©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The High Mobility Group Transcription Factor, SOX4, Transactivates the Human CD2 Enhancer (*)

(Received for publication, September 16, 1994)

David Wotton (§) Richard A. Lake (1) Christine J. Farr (2) Michael J. Owen (¶)

From the  (1)Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom, St. Mary's Hospital Medical School, Department of Immunology, Norfolk Place, London W2 1PG, United Kingdom, and (2)University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A strong T cell-specific enhancer is located 3` to the human CD2 gene. Six sequences within this enhancer are bound by proteins present in T cell nuclear extracts. These sequences share homology with sequences bound by several transcription factors involved in T cell- and lymphoid-specific transcription. The results presented here demonstrate that the human T cell-specific transcription factor, SOX4, is able to bind to one of these regions; further, SOX4 transactivates transcription of a reporter gene via three tandem copies of this sequence. The binding of SOX4 to this site is not via a canonical HMG protein binding sequence, identifying a novel class of binding site for this protein. A second sequence within the CD2 enhancer closely resembles the IL-2 NF-AT site. We show that it is bound by the ets-related factor, Elf1. However, unlike the IL-2 NF-AT sequence, the CD2 NF-AT-like sequence is unable to confer transcriptional inducibility on a reporter gene. Consistent with this result, we show that the observed increase in expression of CD2 protein on the cell surface following T cell activation is a post-transcriptional event.


INTRODUCTION

The human CD2 gene is expressed in all T cells and thymocytes, except for the most immature progenitor cells(1) . Studies using transgenic mice have established that a 28.5-kb fragment containing the human CD2 gene carries all of the necessary information for correct tissue-specific expression(2) . Within this fragment, a weak promoter together with an enhancer that is located 3` to the gene have been identified(3) . These studies have also identified a locus control region (LCR) (^1)within the 3`-enhancer region(4) . These sequences confer copy number-dependent, tissue-specific, position-independent expression of the gene in transgenic mice. Deletion analysis has established that the CD2 enhancer and LCR are both located within about 1.5-kb of flanking sequences immediately 3` to the polyadenylation signal of the gene and may be partially overlapping(3, 5) .

The enhancers of several T cell-specific genes have been well characterized. The identification of transcription factors likely to play important roles in the regulation of expression of these genes has indicated that members of a few key transcription factor families play critical roles in the regulation of expression of a wide range of T cell-specific genes. Ets proteins are important for the regulation of the enhancers of a number of T cell-specific genes, including TcR-alpha and -beta enhancers(6, 7) . Transcription factors with homology to the high mobility group (HMG) of proteins are likely to be important for the T cell specificity of transcription of several genes, such as the CD3 gene (8) and the TcR-alpha and -beta genes(9, 10) . Together with other transcription factors, such as GATA-3 (11, 12) and CRE-binding proteins(13) , it is likely that the binding of combinations of multiple T cell- and lymphoid-specific factors determine the T cell specificity of gene expression and the precise timing of initial expression of T cell-specific genes.

The CD2 gene represents an attractive candidate for studying the control of T cell differentiation by tissue-specific transcription factors, because the promoter and enhancer regions lie close to the gene on a relatively small region of DNA. Moreover, the CD2 LCR and enhancer activities are closely associated at the 3` end of the gene. These considerations prompted us to analyze transcription factor binding to the enhancer. DNase I footprint analysis revealed six protected regions using T cell nuclear extracts(3) . Within these regions several close similarities to sequences bound by transcription factors important in T cell- and lymphoid-specific transcription were observed. Here, we demonstrate that members of two transcription factor families which play important roles in T cell transcription, ets, and HMG, bind to cis elements within the CD2 enhancer and that the HMG protein, SOX4, is able to transactivate the CD2 gene.


MATERIALS AND METHODS

Plasmids and Mutagenesis

The SOX4 expression plasmid was created by cloning the full-length SOX4 cDNA into the pEF-BOS expression vector(14) . The SOX4 bacterial expression vector contained the SOX4 cDNA, lacking the first 17 and last 11 amino acids as an in frame fusion with glutathione S-transferase, within pGEX-KG(15) .

