{'fraction_non_alphanumeric': 0.026596333321037294, 'fraction_numerical': 0.01671031760669888, 'mean_word_length': 5.06216457960644, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor.

T cell-specific expression of the human T cell receptor alpha (TCR- alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR- alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP response element (CRE), and binds a set of ubiquitous nuclear proteins. The T alpha 2 binding site does not contain known transcriptional enhancer motifs. However, it binds at least two nuclear protein complexes, one of which is T cell specific. We now report that although the T alpha 2 nuclear protein binding site displays transcriptional activator activity in the context of the TCR-alpha enhancer, this site alone can function as a potent, T cell-specific transcriptional repressor when positioned either upstream, or downstream of several heterologous promoter and enhancer elements. These results demonstrate that a single nuclear protein binding site can function as a T cell-specific transcriptional activator or repressor element, depending upon the context in which it is located.

T cell-specific expression of the human T cell receptor a (TCRa) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCRa constant region (Cat) gene segment. The minimal TCRa enhancer is composed of two nuclear protein binding sites, Toil and Ta2, that are both required for the T cell-specific activity of the enhancer. The Tat binding site contains a consensus cAMP response element (CRE), and binds a set of ubiquitous nuclear proteins. The Ta2 binding site does not contain known transcriptional enhancer motifs. However, it binds at least two nuclear protein complexes, one ofwhich is T cell specific. We now report that although the Ta2 nuclear protein binding site displays transcriptional activator activity in the context of the TCR-a enhancer, this site alone can function as a potent, T cell-specific transcriptional repressor when positioned either upstream, or downstream of several heterologous promoter and enhancer elements . These results demonstrate that a single nuclear protein binding site can function as a T cell-specific transcriptional activator or repressor element, depending upon the context in which it is located.
T he process ofeukaryotic development involves the tissuespecific and temporally regulated expression of specific subsets of genes. The molecular mechanisms that positively and negatively regulate gene expression during mammalian development are complex, and involve the interaction ofubiquitous, and tissue-specific nuclear proteins with cis-acting transcriptional regulatory sequences known as promoter, enhancer, and silencer elements (1-3) . Mammalian T lymphocyte development in the thymus is an excellent model system for studies designed to better elucidate the molecular mechanisms that control developmentally regulated gene expression. Two subsets of mammalian T cells have been identified that are distinguished by their cell surface expression of heterodimeric antigen receptor molecules (4, 5). All cells of the helper and cytotoxic phenotype express the TCRo/o molecule, whereas a second set of T cells, of unknown function, express the related, but distinct TCR-,y/& These two T cell subsets appear to belong to separate developmental lineages, in that the TCR-a gene is rearranged and expressed only in TCRa/(3+ cells, whereas the TCR 6 gene is rearranged and expressed exclusively in TCR-,y/6' cells (5) .
To better understand the molecular mechanisms that regulate the development of these two T cell subsets, we have attempted to delineate the cis-acting sequences and trans-acting 1443 factors that control the rearrangement and expression of the TCR genes . Previous studies have demonstrated that human and murine TCR a gene expression is regulated by a transcriptional enhancer located 3-4 .5 kb 3' of the single TCR a Ca gene segment (6, 7). This enhancer is required for high level transcription from a TCR Va promoter, and is active only in TCR-a/0+ T cells (7) . Deletion and in vitro mutagenesis analyses, along with DNasel footprint experiments, demonstrated that the minimal TCR-a enhancer is composed oftwo nuclear protein binding sites, Toil and Ta2, that must be separated by more than 15, and less than 85 nucleotides (7,8). The Tal binding site contains a consensus cAMP response element (CRE),' and has been shown to bind a set of ubiquitously expressed CRE binding proteins (CREBs) . In contrast, the Ta2 binding site does not contain previously identified transcriptional enhancer motifs . However, it binds at least two nuclear protein complexes, one of which is T cell specific (8) . In the studies described in this paper, we demonstrate that the Ta2 nuclear protein binding site can function as both a T cell-specific transcriptional activator and repressor, depending upon the context in which it is located.
