACTTCCTCAGAGAGACA-3' and 5'-CAAACCACCTTTACAGCAGATAAGGA-3'; mutation 14, 5'-CTCCGTGAGAATCCTTAAGTGCTGTAAAGGTGGTTTGAT-3' and 5'-TAAGGATTCTCACGGAGACAGAGACAGAGGTGAGGCTC-3'; and mutation 15, 5'-CTTATCTGCTGTAAAGGCAGTTTGATATTAGTCACTTCC-3' and 5'-CCTTTACAGCAGATAAGGATTCTCACGGAGACA-3'.

Sequences of all amplified and mutated DV101S1 promoter fragments were verified by double-stranded dideoxy-DNA sequence analysis.

Transfections

Molt-13 cells (20 x 10⁶) in 0.8 ml RPMI 1640 medium were transfected with 10 µg of pRSVLuciferase reference plasmid (43) and a 3-fold molar excess of test plasmid by electroporation using a gene pulser (Bio-Rad, Richmond, CA) set at 960 µFD and 280 V. Cells were harvested 48 h later, resuspended in 0.1 ml of 0.25 M Tris-HCl (pH 7.5) per 3 x 10⁶ live cells, and extracts were prepared by three cycles of freeze-thaw lysis followed by centrifugation. DNA used in transfections was prepared by CsCl density centrifugation and quantitated by visualization in a gel. Constructs were transfected in singlet or duplicate within an experiment and each experiment with a series of constructs was repeated a minimum of three times.

Luciferase and CAT assays

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

Electrophoretic mobility shift assay (EMSA) and DNase I footprinting

The binding reaction was conducted in a total volume of 20 µl containing 20 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, 6 µg BSA, 2 µg poly(dI-dC), 5 to 12 µg nuclear extract protein, and 0.5–5 ng ³²P-labeled fragment. The assay mixture was incubated for 15 min at 30°C, loaded onto a 4% polyacrylamide gel containing 0.045 M Tris-borate, 1.0 mM EDTA, and 2% glycerol, and electrophoresed at room temperature. For experiments involving inhibition of complex formation, cold fragments or oligonucleotides were added to the binding reaction and incubated for 5 min before the addition of labeled fragment. For EMSAs performed with PEA3 and murine (m) GATA-3 protein produced by in vitro translation, Nonidet P-40 and BSA were excluded from the binding reactions, only 100 to 400 ng poly(dI-dC) was used, and reactions were incubated at either 30°C or 37°C for 15 min before loading them on polyacrylamide gels and running as above. Ab treatment of the fragment 99 EMSA was conducted with monoclonal mouse anti-GATA-3 IgG1 Ab (HG3–31; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL). The reaction procedure consisted of a 15 min 30°C preincubation of 12 µg of Molt-13 nuclear extract and 100, 200, or 400 ng of Ab in reaction buffer, followed by a 15 min 30°C incubation with radiolabeled fragment 99. Reaction mixtures were run on polyacrylamide gels as above.

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

Probes

Fragment 58 is the 113-bp SalI-EcoRI fragment or the 131-bp SalI-BglII fragment from pSP72HincII-MaeIII(-) mini 7, a plasmid derived by blunting the 76 bp EcoRI-MaeIII fragment from pSP72HincII-BamHIpGEM7Z 5'V1(+)(+) and ligating it into the SmaI site of pSP72. Fragment 99 is the 144-bp SalI-EcoRI fragment from pSP72 MaeIII-FokIAirphage3.4(-) mini 2, a plasmid derived by first cloning the 216-bp MaeIII-XbaI DV101S1promoter fragment into the XbaI-SmaI sites of pSP72, then cutting out the 107-bp FokI-SacI fragment from that vector, blunting the ends, and ligating the fragment into the SmaI site of pSP72. The HincII-HinfI probe is the 147-bp SalI-EcoRI fragment from pSP72HincII-HinfI(-) mini 36 derived by blunting the 110-bp EcoRI-HinfI fragment from pSP72HincII-BamHIpGEM7Z5'V1(+)(+) and cloning it into the SmaI site of pSP72. Mutated fragment 58 probes are 113-bp SalI-EcoRI fragments from plasmids pSP72HincII-MaeIII(-) with mutated CRE and pSP72HincII-MaeIII(+) with mutated E box. The mutated HincII-HinfI probe is the 147-bp SalI-EcoRI fragment from pSP72HincII-HinfI(-) with Mut. 1. Mutated fragment 99 probes are 136-bp EcoRI-SalI fragments from plasmids pSP72 MaeIII-FokI(-) with Mut. 2, 3, 7, and 14, respectively. These four plasmids were derived by blunting the 99-bp MaeIII-FokI fragments bearing mutations and ligating them into the SmaI site of pSP72. For use as probes, DNA fragments were labeled at their 5' ends and purified by PAGE.

Nuclear extracts and in vitro translated proteins

Nuclear extracts were prepared by the procedure of Dignam et al. (47). For DNase I footprinting, Molt-13 nuclear extracts were purified by heparin-agarose chromatography. Extract was loaded onto a column and the column 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 1 µg/ml leupeptin. Bound proteins were eluted with the above buffer containing 0.4 M KCl.