Elf1 was expressed from within the T7-plink vector (a gift from R. Treisman) from the T7 promoter. A cDNA (donated by J. Leiden) encoding all but the first 14 amino acids was cloned in frame with a 10-amino acid c-myc epitope.

p142CAT is as described previously (3) and contained the same CD2 enhancer fragment as was used to create the mutants. All footprint deletion mutants were created in pBSKSII+ (Stratagene) containing 2 kb of DNA from 3` of the CD2 polyadenylation site. Mutant fragments were then used to replace the wild type enhancer fragment in p142CAT. Mutants were created using an oligonucleotide-directed in vitro mutagenesis kit (Amersham Corp.). The oligonucleotides for in vitro mutagenesis consisted of two arms, each with an annealing temperature of around 42 °C (calculated as 4 times (G + C) + 2 times (A + T) = T(m)), which hybridized either side of the footprint, looping it out and bringing together flanking sequences to create a restriction enzyme site for easy identification of mutants. The sequences of the oligonucleotides are as follows:

CAT reporter plasmids containing multimerized elements from the CD2 enhancer were created as follows. EMSA oligonucleotides were annealed, phosphorylated using T4 polynucleotide kinase and were ligated into BamHI cut pBSKSII+. Multimers (three, four, or five copies) were removed from pBSKSII+ with HindIII and XbaI and ligated into pBLCAT2(16) . E1x contained four copies of CD2E1; E2x, three copies of CD2E2; E3x, five copies of CD2E3; E4x, four copies of CD2E4; E5x, four copies of CD2E5. The NF-AT-CAT and IL-2-CAT reporters were a gift from D. Cantrell and contained the full IL-2 promoter/enhancer sequences to -2 kb and six copies of the NF-AT sequence, upstream of the minimal IL-2 promoter.

Transfections and CAT Assays

Transfections were carried out using the DEAE-dextran procedure as described previously(3) . All transfections contained a plasmid in which the luciferase gene is activated by the EF-1alpha promoter (pEF-BOS) as a control for transfection efficiency. Cell extracts were assayed for luciferase activity (17) prior to heat treatment, and all results are normalized to the control. CAT activity was assayed by the liquid scintillation method of Sleigh(18) .

Proteins

The SOX4 cDNA was expressed as an in frame fusion with GST in Escherichia coli (XL-1 blue) after induction with 0.1 M IPTG. Bacteria were pelleted by centrifugation at 2500 times g for 10 min, resuspended in 500 µl of extraction buffer (0.1 M KC1, 20 mM HEPES, pH7.9, 20% glycerol, 0.2 mM EDTA, 0.1% Nonidet P-40) in a microcentrifuge tube and sonicated twice for 15 s in a Soniprep at medium amplitude. The bacterial debris was pelleted by centrifugation for 5 min, and the supernatant was frozen in aliquots at -70 °C. To verify the presence of GST-SOX4 fusion protein, bacteria from a 1.5-ml culture were sonicated in 200 µl of NETN (0.1 M, NaCl, 1 mM EDTA, 10 mM Tris-HC1 pH8, 0.5% Nonidet P-40), and the supernatant was incubated at 4 °C for 1 h, with gentle mixing with 20 µl of glutathione-Sepharose (Pharmacia Biotech Inc.), pre-swollen in NETN. The glutathione-Sepharose was pelleted at 6500 rpm for 10 s, and the pellet was washed twice with 1 ml of NETN. The pellet was resuspended in 20 µl of SDS-PAGE loading buffer and analyzed by SDS-PAGE. In vitro transcription and translation were carried out using a Promega TNT kit. S-Labeled protein was fractionated by SDS-PAGE and analyzed by fluorography.

EMSA

Two complementary single-stranded oligonucleotides were synthesized, which, when annealed leave 4-base 5` extensions with the sequence 5`-GATC-3`. Each double-stranded oligonucleotide contained the DNase I-protected sequence plus 5 bp on either side. The sequences of the upper strand of each pair are shown below:

EMSA reactions were carried out in a final volume of 20 µl, containing 1 µg of poly(dI-dC)bulletpoly(dI-dC), 2.5 mM MgCl(2), 40 mM KCl, 5 mM HEPES, pH 7.9, 0.02 mM EDTA, 5% glycerol, and 0.1% Nonidet P-40. Protein (2 µl of bacterial lysate or 0.5 µl of reticulocyte lysate) was incubated with competitor DNA for 5 min at 25 °C prior to the addition of 1 ng of labeled probe. The reaction was continued for 20 min and then fractionated by electrophoresis through 5% acrylamide in 0.25 times TBE (Tris-borate-EDTA).