Ta2 functions as a transcriptional activator domain in the context ofthe intact TCR a enhancer. However, this binding site, alone functions as a T cell-specific transcriptional repressor when positioned either upstream or downstream of a variety of heterologous promoter and enhancer elements. To our knowledge, this is the first report of a single mammalian nuclear protein binding site that can function as a cell lineagespecific transcriptional activator and repressor, and, as such, suggests a novel mechanism for positively and negatively regulating mammalian gene expression from a single transcriptional enhancer element that is composed of multiple nuclear protein binding sites . Plasmids. The pSPCAT plasmid containing the minimal SV40 promoter immediately 5' of the bacterial chloramphenicol acetyl transferase (CAT) gene, and the pSPI1CAT plasmid which is pSPCAT that contains the 4F2 heavy chain (4F2HC) enhancer 5' of the minimal SV40 promoter have been described previously (9,10) as have the pRSVCAT (11) and p4F2-589CAT (12) plasmids in which CAT transcription is under the control of the Rous sarcoma virus long terminal repeat (LTR) and 4F2HC promoters, respectively. The pSPcwnhCAT plasmid which is pSPCAT that contains the 116-bp BstXI/Dral human TCR-a enhancer fragment 3' of the CAT gene has also been described previously (7). The promoterless pCATBasic plasmid was obtained from Promega-Biotec (Madison, WI) .

Materials and Methods
Synthetic Oligonucleotides and In Vitro Mutagenesis. Complementary synthetic oligonucleotides corresponding to the wild-type  (7). The nucleotide sequences of the previously described (7) Toil and Ta2 nuclear protein binding sites are shown above and below the map, respectively. The CRE within the Tal binding site is boxed and labeled . The sequence of a mutant Ta2 oligonucleotide (mTa2) that fails to bind nuclear proteins (8) is shown in the bottom line of the panel. Synthetic Ta2 and mTa2 oligonucleotides corresponding to the sequences shown were synthesized with BamHI and Bg111 overhanging ends, and used in the functional studies described in C and D below. (B) The effects of mutations of the Ta2 binding site on the activity of the TCR-a enhancer. The wild-type TCR-a enhancer or a mutant enhancer (M) containing 11 nucleotide substitutions in the TO binding site (mTa2, A above) were cloned into the BamHI site 3' of the minimal SV40 promotor/CAT cassette in the pSPCAT vector (9) and 10-,Ug of each of the resulting plasmids along with 2 Ag of the pRSV#gal reference plasmid were transfected into human Jurkat T cells. The data are displayed as CAT activities relative to that produced by the plasmid containing the wild-type TCR-a enhancer that resulted in 34% acetylation. (C) The effects of the Ta2 nuclear protein binding site on transcription from the minimal SV40 promoter and 4F2HC enhancer. Two copies of synthetic oligonucleotides corresponding to the wild-type (Ta2) or mutant (mTa2) Ta2 binding sites (see A above) or three copies of the T05 nuclear protein binding site of the human TCR-O enhancer (TAGAACTTCAGAGGGGAGGG) were cloned into the BamH1 site 3' of the minimal SV40 promoter and the 500 by 4F2HC enhancer (4F2 Enh) in the pSPI1CAT plasmid (10). 10 ug of each of the resulting plasmids along with 2 leg of the pRSV#gal plasmid was transfected into Jurkat T cells and CAT, and S-galactosidase activities were determined as described in Materials and Methods. The data are displayed as CAT activities relative to that produced by the pSP11CAT plasmid that produced 3% acetylation. (D) The effects of position on the transcriptional repressor function of the Ta2 nuclear protein binding site. Four copies of the synthetic TO oligonucleotide (see A) were cloned into the BamHI site 3' of the 4F2HC enhancer and minimal SV40 promoter/CAT gene or the Sacl site 5' of the 4F2HC enhancer and SV40 promoter/CAT gene in pSP11CAT, and 10 ug of the resulting plasmids along with 2 Rg of the pRSVfgal plasmid were transfected into Jurkat cells. The data are shown as CAT activities relative to those produced by the pSPIlCAT control plasmid that produced 7% acetylation.