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

Oligonucleotides

ds oligonucleotides used for EMSA inhibition experiments were prepared by annealing equal amounts of unlabeled complementary oligonucleotides followed by purification on 15% polyacrylamide gels. They contained the following sequences and their complementary strands: CRE, 5'-GATCCTGGGGGCGCCTCCTTGGCTGACGTCAGAGAGAGAGTTAACG-3', from the rat somatostatin promoter (50); GATAglobin, 5'-GATCTCCGGCAACTGATAAGGATTCCCTG-3', from the mouse 1-globin promoter (51); GATA-V1, 5'-TAGGATAGCCCTGAGATAACGCGAATATTCTC-3'; Ets, 5'-AATATTGAGCTCGGAGAGCGGAAGCGCGCGAACTCGAG-3', from the Moloney murine sarcoma virus long terminal repeat (52); AP1, 5'-GATCCCCCGGATGAGTCATAGCTTATCGATACCG-3'; E box, 5'-GATCAATATTGAGCTCGGATTGCCACATGTCTCGACGCGAACTCGAG-3'; TCF-1, 5'-GATCCAGGGAATCCAATTCTCTGGGCTTGCCGGA-3'; Start Site, 5'-CTCGAGTTCGCGAGGATTCTCATCCGAGCTCAATATT3'; and Octamer, 5'-ATGAATATGCAAATCAGGTG-3', from the BCL₁ V_H gene promoter (53).

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

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transcription initiates at six sites upstream of the murine TCRDV101S1 gene segment

To initiate analysis of the TCRDV101S1 promoter, 1234 upstream bp were sequenced beginning from the 53rd residue of the DV101S1 gene segment (Fig. 1A). S1 nuclease protection analysis was then performed to define the RNA start site (Fig. 1B). A 432-bp SstI-RsaI 5' end-labeled fragment was used to protect RNA from two DV101S1-expressing T cell hybridomas and a DV101S1 expressing T cell line. Six protected bands (110, 116, 118, 133, 138, and 141 bp) were consistently observed with each of these RNA samples, indicating multiple transcription initiation sites (Fig. 1B, lanes 4–6). In contrast, RNA from three negative controls (a DV104S1⁺ and a DV2S8⁺ T cell hybridoma, and an ß T cell line) did not produce any protected bands (Fig. 1B, lanes 1–3). These findings were confirmed using RNase protection analysis of a uniformly [³²P]CTP-labeled RNA probe (data not shown). The six transcription initiation sites fall into two clusters of three (Fig. 1A, Table I), with the strongest start site (number 3, solid arrow in Fig. 1A) associated with one cluster and the second strongest start site (number 5, open arrow with the asterisk above it in Fig. 1A) associated with the other. The strongest start site is located 91 bp from the translation initiation codon and was designated as +1.

Several potential initiator (Inr) sequences are present in the -20 to +66 region encompassing the DV101S1 transcription start sites. Using the Inr consensus derived through sequence comparison of various start site regions (5'-KCABHYBY-3' (K = G or T; B = C, G, or T; H = A, C, or T; Y = C or T); significant similarity cut-off value = 81.4% (56)) there are nine potential Inr sequences in this region. Four of these nine coincide with experimentally identified transcriptional start sites (Table I, start sites 1, 2, 5, and 6). The others are located at -18, +35, +43, +56, and +59. Using a different Inr consensus, one derived by functional studies (5'-YYANWYY-3' (57)) and requiring a 5/7 bp match, an additional seven potential Inrs can be found within this region. These elements are at -17, -15, -14, -12, +2, +6, and +29. However, if the critical bases in the experimentally defined Inr consensus and the location of the pyrimidines are considered (57, 58), then only the additional -12 and +2 potential Inrs are likely functional Inrs (57, 58).

Sequences between -86 and +66 contribute substantially to TCRDV101S1 promoter activity in the presence of the TCRD Enh

To map the region containing the functional elements of the DV101S1 promoter, the -2100 to +368 DV101S1fragment (numbering relative to the transcription start site) was inserted into a CAT reporter construct for transient transfection studies in human T cells (Molt-13). This vector did not yield detectable CAT activity (data not shown), so the homologous murine core TCRD Enh (59) was added to the construct. Detectable CAT activity was obtained with this vector (Fig. 2A), and a series of constructs was then made with deletions at the 5' end of the DV101S1 fragment utilizing restriction enzyme sites (Fig. 1A). These plasmids were electroporated with the reference plasmid, pRSVLuciferase, into Molt-13 cells and CAT activity was determined in cell extracts prepared 48 h after DNA transfection (Fig. 2A). CAT activity was used as a measure of the transcriptional activity of the constructs, but CAT activity may not accurately reflect true promoter activity because we did not quantitate correctly initiated DV101S1 transcripts. Furthermore, the CAT activity obtained will depend upon the stability of the RNA and the efficiency of CAT translation. The presence of multiple translation initiation sites after the DV101S1 transcriptional start site in the reporter gene constructs complicates the translation issue. The context of the CAT translation initiation site (AUG^CAT) is the strongest of the multiple translation initiation sites present in mRNA derived from the DV101S1 promoter reporter constructs (Fig. 2; 61 ; however it is the fourth AUG codon from the 5' end of the mRNA and varies as to whether it is in the same or different reading frame as the first translation initiation codon, AUG^Vd1, and to whether an in-frame terminator codon occurs upstream of it. Hence, it is best to score the constructs in Fig. 2 only as yielding or prohibiting CAT activity and to compare variabilities in CAT activity only within constructs that have the translational start sites similarly oriented (grouped together in Fig. 2; Refs. 61 and 62).