Cloned Human Antigen-reactive T Lymphocytes

Human influenza hemagglutinin (HA)-reactive T cells were cloned by limiting dilution in the presence of irradiated (5000 rad) autologous peripheral blood mononuclear cell, antigen, and IL-2 (10% v/v; Lymphocult T, Biotest Folex). T cells were expanded by stimulation with antigen and filler cells every 7 days. After stimulation, the cells were expanded with IL-2 on the 3rd or 4th day as described previously(19) . The cells were rested 7-8 days after the last addition of filler cells and antigen before use. Cells were harvested at various times after stimulation, washed by centrifugation, and either analyzed for cell surface changes or lysed for RNA extraction.

Determination of Phenotypic Changes by Fluorescence Flow Cytometry

T cells were stained directly using saturating concentrations of fluorescein-conjugated murine monoclonal antibodies. Anti-Leu4 (CD3), anti-interleukin-2 receptor (CD25), and a mouse IgG1 control were bought from Becton Dickinson (Oxford, UK). Propidium iodide exclusion was used to identify viable cells. Cell populations were analyzed by flow cytometry using an Epics-Profile II (Coulter, Luton, UK). Mean fluorescence intensity was measured on a linear scale. All viable cells were included in each determination, and in each case, the fluorescence profile was unimodal.

Quantitation of mRNA by Northern Analysis

RNA was prepared essentially according to the method of Chomczynski and Sacchi (20) and resolved on 1% agarose-formaldehyde gels. The RNA was blotted onto Hybond membrane (Amersham Corp.) under conditions recommended by the supplier. Blots were incubated with random primed probes(21) , washed, and subjected to autoradiography.


RESULTS

Comparison of the CD2 Footprinting Regions with Known Enhancer Motifs

The footprinting regions of the human CD2 enhancer (3) were analyzed for known transcription factor binding motifs. Comparison of the sequences of the six protected regions with sequences of known protein binding motifs revealed several close homologies. As shown in Fig. 1, CD2E1, CD2E3, CD2E4, and CD2E5 share homology to the WWCAAAG sequence recognized by HMG proteins such as TCF-1, TCF-1a, and SRY(8, 22, 23) , although none of these HMG-like sites perfectly matches the consensus. Two copies of the CAAAG sequence are present within CD2E5, one in each of two inverted repeats.


Figure 1: Comparison of the CD2 enhancer footprints with known protein-binding sequences The CD2E1, -3, -4, and -5 sequences share some homology with the binding site for the HMG family of proteins(8, 22, 23) . The CD2E6 footprint is a consensus CRE(32) . CD2E4 has homology to the NF-AT sequence of the IL-2 gene(28) , as well as to the consensus binding site for the ets family of transcription factors(24, 25, 26) . CD2E5 contains a consensus LyF-1 site (31) and CD2E1 has a sequence capable of binding to GATA-3(33, 34) . Regions of homology with consensus sites (above) are shaded gray. Sequences shown in lower case letters were not clearly within the protected region.



CD2E4, in addition to its HMG site homology, also contains the central region (MGGAW) of the binding site for the ets family of transcription factors(24, 25, 26) . The sequence of the CD2E4 ets site is similar to that bound by the ets-like factor Elf1(27) . Interestingly, the ets site of CD2E4 is located within a region of extensive homology (15/19 nucleotides over the central region) to the NF-AT site of the IL-2 enhancer, which has been shown to play a major role in the inducibility of IL-2 expression(28) . However, the region of CD2E4 homologous to the 3` region of the NF-AT-like site was not protected in DNase I footprint analysis and contains three mismatches within the last 4 protected bases of CD2E4. Present evidence suggests that Fos/Jun heterodimers bind to the 3` part of the NF-AT site(29, 30, 31) . These differences may have critical effects on the activity of CD2E4 compared to that of the NF-AT sequence.