1444
Nuclear Protein Binding Site Functions as Transcriptional Activator or Repressor Tal and Ta2 nuclear protein binding sites, and a mutant Ta2 nuclear protein binding site (rnTce2, see Fig. 1 A) were synthesized with BamHI/BgIII overhanging ends on a DNA synthesizer (model 380B; Applied Biosystems, Foster City, CA), and annealed before cloning into the pSPCAT, pSPI1CAT, p4F2CAT, and pRSVCAT plasmids. Thecopy number cloned into each plasmid was confirmed by dideoxy DNA sequence analysis. A minimal human TCR-at enhancer containing 11 nucleotide substitutions within the Tact nuclear protein binding site (see Fig. 1 A) was produced by oligonucleotide-mediated gapped heteroduplex mutagenesis as described previously (10) and verified by dideoxy sequence analysis. Transfections and CAT Assays. Jurkat, K562, and clone 13 cells were transfected using a modification ofthe DEAF-dextran method as described previously (7). Peer cells were transfected by electroporation (7) . To control for differences in transfection efficiencies, all transfections contained 2 ttg of the pRSVOgal reference plasmid. Cells were harvested 48 h after transfection, and cell extracts normalized for protein content using a commercially available kit (Biorad, Richmond, CA) were assayed for both 0-galactosidase and CAT activities as described previously (7).

Results
The Ta2 Nuclear Protein Binding Site Can Function as a Transcriptional Activator and Repressor. To determine whether the Toil and Ta2 binding sites by themselves were necessary, or sufficient for TCR-a enhancer activity, two types of experiments were performed. First, the Tat and Ta2 binding sites were mutated so as to abolish nuclear protein binding as assayed by electrophoretic mobility shift analyses. Mutation or deletion of either the Toil (8) or Ta2 (reference 8, and Fig. 1 B) binding sites essentially abolished enhancer activity, indicating that both the Tat and Ta2 sites function as transcriptional activator elements in the context of the TCR a enhancer. In a reciprocal set ofexperiments, synthetic oligonucleotides corresponding to the Toil and Ta2 binding sites were cloned into the BamHI site of the pSPCAT reporter plasmid (9), 3' of the minimal SV40 promoter and the bacterial CAT gene, and transfected into Jurkat TCR a/a+ T cells. In the absence of stimulation ofprotein kinase A, two copies of the Tat oligonucleotide had no significant effect on CAT transcription (data not shown) . Surprisingly, however, two copies of'the Ta2 oligonucleotide appeared to completely suppress CAT transcription (data not shown), although the magnitude of this repression was difficult to quantitate because of the low basal levels of transcription from the minimal SV40 promoter in this plasmid . To better assess the negative regulatory activity of the Ta2 element, two copies of the synthetic Ta2 oligonucleotide were cloned into the BamHI site ofthe pSPHCAT vector (10) that contains the previously described 4F2 heavy chain (4F2HC) enhancer 5' of the minimal SV40 promoter/CAT cassette (Fig. 1 C). This plasmid was chosen as a reporter for Ta2 transcriptional repressor activity because we have demonstrated previously that the 4F2HC enhancer is highly active in a wide variety of lymphoid and non-lymphoid human tumor cell lines (10). As shown in Fig. 1 C, two copies ofthe Ta2 binding site resulted in an 84% reduction in CAT activity in Jurkat cells as compared with the pSPI1CAT control plasmid . This effect was 1445 Ho and Leiden specific for the Ta2 oligonucleotide because it was not observed when two copies of a mutant Ta2 (mTa2) oligonucleotide ( Fig. 1 A) that fails to bind T cell-specific nuclear proteins (8), or three copies ofthe T05 oligonucleotide that corresponds to a nuclear protein binding site from the human TCR-O transcriptional enhancer (13) were cloned into the BarnHI site of pSPI1CAT (Fig. 1 C).