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Deletional analysis of the 2.5-kb DV101S1 promoter fragment reveals that 152 bp is sufficient to generate high levels of promoter activity. Various DV101S1 promoter region fragments were fused to the CAT reporter gene and tested for CAT activity in the presence of the homologous TCRD Enh by transient transfection into Molt-13 cells. Transfection efficiencies were controlled by transfecting pRSVLuciferase along with the test plasmid. The promoterless plasmid, pGEM7ZCATTCRDEnh(+), served as a negative control and pRSVmCAT as a positive control in each experiment. Within each experiment, extract volumes were normalized for luciferase activity before analysis of CAT activity. The percent acetylation with the negative control was then subtracted from measurements of test constructs before their CAT activities were evaluated relative to that of the full-length parent construct. A, Results with the -2100 to +368 promoter fragment progressively deleted at the 5' end. Four experiments are presented, with the data expressed as the percent of CAT activity displayed by the -2100 to +368 promoter reporter construct. pGEM7ZCATTCRDEnh(+) produced 1.0, 0.9, 1.2, and 0.2% acetylation, and the -2100 to +368 construct produced 8.7, 3.3, 11.5, and 3.5% acetylation in experiments 1 through 4, respectively. Mean percentages of CAT activity are listed in the last data column. B, Results with the -86 to +368 DV101S1 promoter fragment progressively deleted at the 3' end. Three experiments are presented with the data expressed as the percent of CAT activity displayed by the -86 to +368 promoter reporter construct. pGEM7ZCATTCRDEnh(+) produced 0.2, 0.2, and 0.2% acetylation, and the -86 to +368 construct produced 35.0, 22.4, and 22.3% acetylation in these three experiments, respectively. Mean percentages of CAT activity are listed in the last data column. The orientation of translational start sites in mRNA presumed to be derived from the reporter gene constructs is diagramed in the right hand side of the tables in A and B. Constructs yielding identical mRNA are blocked together. Parentheses around an AUG indicate that the codon is out of frame with the 5'-proximal AUGDV101S1 (designated AUGVd1). STOP indicates a terminator codon in frame with AUGVd1. Base pairs between translation initiation codons or between translation initiation and termination codons are indicated. The sequences of the four translation initiation sites found in mRNA from the reporter gene constructs in order of their predicted strength (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.

To determine the 3' boundary of the minimal functional DV101S1 promoter, a series of constructs was made with deletions at the 3' end of the -86 to +368 DV101S1 fragment. Deletions were made by using restriction enzyme sites or the PCR (Fig. 1A), and constructs were tested for CAT activity by transient transfection in Molt-13 cells (Fig. 2B) as above. A deletion of 277 nt to just 5' of the methionine translation initiation codon in the first leader exon, +91, had no appreciable affect on CAT activity. Further, when an additional 25 nt were removed to +66, the promoter fragment yielded activity equivalent to that obtained with the -86 to +368 construct. However, with the furthest 3' truncation, +6, no significant CAT activity was obtained. These results show that sequences within +6 and +66 are essential for expression of DV101S1 and define the 3' endpoint of the minimal functional DV101S1 promoter as +66 nt from the mRNA start site.

Molt-13 nuclear factor(s) specifically bind to the minimal DV101S1 promoter region

To identify proteins regulating the activity of the minimal TCRDV101S1 promoter, EMSAs were performed using two ³²P-labeled fragments that comprise all of the -86 to +66 DV101S1 promoter region (Fig. 3A). These fragments are the 58-bp HincII-MaeIII fragment, representing -86 to -29, and the 99-bp MaeIII-FokI fragment representing -33 to +66. Numerous retarded DNA-protein bands were observed in Molt-13 nuclear extracts with fragment 58 (Fig. 3B, lane 7). To establish the specificity of the DNA-protein interactions, unlabeled fragment 58 (Fig. 3B, lanes 2–6) and an irrelevant fragment (Fig. 3B, lanes 8–12), were used as inhibitors in the binding assay. Four specific retarded protein-DNA bands were observed (arrows in Fig. 3B).