In addition to HMG site homology, the CD2E5 element also contains a consensus LyF-1 binding site (YYTGGGAGR)(32) . LyF-1 binding activity has been demonstrated to be present at high levels only in B and T cells(32) , although no proteins that specifically bind to this sequence have been identified. The CD2E6 footprint perfectly matches the consensus cyclic AMP response element(33) .

Members of the GATA family of transcription factors have recently been shown to recognize sequences other than the canonical WGATAR(34, 35) . The T cell-specific human GATA-3 protein is able to bind with high affinity to the sequence TGATTA(35) , which is present within CD2E1.

Deletion Analysis of the CD2 Enhancer

The human CD2 enhancer has been shown previously to be highly active in transient transfection assays in the Jurkat human T cell line. To determine the relative contribution of each of the six cis elements within the enhancer to this activity, constructs were created in which each of the elements had been deleted individually (Fig. 2A, constructs DE1-DE6). Jurkat cells were transfected with each of the deletion mutants together with a transfection control plasmid containing the firefly luciferase reporter gene(17) , and the activity of each construct was compared to that of the full enhancer. As shown in Fig. 2B, deletion of CD2E6 had no significant effect on enhancer activity, suggesting that this element plays little role in enhancer activity as assayed in transient transfection assays. In contrast, deletion of each of the other elements (CD2E1-5) had significant effects on activity. In particular, deletion of CD2E1, -2, -3, and -4 had profound effects on enhancer activity, these constructs (DE1-DE4) showing only 20-35% of the activity of the full enhancer. Deletion of CD2E5 had a more minor effect on enhancer activity. We, therefore, conclude that an essential core of the CD2 enhancer appears to comprise the elements CD2E1-CD2E4.


Figure 2: Activity of reporter constructs in which each of the footprinting regions has been deleted. A, a restriction map of the enhancer is shown, and the positions of the six footprints are indicated as gray boxes(3) . Six constructs (DE1-6) in which each of the footprinting regions has been deleted individually are shown. B, 10 µg of each of the six enhancer deletion mutants or of the full enhancer (p142) were transfected into J6 cells, together with 10 µg of pEF-BOS-luciferase. After 40 h, cells were assayed for CAT and luciferase activity. Results, corrected for luciferase activity, are presented as a percentage of the activity of the full enhancer (p142, 24,550 cpm; background, 350 cpm.).



Inducibility of the CD2 Enhancer

As shown in Fig. 1, the CD2E4 element shows a high degree of similarity to the NF-AT sequence that is essential for the inducibility of the IL-2 gene in activated T cells(28) . To determine whether the CD2E4 element conferred inducibility to the CD2 enhancer, the mouse T cell line EL-4 was transfected with p142CAT, with a reporter construct containing the CD2 enhancer from which the CD2E4 element had been deleted (DE4) or with a reporter construct in which the expression of the CAT gene is driven by four copies of the CD2E4 element (E4x). The transfected cells were divided into four pools and were incubated in normal growth medium or medium containing PMA, ionomycin, or both PMA and ionomycin. As shown in Fig. 3, no induction of expression of any of these constructs was observed with either PMA or ionomycin. In contrast, when EL4 cells were transfected with reporters in which expression of the CAT gene is driven by the human IL-2 promoter and enhancer or by a multimer of the NF-AT sequence, efficient activation of both constructs occurred upon addition of the phorbol ester PMA together with the calcium ionophore ionomycin. These results demonstrate that, despite the similarity between the CD2E4 element and the IL-2 NF-AT sequences, CD2E4 fails to act as an inducible element in the same way as the NF-AT element.


Figure 3: Inducibility of the CD2 enhancer in EL-4 cells. Cells were transfected with 10 µg of pEF-BOS-luciferase and 10 µg of the IL-2-CAT or NF-AT-CAT constructs (in which expression of the CAT gene is driven by the IL-2 promoter/enhancer or six copies of the NF-AT site upstream of the minimal IL-2 promoter, respectively) or with the CD2 enhancer constructs indicated. p142 contains the full CD2 enhancer, the E4x construct contains four copies of the CD2E4 element upstream of the tk promoter and DE4 is p142 with the CD2E4 element deleted. Following transfection, cells were incubated for 40 h in normal medium or in medium supplemented with PMA (10 ng/ml), ionomycin (1 µg/ml), or PMA and ionomycin together (10 ng/ml and 1 µg/ml). Cells were assayed for CAT and luciferase activity. Results (luciferase corrected) are presented as the fold increase in activity relative to cells grown in normal medium. Basal activities, in counts/min, were IL-2-CAT, 500; NF-AT-CAT, 400; p142, 43,900; E4M, 1250; DE4, 12,600; pBLCAT2, 300; background, 200.