Ta2 Transcriptional Repressor Activity Is Position Independent. We have demonstrated previously (7) that the transcriptional activator properties of the TCR a enhancer are independent ofthe position and orientation of the enhancer element . To determine whether the transcriptional repressor activity of the Ta2 binding site was similarly position independent, we compared the activity of four copies of Ta2 cloned 5' of the 4F2HC enhancer and minimal SV40 promoter/CAT cassette to the activity of the same sites cloned 3' of the CAT gene in this vector (Fig. 1 D). The ability of Ta2 to repress transcription was slightly reduced when the Ta2 sites were cloned 5' of the promoter and enhancer as compared with 3' of the CAT gene (66% reduction vs. 92% reduction). Nevertheless, the transcriptional repressor effect was relatively independent of the position of the Ta2 binding sites .
Ta2 Represses Transcription from Multiple Heterologous Promoters and Enhances. Previous studies (7) have demonstrated that the TCRa enhancer is able to confer T cell-specific transcriptional enhancement upon both a Va promoter and several heterologous promoters. To determine whether the Ta2 element alone was capable of repressing transcription from multiple distinct promoter and enhancer elements in Jurkat cells, four copies of a synthetic Ta2 oligonucleotide were cloned 3' ofCAT reporter genes that were under the control of the Rous sarcoma virus (RSV) promoter/enhancer, or the 4F2 heavy chain (4F2HC) promoter, a G + C rich "housekeeping" promoter that lacks a TATA box and initiator sequence (14), but contains multiple SP1 binding sites (15). These plasmids were transfected into human Jurkat T cells, and the resulting CAT activities were compared with those produced by control plasmids lacking the Ta2 binding sites (Fig. 2). Four copies of Ta2 significantly reduced transcription from each of these promoter/enhancer combinations (>90% reduction), demonstrating that the transcriptional repressor activity of Ta2 was not unique to the SV40 promoter or to the 4F2HC enhancer, and that this activity was not restricted to TATAcontaining promoters . In additional experiments, four copies of the Ta2 binding site also inhibited transcription from a minimal SV40 promoter/TCR a enhancer by >98% (data not shown) .

The TranscriptionalRepressorActivity of T62 Is T Cell-specific
The TCRa enhancer is active only in TCR a/a+ T cells (7), and previous studies have demonstrated that the Ta2 enhancer motifbinds at least two nuclear protein complexes, one of which is present only in TCR-a/(3+ and TCR-y/S+ T cells (8) . To determine whether the transcriptional repressor activity of the Ta2 binding site was restricted to a specific cell lineage, plasmids containing four copies of the Ta2 binding site cloned 3' ofthe 4F2HC enhancer and SV40 promoter/CAT cassette were transfected into Jurkat TCR a/a+ Figure 2 . The Ta2 element can repress transcription from multiple heterologous promoter and enhancer elements . Four copies of a synthetic Tat oligonucleotide (see Fig . 1 A) were cloned into the BamHI site 3' of the 4F2HC enhancer and minimal SV40 promoter/CAT cassette in PSPIICAT (10) (A), the RSV LTR/CAT cassette in pRSVCAT (11) (B), or the 589-bp 4F2HC promoter (4F2 Pr)/CAT cassette in p4F2-589CAT (12) (C) . 10 fcg of each of the resulting plasmids along with 2 ug of the pRSV,6gal reference plasmid were transfected into Jurkat T cells, and CAT and fl-galactosidase activities were determined as described in Materials and Methods. The data are displayed as CAT activities relative to those produced in transfections with the appropriate control plasmids lacking the Ta2 elements . Figure 3 . The Ta2 element functions as a T cell-specific transcriptional repressor. 10 Etg of the pSPCAT, pSP11CAT, and pSPIICAT(Ta2)4 plasmids shown schematically in the top panel along with 2 j+g of the pRSVsgal reference plasmid were transfected into Jurkat TCR-a/g' T cells, Peer-,y/6' T cells, clone 13 EBVtransformed B cells, and K562 chronic myelogenous leukemia cells, and cell extracts were assayed for both CAT and ,B-galactosidase activities as described in Materials and Methods . The data are displayed as CAT activities relative,to those produced by the pSPHCAT plasmid that were 7, 8, 3, and 6% total acetylation, respectively, in the Jurkat, Peer, clone 13, and K562 cell lines.