View larger version (55K): [in this window] [in a new window]   FIGURE 3. Factor(s) in Molt-13 nuclear extracts specifically bind to the minimal DV101S1 promoter. A, Restriction endonuclease map of the minimal DV101S1 promoter. Relevant enzymes are noted as well as the fragments used as probes. Fragments are referred to here and in subsequent figures by their length in base pairs. B, EMSA analysis of fragment 58. 32P-labeled fragment 58 was incubated without (lane 1) or with (lanes 2–12) Molt-13 nuclear extract. A 5-, 10-, 25-, 50-, and 100-fold molar excess of unlabeled fragment 58 and the irrelevant 265-bp BalI-EcoRI CAT coding region fragment was added to the binding assay in lanes 6–2 and 8–12, respectively. Migration of the arbitrarily numbered four retarded species is indicated on the left. C, Summary of the fragment 58 nuclear protein-binding activities detected in different lymphoid cell nuclear extracts. D, EMSA using 32P-labeled fragment 99 and Molt-13 nuclear extract (lanes 2–14); lane 1 received no extract. A 1-, 5-, 10-, 25-, 50-, and 100-fold molar excess of unlabeled fragment 99 and the irrelevant 265-bp BalI-EcoRI CAT coding region fragment was added to the binding assay in lanes 7–2 and 9–14, respectively. Migration of the arbitrarily numbered four retarded species is indicated on the left. E, Summary of the fragment 99 nuclear protein-binding activities detected in different lymphoid cell nuclear extracts.

EMSAs were also performed with the two probes using nuclear extracts from other cell lines including TCR- 70BET104 and 33BTE125.5, TCR-ß EL4 and AKR117, pre-B 38B9 and PD31, and mature B M12.4. The results (summarized in Fig. 3, C and E) demonstrated that the probe 99 nuclear protein binding species 4 was T cell specific (Fig. 3E).

The CRE, Ets, E Box, GATA, and two elements in the 5' untranslated leader bind Molt-13 nuclear factor(s)

To identify the binding sites of the proteins interacting with the DV101S1 minimal promoter, this region was searched initially for consensus recognition sequences of transcription factors involved in gene regulation during T-cell development (63, 64) and later for consensus recognition sequences listed in the National Center for Biotechnology Information transcription factor database (Fig. 4D; 68 . Duplex oligonucleotides containing most of the observed transcription factor binding motifs were then used as competitors in EMSAs. A CRE, two E boxes, and an Ets site were observed in fragment 58 at -62, -52, -47, and -39 nt from the mRNA start point, respectively (Fig. 4D). Duplex oligonucleotides containing these consensus sequences were used as inhibitors in EMSAs with ³²P-labeled fragment 58 and Molt-13 nuclear extract (Fig. 4, A and B). An oligonucleotide containing a CRE site from the rat somatostatin promoter (50) inhibited the formation of migration retarded species 1 and 4 with this probe in a titratable manner (Fig. 4A, lanes 2–7), whereas formation of species 2, 3, and 4 was inhibited in a titratable manner by an oligonucleotide containing the region -56 to -43 of the DV101S1 promoter (E box oligo; Fig. 4B, lanes 2–7). An oligonucleotide containing an Ets site from the Moloney murine sarcoma virus long terminal repeat (52) did not inhibit formation of any of the migration retarded species (Fig. 4A, lanes 9–14) despite the presence of the consensus Ets binding site in the fragment. Two irrelevant duplex oligomers, one containing a GATA site from the mouse ₁-globin promoter (GATA-globin oligo; 66 and the other containing an AP1 site, also had no inhibitory capacity (data not shown).

View larger version (68K): [in this window] [in a new window]   FIGURE 4. Synthetic oligonucleotides containing CRE, E Box, or GATA elements inhibit interaction of Molt-13 nuclear protein with radiolabeled DV101S1 promoter fragments. A and B, 32P-labeled fragment 58 was incubated without (A and B, lane 1) or with (A, lanes 2–14; B, lanes 2–8) Molt-13 nuclear 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 99 was 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 excess of the ds GATA-globin oligonucleotide (lanes 7–2, respectively) or an irrelevant ds oligonucleotide containing a TCF-1 site (lanes 9–14). D, Schematic of the minimal functional DV101S1 promoter with computer-identified transcription factor consensus recognition sequences boxed. Potential TATA boxes are underlined (see Table I). AP1, 5'-TGASTMA-3' (M = A or C) (65); CRE, 5'-TGANNTCA-3' (64); E Box, 5'-CANNTG-3' (N = any nucleotide) (64); Ets, 5'-SMGGAWGY-3' (65); GATA, 5'-WGATAR-3' (66); Sp1, 5'-KRGGCKRRK-3' (65); and viral Enh core, 5'-GTGGWWWG-3' (67).

To further examine nuclear protein binding sites in the minimal DV101S1 promoter region, fragments 58 and 99 were subjected to DNase I protection studies using Molt-13 nuclear extract. Both fragments were labeled on either strand at the 5' end and four protected regions were identified (Fig. 5). All four protected regions were detected on only one of the DNA strands, and three had at least one DNase I hypersensitive site at their boundary. The first protected region overlaps the 3' end of the E box motif, protecting nt -48 to -42 (Fig. 5A). Another protected region spans the Ets site, protecting nt -39 to -33 (Fig. 5A). The third protected region overlaps the major transcription initiation site and two potential Inr elements, protecting nt +1 to +10 (Fig. 5B). Finally, the last protected region is downstream of the major transcription initiation site and overlaps one potential Inr, protecting nt +17 to +21 (Fig. 5C).