Consistent with this observation are the results of experiments in which the steady state levels of CD2 mRNA were determined by Northern analysis following T cell activation. The classical phenotypic changes consistent with T cell activation include the up-regulation of CD2 and CD25 surface levels and the down-regulation of surface CD3 expression (36) . Thus, incubation of the human T helper clone, HA1.7, with activating CD3-specific antibodies, staphylococcal enterotoxin B (SEB), or an influenza HA peptide results in a 2.3-3.1-fold increase in CD2 surface expression, together with an increase in surface expression of CD25 and a decrease in the level of surface CD3 expression (Table 1). No significant changes in the levels of surface expression of these molecules were observed when the SEC2 toxin was used. When HA1.7 cells were activated similarly, the steady state levels of CD2 transcripts failed to change by a factor greater than that of the GAPDH or ribosomal S26 mRNA controls (Fig. 4). In contrast, the levels of TNF-alpha transcripts were dramatically induced in the presence of CD3-specific antibodies, SEB, or HA peptide relative to the levels in unstimulated cells (Fig. 4).




Figure 4: Northern analysis of RNA from HA1.7 cells before and after antigen stimulation. Cytoplasmic RNA isolated from cloned T cells was Northern blotted and hybridized sequentially to probes from the CD2, TNF-alpha, GAPDH, and ribosomal S26 genes. Cells (2 times 10^6 per treatment) were exposed to various T cell receptor ligands for times as indicated (in hours). HA is the minimal peptide epitope (HA 307-319); HDM is a peptide which does not bind to the HA1.7 TcR; SEB is an enterotoxin from Staphylococcus aureus that binds to the HA1.7 TcR, whereas the closely related toxin SEC2 does not; anti-CD3 represents cells treated with activating antibodies to the CD3 components of the TcR complex. RNA from cells incubated in normal growth medium is also shown.



Thus, under conditions in which HA1.7 cells are activated (as judged by surface phenotypic changes and increased TNF-alpha mRNA levels) no increase in the level of CD2 transcripts is observed, despite the increased level of cell surface expression of the CD2 molecule. Taken together, these results demonstrate that the increase in surface CD2 expression upon T cell activation is not a consequence of increased CD2 enhancer activity or steady state mRNA levels, and is, therefore, likely to be due to post-translational events.

Binding of Elf1 to the CD2 Enhancer

The presence of the AGGAA sequence within the CD2E4 footprint suggests that this region may be bound by a member of the ets family of transcription factors. The most likely candidate for binding to CD2E4 is Elf1, which has been shown to bind preferentially to sequences in which the GGA motif is followed by AAA, as in the NF-AT element(27) . Elf1 protein was produced by in vitro transcription and translation in the presence or absence of [S]methionine. The labeled reaction products were analyzed by SDS-PAGE to assay for production of protein (data not shown) and unlabeled Elf1 was used in EMSA analysis. As shown in Fig. 5(lane 2), a complex of retarded mobility was formed when a radiolabeled oligonucleotide containing the CD2E4 sequence was incubated with in vitro translated Elf1 prior to electrophoresis. In contrast, no binding to CD2E4 was observed when unprogrammed lysate was used (lane 1). When a 100-fold excess of unlabeled CD2E4 competitor oligonucleotide was used in the reaction, a complete loss of complex formation was observed, whereas addition of CD2E1 failed to compete fully for binding (lanes 3 and 4). Similarly, a competitor in which the AGGAA of CD2E4 had been altered to ATTAA (E4mut; lane 7) failed to abolish the formation of the CD2E4bulletElf1 complex, strongly suggesting that binding of Elf1 to CD2E4 was dependent on the integrity of the ets site. An oligonucleotide containing the NF-AT sequence competed for binding of Elf1 to CD2E4, although the level of competition was less than that seen when unlabeled CD2E4 was used. The betaE2 element of the TcR-beta enhancer, which binds to ets 1 (7) , was unable to compete for binding of Elf1 (lane 9). Consistent with this observation, no binding of baculovirus-produced ets 1 to CD2E4 was observed (data not shown). These data suggest that the CD2E4 footprinting region is bound in vivo by Elf1.