T cells, Peer TCR-,y/6' T cells, clone 13 EBV transformed B cells, and K562 chronic myelogenous leukemia cells, and their activities were compared with those produced by the same plasmids lacking the Ta2 binding sites (Fig . 3) . Significant repression of transcription (92 and 98% reductions, respectively) was observed in the Jurkat and Peer T cells. In contrast, no repression was observed in either the K562 or clone 13 cells. A similar pattern of T cell-specific repression was observed after transfection of a plasmid containing four copies of the Ta2 binding site cloned 3' of an RSV promoter/enhancer/CAT cassette (data not shown) . Taken together, these results demonstrated that the transcriptional repressor activity of the Ta2 element is T cell specific, and suggested that this activity may be mediated by the ability of this element to bind one or more T cell-specific nuclear proteins.
The Transcriptional Repressor Activity of Ta2 is a cis-acting Eject and Is Proportional to the Number ofCopies of the Ta2 Binding Site. The transcriptional activator function of many enhancer elements is directly proportional to the number of copies of these elements that are present in a given reporter plasmid . To determine whether the transcriptional repressor activity of the Ta2 element was similarly proportional to its copy number, one to four copies of the synthetic Ta2 oligonucleotide were cloned into the BamHI site 3' of the RSV promoter/enhancer/CAT cassette, and these plasmids were transfected into Jurkat T cells (Fig. 4). These experi- 1447 Ho and Leiden meats demonstrated that the transcriptional repressor activity of the Ta2 element was directly proportional to the number of copies of Ta2 with 94% repression of transcription observed with four copies of Ta2. One mechanism that could account for the transcriptional repressor activity of the Ta2 element is that this element is able to compete efficiently for the binding of generalized transcription factors, thereby causing an overall decrease in transcription in the transfected cells. To test this possibility, all transfections contained 2 pg of the pRSV#gal reference plasmid in which (3-galactosidase gene expression is under the control of the RSV promoter/enhancer. As shown in Fig.  4, 0-galactosidase activity was essentially identical in all of the transfections, regardless of the number of copies of Ta2 contained within the CAT reporter plasmid . Similar results were obtained in all of the transfections shown in Figs . 1-3 (data not shown) . Thus, the transcriptional repressor activity of Ta2 is a cis-acting effect, and is not simply the result of generalized decreases in transcriptional activity in cells transfected with Ta2-containing plasmids .
A second mechanism that could account for the apparent transcriptional repressor activity of the Ta2 binding site is that this enhancer element, when polymerized, can also function as a promoter, thereby artifactually reducing CAT transcription in our transient transfection assays by a mechanism of promoter interference. To rule out this possibility, four copies of the Ta2 binding site were cloned in both orienta- Figure 4. The effect of copy number on the transcriptional repressor activity of the Ta2 element. One to four copies of the synthetic Ta2 oligonucleotide were cloned into the BamHl site of pRSVCAT (see Fig. 2 B) 3' of the RSV LTR/CAT cassette and 10 Ag of the resulting plasmids, along with 2 Eag of the pRSV/Igal reference plasmid, were transfected into Jurkat T cells. CAT (solid ban) and 0-galactosidase (slashed bars) activities were determined as described in Materials and Methods . The CAT activities were normalized to that produced by the pRSVCAT control plasmid that produced 27% total acetylation .
tions immediately 5' of a promoterless CAT gene in the pCAT-Basic plasmid, and the resulting plasmids were transfected into Jurkat T cells (Fig.5) . Four copies of the Ta2 binding site had no effect on CAT activity as compared with the promoterless pCATBasic control plasmid . Thus, the element does not display significant promoter activity in this system .