View larger version (63K): [in this window] [in a new window]   FIGURE 5. DNase I protection analyses of the migration retarded species observed with Molt-13 extract and fragments 58 and 99 from the DV101S1 promoter. A, Fragment 58 5' end-labeled to establish coding strand contacts. B, Fragment 99 5' end-labeled to establish noncoding strand contacts. C, Fragment 99 5' end-labeled to establish coding strand contacts. Lanes 1 and 5 in A and B and lane 3 in C represent the G+A-specific modification/cleavage reaction conducted on the respective fragments for orientation. Lanes 2 and 4 in A and B, and lane 2 in C represent the pattern of DNase I cleavage of the unbound DNA fragment (digested in the presence of nuclear proteins). Partial sequence of the fragments are given. *, hypersensitivity sites; •, complete protection.

The demonstration that a T cell-specific nuclear factor binds the GATA element at -5 prompted us to examine whether the GATA-3 transcription factor binds this element. GATA-3 is one of a family of six transcription factors that bind the GATA consensus recognition sequence (69, 70, 71). It is the predominant member expressed in T cells and has been shown previously to be important in regulation of TCR A, B, and D genes as well as the CD8 gene (49, 72, 73, 74, 75, 76). GATA-3 binding to the TCRDV101S1 promoter site was first examined by comparing the mobility of the species 4 complex generated with fragment 99 and Molt-13 nuclear extract with that of the complex generated with this probe and mGATA-3. Nonradioactive mGATA-3 was produced by in vitro translation and production was confirmed by performing parallel reactions in the presence of [³⁵S]methionine and analyzing reaction products on a SDS-polyacrylamide gel (data not shown). In vitro-translated products yielded a GATA-3 protein of 48 kDa when the sense mRNA was synthesized from p34 mc5b8 (49) with the T7 polymerase, but no radioactively labeled protein was made when the antisense mRNA was synthesized with the SP6 polymerase (data not shown). The other control, PEA3, an irrelevant transcription factor belonging to the Ets gene family (48), yielded a major species of 68 kDa from the sense mRNA as expected (data not shown). The in vitro-translated mGATA-3/fragment 99 nucleoprotein complex (Fig. 6A, lanes 4and 9) comigrates with specific T cell nuclear extract/fragment 99 complex species 4 that is inhibitable with the duplex GATA-globin oligonucleotide (Fig. 6A, lanes 7and 11–13) supporting the notion that it is GATA-3 in the T cell nuclear extracts that binds this site. Additional support derives from the observation that mutation of the GATA site in fragment 99 (Mut. 2) disrupts formation of Molt-13 nuclear extract/DNA complex species 4 and significantly decreases binding of in vitro translated mGATA-3 to this promoter region fragment (Fig. 6A, lanes 8 and 10, respectively).

View larger version (54K): [in this window] [in a new window]   FIGURE 6. mGATA-3 binds to the GATA site in the DV101S1 promoter region. A, EMSAs performed with 32P-labeled wild-type DV101S1 promoter region fragment 99 (W; lanes 1–5, 7, 9, and 11–15) or 32P-labeled fragment 99 bearing mutation 2 (m; lanes 6, 8, and 10) and unlabeled, in vitro translated proteins from sense PEA3 RNA (lane 2), antisense mGATA-3 RNA (lane 3), sense mGATA-3 RNA (lanes 4, 9, and 10), or Molt-13 nuclear extract (lanes 7, 8, and 11–15). In lanes 1, 5, and 6, radiolabeled fragments were incubated without in vitro-translated protein or nuclear extract. Binding was competed with a 50- and 100-fold molar excess of the double-stranded GATA-globin oligonucleotide (lanes 12 and 13) or the Ets containing double-stranded oligonucleotide (lanes 14 and 15). Arrows indicate the specific migration retarded species seen with mGATA-3 protein and fragment 99 and complex 4 previously seen with the Molt-13 nuclear extract/fragment 99 binding reaction. B, EMSAs with 32P-labeled DV101S1 promoter region fragment 99 and Molt-13 nuclear extract (lanes 2–8); binding reactions in lanes 1 and 9–14 contained no extract. Nuclear extract was preincubated with 100, 200, or 400 ng 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 arrow indicates the specific complex 4 previously seen with the Molt-13/fragment 99 binding reaction.

The CRE, Ets site, 5' TATA box, and nucleotides in the region -8 to +33 are essential for maximal DV101S1 promoter function in the presence of the TCRD Enh