Figure 5: Binding of Elf1 to CD2E4. Radiolabeled CD2E4 oligonucleotide (0.5 ng) was incubated with 2 µl of Elf1 translated in vitro (lanes 2-9) or with unprogrammed reticulocyte lysate (lane 1). Competitor oligonucleotides, at 100-fold molar excess, were preincubated with protein for 5 min before the addition of probe. The competitors used are indicated, E2 and E4 are the CD2E2 and CD2E4 footprinting regions; E4mut is a mutant version of the CD2E4 footprint in which the AGGAA sequence was replaced with ATTAA; NF-AT contains the NF-AT site from upstream of the human IL-2 gene; betaE2 is the ets-1-binding betaE2 element from the TcR-beta enhancer. Complexes were resolved by electrophoresis through 5% polyacrylamide gel. The position of the Elf1bulletCD2E4 complex is indicated.



Transactivation of CD2E2 by Human SOX4

The CD2E1, -3, -4, and -5 elements each contain partial matches to the consensus sequence for binding of HMG proteins. To determine the effects of expression of human SOX4, a recently identified HMG protein expressed at high levels in T cells (37) on the activity of these elements, an expression plasmid encoding the SOX4 protein was co-transfected with CAT reporter constructs containing three to five copies of each of the elements, CD2E1-5 (constructs E1x-E5x). As shown in Fig. 6, SOX4 failed to transactivate the CD2E1, -E3, -E4, and -E5 elements. Indeed, the activity of E1x, E3x, E4x, and E5x was decreased when high amounts of SOX4 expression plasmid were used, although this decrease was judged to be nonspecific, because the activity of the pBLCAT2 reporter plasmid also decreased by a similar amount. However, the activity of the CD2E2 construct, containing three copies of CD2E2 (E2x), was activated by up to 2.4-fold with a ratio of 2:1 of expression to reporter plasmid. When this experiment was repeated using the erythroleukemic cell line K562, in which the CD2 enhancer is weakly active, the E2x construct was activated by 5.6-fold with the highest level of SOX4 plasmid. In contrast, none of the other constructs was activated by SOX4 in K562 cells. A reporter construct containing a single copy of the CD2E2 element was not significantly activated by co-expression of the human SOX4 protein (data not shown).


Figure 6: Coexpression of SOX4 with multimerized elements from the CD2 enhancer. Cells were transfected with 5 µg of one of the CAT reporter constructs, containing multimerized elements from the CD2 enhancer upstream of the tk promoter, together with 5 µg of pEF-BOS-luciferase and 0-10 µg of pEF-BOS-SOX4. The amount of DNA in each transfection was kept constant by adding pEF-BOS without an insert in place of pEF-BOS-SOX4. 40 h after transfection, cells were assayed for CAT and luciferase activities. Results are presented (normalized to luciferase) relative to the CAT activity of each reporter (set to 1) in the absence of cotransfected SOX4. The activity of the constructs containing multimerized elements in the absence of SOX4 was 2150-8050 cpm in J6 and 3450-9100 cpm in K562, background 450 cpm.



To determine whether the transactivation by SOX4 is a consequence of direct binding to CD2E2, the SOX4 cDNA was cloned into the vector pGEX-KG as an in-frame fusion with the glutathione S-transferase gene(15) . Production of GST-SOX4 fusion protein in bacteria was induced with IPTG and bacterial cell extracts were prepared. Radiolabeled CD2E2 oligonucleotide was incubated with bacterial extracts prepared from induced or uninduced cells and complexes were analyzed by electrophoresis. As shown in Fig. 7(lane 1), when radiolabeled CD2E2 was incubated with extract from uninduced bacteria, only very weak complexes were observed, possibly due to some expression of SOX4 in the absence of IPTG induction. However, a complex of reduced mobility was clearly visible when extract from IPTG-induced bacteria was used (lane 2). Formation of this complex was severely reduced by including a 50- or 100-fold excess of unlabeled competitor CD2E2 oligonucleotide in the reaction (lanes 5 and 6). However, the CD2E1, E3, E4, and E5 oligonucleotides all failed to compete for binding of SOX4 (Fig. 7, lanes 3, 4, and 7-12).