Discussion
The studies described in this report have demonstrated that a single nuclear protein binding site from the human TCR-a enhancer can function as both a T cell-specific transcriptional activator and repressor, depending upon the context in which this element is located . The finding that Ta2-mediated transcriptional repressor activity is T cell specific, when taken together with previous observations (8) that the Ta2 element binds at least one T cell-specific nuclear protein complex, suggests that nuclear protein binding may be responsible for the Tc12 transcriptional repressor activity. This hypothesis is supported by the finding that Tc12 mutations that abolished nuclear protein binding also abolished transcriptional repressor activity. The fact that the Ta2 element is able to repress transcription from multiple, apparently unrelated, enhancer elements suggests that repression may be mediated by direct effects of Ta2 and its cognate nuclear proteins on the ability of the transcription complex to form at the promoter, or to initiate transcription once formed . These effects apparently do not require either a functional TATA box, or an initiator sequence in that Tc12 significantly repressed transcription from the 4F2HC promoter that lacks these elements.
Previous studies in other systems have demonstrated that a single nuclear protein can either activate or repress transcription by binding to distinct cis-acting regulatory elements (16,17) . In addition, one previous report (18) has demonstrated that two different sequence motifs that are involved in silencing transcription of the yeast silent mating type loci can activate transcription when linked to heterologous promoters. To our knowledge, however, this is the first report of a single cis-acting element that can both activate and repress transcription in a tissue-specific fashion in a mammalian system . Our results raise two questions concerning Figure 5 . lack of promoter activity of the Ta2 element . Four copies of the synthetic Tc 12 oligonucleotide were cloned in both orientations into the Hindlil site immediately 5' of the CAT gene in the promoterless pCATBasic plasmid. 10 Pg of the resulting plasmids along with 2 ug of the pFtSVsgal reference plasmid were transfected into Jurkat T cells. The data are shown as CAT activities relative to that produced by the promoterless pCAT Basic control plasmid (which produced 0 .17% acetylation) following correction for differences in transfection efficiencies . The pSPaxnhCAT plasmid was included as a positive control in a parallel transfection and produced 24% total acetylation (data not shown) . the general phenomena of transcriptional repression and activation . First, what is the molecular mechanism that accounts for the bifunctional transcriptional regulatory potential of the Ta2 element in T cells? And second, what is the physiologic significance of these findings in terms of developmentally regulated gene expression?
One mechanism that could account for the positive and negative regulatory potential of the Tc12 nuclear protein binding site is that different sets of proteins are able to bind to this site, depending upon the context in which it is located. Thus, one or more transcriptional activator proteins might bind to Ta2 in the context of the wild-type TCR a enhancer, while a distinct set of transcriptional repressor proteins might bind preferentially to the Ta2 element alone . There is an equally tenable, and perhaps more interesting possibility that could account both for the ability of a single nuclear protein to activate and repress transcription when bound to different cis-acting sequence elements, and for the ability of a single cis-acting sequence element to mediate both transcriptional activation and repression . Specificially, some transcriptional regulatory proteins might be capable of binding to cis-acting regulatory sequences with two different conformations, one of which mediates transcriptional activation, and the other of which causes transcriptional repression . Interconversions between these two conformations could be caused either by the binding of such bifunctional regulatory proteins to different sequence motifs, or alternatively, by interactions of such bifunctional proteins with additional proteins bound to adjacent cis-acting elements. Thus, for example, interactions between proteins bound to the Ta2 and Tail sites of the TCR a enhancer might result in a Tc12 conformation that activates transcription, while binding of the same proteins to Ta2 elements alone might result in a transcriptional repressor conformation. Our ability to distinguish between these different potential mechanisms requires a better understanding of the Tat and Tc12 binding proteins .
Finally, it is worth noting that, regardless of the molecular mechanisms that account for the dual transcriptional regulatory activities of the Ta2 element, our results suggest that a transcriptional enhancer that is composed of multiple nuclear protein binding sites can be converted to a transcriptional silencer by altering the levels of expression of the relevant transcriptional activator proteins. Thus, for example, the