The functional significance of cis-elements in the DV101S1 promoter identified by either computer search, oligonucleotide inhibition of gel retarded species, or DNase I protection studies was assessed by site-directed mutagenesis. Fig. 7A shows the mutations introduced into elements in the reporter construct containing -86 to +368 of the DV101S1 promoter and the core TCRD Enh. The effects of the mutations on CAT activity and on protein binding are shown in Figs. 7B and 8, respectively. Transient transfection experiments of mutated constructs into Molt-13 cells showed that mutation of the CRE element (Mut. 9A) reduced CAT activity to 40% of the level obtained with the wild-type sequence. Mutation of the Ets site and 8 upstream nucleotides, including 1 nt within the E box element (Mut. 1) also decreased CAT activity, but only to 83% of the level obtained with the wild-type construct. Results with the E box mutation (Mut. 11A) demonstrated that this element is not important for the functionality of the promoter. Thus, the CRE binding site is an essential upstream DV101S1 promoter element and the Ets binding site is necessary for full transcriptional activity. Replacing 5 nt at positions -27 to -23 (Mut. 7) reduced CAT activity by more than 90%, confirming the importance of all or some of these nucleotides in TCRD gene transcription. Nucleotides -27 to -23 fall within the 5' putative TATA box, and the replacement nucleotides reduce the similarity to the TATA consensus from 70.4% to 37.9%. Thus, this TATA box may be important for DV101S1 promoter activity, but nt -27 and -26 also fall within an AP1 element; without an independent mutation it is unclear which element(s) the -27 to -23 replacement mutation is affecting. However, under the conditions of our binding assay, no AP1-inhibitable nuclear factors bound to the DV101S1 minimal promoter. Thus, the likely element affected by mutation 7 is the TATA box and the inability of the putative 3' TATA at -98 bp, relative to the translation initiation site, to compensate indicates that the 3' TATA-like sequence does not direct transcription initiation from DV101S1 start site numbers 4 to 6. Nucleotides -16 and -17 (Mut. 15) were replaced to test the importance of the DV101S1 viral Enh core site. These particular base substitutions have previously been used to demonstrate the importance of a viral Enh core site for activity of the TCRG Enh (77) and in the DV101S1 promoter; they do not significantly alter the overlapping 5' TATA box (similarity to the consensus was lowered by <1%). Results with mutation 15 demonstrated that the viral Enh core is not important for DV101S1 promoter activity. In contrast, nucleotides within the region -8 to +11 are critical for DV101S1 gene expression since substituting 17 of these 19 nt surrounding the major transcription initiation site (Mut. 2) reduced CAT activity by >70%. Mutation 2 disrupts six potential Inr sequences and the GATA consensus sequence and to independently evaluate the importance of the GATA site, nt -4 and -3 were substituted within the DV101S1 minimal promoter (Mut. 14). The second and third nucleotides of the GATA consensus site were altered since changing these nucleotides within other TCR gene GATA sites revealed the functional significance of these elements (72, 73). Further, the replacement nucleotides only serve to increase the similarity of the two overlapping Inr sequences to the consensus Inr sequence (1.2%). Mutation 14 did not alter DV101S1 promoter activity, implying that this GATA site is not necessary for DV101S1 promoter function. Finally, nucleotides within the region +12 to +33, downstream of the major transcription start site (Mut. 3), are essential for DV101S1 gene expression in vivo as substitution of these 22 bases reduced CAT activity by 89%. Nucleotides within the 5' untranslated region could affect TCRD transcriptional activity, but the lower CAT activity levels could also reflect a reduction in the transfected cells of translation efficiency and/or mRNA stability.

View larger version (30K): [in this window] [in a new window]   FIGURE 7. Mutational analysis identifies five cis-regulatory elements important for DV101S1 promoter activity. A, Schematic of the -86 to +368 DV101S1 promoter region with computer identified transcription factor consensus recognition sequences boxed, potential TATA boxes underlined, transcriptional start sites indicated, and introduced element mutations listed below the wild-type sequence. Base changes are noted, and a (-) indicates sequence identity. Mutations were introduced into cis-regulatory elements individually in the reporter gene construct containing -86 to +368 of the DV101S1 promoter. B, Promoter activity of the mutated reporter gene constructs assessed by transient transfection into Molt-13 cells. Transfection efficiencies were controlled by transfecting pRSVLuciferase along with the test plasmid. The promoterless plasmid, pGEM7ZCATTCRDEnh(+), served as a negative control and pRSVmCAT as a positive control in each experiment. Within each experiment, extract volumes were normalized for luciferase activity before analysis of CAT activity. The percent acetylation with the negative control was then subtracted from measurements of test constructs before their CAT activity was evaluated relative to the CAT activity of the wild-type -86 to +368 construct. Reported are the percentages of the wild-type -86 to +368 construct’s CAT 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(+) produced 0.2, 0.6, 0.2, 0.3, 0.2, 1.1, 1.1, 1.3, 1.3, and 1.3% acetylation, and the -86 to +368 wild-type construct produced 9.0, 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.

To ensure that the lack of an effect of a mutation in a protein binding site on DV101S1 promoter activity is due to the unimportance of that element rather than to the failure of that mutation to disrupt protein binding, EMSAs were performed with Molt-13 nuclear extracts and ³²P-labeled DV101S1 promoter region fragments containing individual mutations (Fig. 8). Mutation 11A (E box), mutation 14 (GATA), and mutation 15 (viral Enh core) were the only mutations that failed to affect DV101S1 promoter activity. The E box mutation did disrupt formation of migration retarded species 1, 3, and 4 normally observed with Molt-13 nuclear extract and fragment 58 (Fig. 8A, lane 6). Migration retarded species 2, 3, and 4 are the Molt-13/fragment 58 nucleoprotein complexes inhibitable with a ds E box containing oligonucleotide (Fig. 4B). Likewise, the GATA mutation disrupted the Molt-13 nuclear extract/fragment 99 complex that is inhibitable with a ds GATA containing oligonucleotide, species 4 (Fig. 8C, lane 9). Thus, our previous conclusion that the E box and the GATA site are unimportant for DV101S1 gene expression in Molt-13 cells is reasonable. However, it is not possible to definitively rule out the functional significance of the viral Enh core in DV101S1 promoter activity as the binding of nuclear factors to either the wild-type or mutated DV101S1 viral Enh core was not examined in this study.

