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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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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 VH gene promoter (53).
All oligonucleotides were made on an Applied Biosystems (Foster City, CA) model 380A oligonucleotide synthesizer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To initiate analysis of the TCRDV101S1 promoter, 1234
upstream bp were sequenced beginning from the 53rd residue of the
DV101S1 gene segment (Fig. 1
A). 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.
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).
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 (AUGCAT)
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,
AUGVd1, 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).
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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).
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D demonstrates that nuclear extract from Molt-13
cells also contains factor(s) which specifically bind to fragment 99.
Four of the migration retarded species observed with Molt-13 nuclear
extract and fragment 99 (Fig. 3D, lane 8)
could be specifically inhibited by unlabeled fragment 99 (Fig. 3D, lanes 2–7), whereas the irrelevant
fragment (Fig. 3D, lanes 9–14) did not
inhibit formation of these species.
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).
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C, lanes 2–7). Because
the GATA-globin oligo is identical to fragment 99 at 6 bp immediately
3' of the GATA site, a duplex oligonucleotide containing only the
DV101S1 GATA site (GATA-V1 oligo) and a duplex
oligonucleotide containing the 10-bp region immediately 3' of the GATA
site (+1 to +10; Start site oligo), were used as competitors in EMSAs.
Interference of formation of species 4 was again seen with the
GATA-V1 oligo, but all migration retarded species were observed with
the Start site oligonucleotide (data not shown), confirming that a GATA
binding factor interacts with fragment 99. An oligonucleotide
containing an AP1 site did not interfere with the formation of any of
the four migration-retarded species (data not shown). Also, two
irrelevant duplex oligonucleotides, one containing a potential TCF-1
site (5'-YCTYTKWW-3'; 63 from the DV101S1 intronic
region +191 to +201 (Fig. 4C, lanes 9–14)
and the other an Ets site (data not shown), did not interfere with the
formation of any of the 4 migration retarded species.
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).
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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).
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B).
Species 4 was ablated with anti-GATA-3 mAb (Fig. 6B,
lanes 3–5) but not with the control Ab (Fig. 6B, lanes 6–8), establishing that GATA-3
specifically binds to the GATA site at -5 within the
TCRDV101S1 promoter. 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.
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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.
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C, lane
8) normally observed with wild-type fragment 99 and Molt-13
nuclear extract (Fig. 8C, lane 6).
Mutation 3 (the +12 to +33 region) also partially disrupted formation
of migration retarded species 1 (Fig. 8C, lane
10). The Ets site mutation (Mut. 1) was examined using the
93-bp HincII-HinfI fragment (see Fig. 1) since
the mutation destroys the MaeIII restriction site. This
fragment routinely yields five specific migration retarded species
(data not shown, arrows in Fig. 8B, lane
3), but when the Ets site was mutated only retarded species
5 was generated (Fig. 8B, lane 4).
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.
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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 VH and VL 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.
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Acknowledgments |
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Footnotes |
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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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