View larger version (68K): [in this window] [in a new window]   FIGURE 8. Mutating cis-regulatory elements within the DV101S1 promoter does not always alter Molt-13 nuclear protein binding. EMSAs performed with Molt-13 nuclear extract and 32P-labeled wild-type or mutated DV101S1 promoter region fragments. A, Wild-type and mutated fragment 58 as probes. B, Wild-type and mutated 92-bp HincII-HinfI fragment (-86 to +4) as probes. The 92-bp HincII-HinfI fragment (see Fig. 1) had to be used since mutation 1 destroys the MaeIII restriction site. C, Wild-type and mutated fragment 99 as probes. The presence (+) or absence (-) of Molt-13 nuclear extract and the radiolabeled fragments used in the binding reactions are indicated above the lanes. Arrows and numbers, to the right in each panel, indicate the specific migration-retarded species observed with the wild-type fragment.

The last two mutations that significantly reduced promoter activity, mutations 7 (TATA box/AP1) and 9A (CRE), unexpectedly did not alter binding of nuclear proteins to DV101S1 promoter fragments (Fig. 8C, lane 7, and Fig. 8A, lane 5, respectively). This is actually reasonable in the case of the TATA box/AP1 mutation because no AP1 inhibitable migration retarded species were observed with wild-type fragment 99, and it has been impossible to detect TFIID by gel retardation with crude extracts (78). However, the results with the CRE mutation (Mut. 9A) are more difficult to explain and may imply that complex interactions occur between factors that bind to the multiple regulatory elements in the DV101S1 promoter.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study has defined the 5' DV101S1 cis-sequences necessary and sufficient for D Enh dependent basal promoter activity to investigate the regulation of ordered TCRD V rearrangements during ontogeny. Several findings are inconsistent with a previous characterization of this promoter (79). First, although we confirmed the presence of multiple DV101S1 transcription start sites, the major DV101S1 initiation site was mapped 13 bp further downstream than where it was mapped previously (79). This leads to a TATA box being properly positioned upstream from the major transcription start site and the importance of this element in DV101S1 gene expression was confirmed by mutational analyses. Therefore, the DV101S1 promoter is properly identified as a TATA-box-containing rather than a TATA-box-deficient promoter (79). Second, with regard to regulatory elements located upstream of the DV101S1 start site, we also identified the CRE and Ets sites as functionally significant promoter elements. However, we did not find that the Ets site is critical for DV101S1 promoter activity. Mutation of the Ets site reduced DV101S1 promoter activity by 17% in the human T cell line, Molt-13. In contrast, Punturieri et al. (79) reported that mutation of the Ets site completely abolished DV101S1 promoter activity in the murine DV101S1⁺ dendritic epidermal T cell hybridoma, T245/BW. Furthermore, we failed to demonstrate any functional significance of the -52 E box, mutation of which, Punturieri et al. (79) showed, reduced DV101S1 promoter activity by 40% in T245/BW cells. The reasons for these discrepancies remain unclear, but may be related to the different cells used for transfection analyses (see below).

We observed that the 5' untranslated leader region and the nucleotides around the major transcription start site are involved in DV101S1 gene expression. The importance of both of these regions was not noted previously (79), and their identification here was allowed by the nature of our mutations. Transient transfection analyses of 3' deletion mutants initially revealed the necessity of elements within the 66 nt immediately downstream of the major RNA start site. Analyses of mutations replacing the sequences -8 to +11 (Mut. 2) and +12 to +33 (Mut. 3) then confirmed the importance of nucleotides within the first 33 bases downstream of the major CAP site for in vivo DV101S1 gene expression. The functional significance of the +34 to +66 region was not directly addressed by our mutational studies so we are unsure of the contribution of elements within this region (e.g., Sp1) to DV101S1 gene expression. However, all of the reporter constructs used by Punturieri et al. (79) included only 37 nt of downstream DV101S1 sequence, relative to our major transcription start site, so presumably, this region is unimportant for promoter activity. Several other eukaryotic genes have been found to have regulatory elements within their 5' untranslated leader regions including RAG-1 (80). Interestingly, the +12 to +25 region from the DV101S1 promoter is highly homologous to the +11 to +25 region from the RAG-1 promoter (80). Thirteen of the DV101S1 nucleotides match the RAG-1 nucleotides, if a 1 base gap is introduced. Regulatory elements within the 5' untranslated leader region could operate at either the DNA or RNA level, and affect either transcription (81, 82, 83) or posttranscriptional processes (84, 85, 86). DNase I protection analyses showed that nuclear factors bind to the DV101S1 downstream sequence. Protected regions were observed at +1 to +10 and +17 to +21. These binding motifs overlap potential Inr sequences, but no obvious similarities with other known consensus sequences were found. Mutational analyses demonstrated that binding of factor(s) to the +1 to +10 and +17 to +21 regions was integral for in vivo DV101S1 gene expression. This observation supports the notion that the DV101S1 downstream sequence influences transcriptional efficiency, but additional studies examining steady-state mRNA levels and mRNA stability are required to thoroughly address this issue.

The mutant substituting the wild-type DV101S1 -8 to +11 sequence revealed the critical nature of nucleotides near the major transcription start site for in vivo DV101S1 gene expression. Reporter gene activity was reduced by 70% suggesting that the start site region either acts as an Inr (58, 87) or contains a binding site for an essential activator (88). Inrs are basal control elements that overlap transcription start sites and serve to direct RNA polymerase II to a precise transcription start site in TATA-less promoters (89), and to increase promoter strength in TATA-containing promoters, if they are located 25 bp downstream of the TATA box (89, 90, 91). Both general transcription factors (92, 93, 94) and specific activator proteins (Ref. 95, reviewed in 58 have been shown to recognize Inrs, but their mechanism of action remains unclear. Consistent with the idea that the DV101S1 start site possesses functional Inr activity is the observation that the start site mutation disrupts six potential Inr sequences, identified using the two available loose consensus sequences (56, 57). However, before it can be concluded that an Inr element functions as a basic regulator for TCRDV101S1 promoter activity, it needs to be shown that the DV101S1 start site functions as a transcriptional positioning element.

Currently, only the DV101S1 and AV11S1 gene promoters have been characterized from the TCR A/D locus (this paper; Refs. 79 and 96). Upstream sequence information is available, however, for another TCRD V gene segment (DV105S1; 97 , and three TCRA Vgene segments (AV1S3, AV12S1, and AV3S1; Refs. 96, 98, and 99) allowing a comparison of the trans-acting factor binding motifs within these promoter regions. Analysis of the AV11S1 promoter identified a single element, the GT box, critical for AV11S1 promoter activity (96), and this element is shared by two of the three other TCRA V segment promoters (AV1S3 and AV12S1), but not by either of the two TCRD V segment promoters. Thus, the GT box may be a conserved element in TCRA V promoters similar to the conserved decanucleotide found in 13 of 14 characterized murine TCRB V promoters (100) and the conserved octanucleotide, and its inversion, found in all Ig V_H and V_L promoters respectively (101). Neither the CRE or Ets site are conserved TCRD V promoter elements as they are present 5' of AV1S3 and AV11S1, and absent 5' of DV105S1. The viral Enh core sequence is uniquely shared by the two TCRD V upstream sequences, but preliminary studies reported here suggest that this element may be unimportant for DV101S1 promoter activity. Thus, all of the TCRD V promoter elements may be heterogeneous.

An interesting observation from this study is that not all of the protein-binding sites within the murine DV101S1 minimal promoter are important for Enh-driven TCRD gene transcription in Molt-13 cells. EMSA analyses showed that the GATA consensus at -5 is recognized by recombinant GATA-3 as well as GATA-3 in Molt-13 nuclear extracts. However, a 2-base substitution in the GATA site that abolished GATA-3 binding had no effect on DV101S1 promoter activity. Likewise, oligonucleotide competition experiments demonstrated that ubiquitously expressed nuclear proteins bind the DV101S1 -52 E box, but a complete substitution of the E box abrogating protein binding did not alter DV101S1 promoter activity. It is conceivable that the GATA and E box sites function at a stage in T cell development different from that represented by Molt-13. For example, Molt-13 is well beyond the predicted stage for Enh-independent DV101S1 germline transcription. On the other hand, GATA-3 and E box binding proteins may be exclusively involved in the developmentally regulated selection of DV101S1 for rearrangement in early mouse ontogeny since cis-elements of the DV101S1 promoter that are essential for targeting gene rearrangement may not be the same as those essential for transcriptional activation (24, 25). The possibility that the GATA site is required for the DV101S1 promoter to mediate V(D)J recombinational accessibility is particularly intriguing given that a GATA motif is only present upstream of the DV101S1 segment out of the two TCRD and four TCRA V segment promoter regions compared (see above), and GATA-3 is transcribed as early as day 13/14 of gestation (42, 102), coincident with the onset of TCRDV101S1 rearrangements (11). Regardless, our original results showing the lack of an effect of certain mutations on DV101S1 promoter activity in Molt-13 cells should be confirmed with a murine DV101S1⁺ cell line.

	Acknowledgments

 
We thank Drs. Y. H. Chien, J. Hassell, and R. Sen for phage, cosmid, and plasmid DNA; Drs. R. Baer, W. Born, A. Clausell, J. Forman, and D. Yuan for cell lines; Dr. K. Meek for providing laboratory space to complete a portion of the project; and Dr. K. Arizumii for critically reading the manuscript.

	Footnotes

 
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 the National 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 for Cellular and Molecular Biology, The University of Texas at Austin, Experimental Science Building, Room 534A, 24th and Speedway, Austin, TX 78712-1095. E-mail address:

⁴ Abbreviations used in this paper: Enh, enhancer; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; Inr, initiator; m, murine; CRE, cAMP responsive element; nt, nucleotide.

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

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