Figure 7: Binding of SOX4 to CD2E2. Radiolabeled CD2E2 oligonucleotide was incubated with 1 µl of bacterial lysate (lane 1), or lysate from bacteria which carried an inducible GST-SOX4 expression vector and had been induced with IPTG for 4 h prior to lysis (lanes 2-12). Competitor oligonucleotides (as indicated) were incubated with the lysate for 5 min prior to the addition of probe. Complexes were resolved by electrophoresis through 5% polyacrylamide. The position of the SOX4bulletCD2E2 complex is indicated.



No match to the canonical HMG binding site is present within CD2E2. To determine the sequence within CD2E2 to which SOX4 binds, several mutant oligonucleotides were used (see Fig. 8B). As shown in Fig. 8A, when 7 bases were removed from the 3` end of the CD2E2 oligonucleotide (CD2E2-Delta3`), binding to SOX4 was abolished. No retarded complexes are visible with this oligonucleotide (lane 6); similarly, when radiolabeled CD2E1, -E3, -E4, or -E5 were incubated with SOX4, no retarded bands were visible (Fig. 8A, lanes 1-4), confirming the results shown in Fig. 7. In contrast, SOX4bulletCD2E2 complexes are clearly visible (lane 5). Within CD2E2, and overlapping the region not present in CD2E2-Delta3`, is the sequence AACAATA (in CD2E2-Delta3`, only AACA is present). As this is the sequence within CD2E2 which most closely resembles the WWCAAAG to which HMG proteins bind, oligonucleotides with specific changes in this sequence were created (see Fig. 8B). To test the binding of these mutant sites to SOX4, the six oligonucleotides were radiolabeled and incubated with SOX4. As shown in Fig. 8A, some binding of SOX4 to CD2E2-C7 was observed (lane 12). Of the other mutants, only CD2E2-G3 bound SOX4 extremely weakly (lane 9). None of the other four mutant sites showed any binding to SOX4. Interestingly, CD2E2-T1T2 (TTCAATA) failed to bind SOX4, whereas other HMG proteins are capable of binding sequences with either A or T residues at the first two positions.


Figure 8: Binding of SOX4 to mutant CD2E2 sites. EMSA reactions are as for Fig. 7. A, SOX4-containing lysate was incubated with radiolabeled oligonucleotides containing each of the footprinting regions CD2E1, -3, -4, and -5 (lanes 1-4), CD2E2 (lane 5) or an oligonucleotide lacking the 3` part of the CD2E2 footprint (CD2E2-Delta3`; lane 6). B, radiolabeled mutant CD2E2 oligonucleotides as indicated were incubated with SOX4-containing lysate. Complexes were resolved by electrophoresis through 5% acrylamide. C, the mutant CD2E2 oligonucleotides used are shown. Dashes indicate bases the same as those in the wild-type CD2E2 (above). The SOX4-binding site is boxed.



Taken together, these results demonstrate that the SOX4 protein can bind to and activate transcription via the AACAAT-containing CD2E2 element. Interestingly, no binding or transactivation was observed with any of the other CD2 enhancer elements which have some homology with the canonical HMG protein binding site.


DISCUSSION

The results presented here have identified four cis-acting elements (CD2E1-E4) within the CD2 enhancer that play the major role in its activity in T cells. These elements contain consensus binding motifs for transcription factor families that have been shown to be important in the transcription of T cell specific genes. A summary of the transcription factor binding sites and the proteins shown, by this study, to bind to the CD2 enhancer is shown in Fig. 9.


Figure 9: Enhancers of T cell specific genes. The transcriptional enhancers from the human CD2 gene and from the human TcR-alpha(42, 43) , beta (7, 44, 45), (46), and CD8alpha (47) genes are represented schematically. Sequence elements are represented as ovals, all are within regions important for activity in transient transfection assays. All elements except those from the CD8alpha gene were identified by DNase I footprinting. The importance of the elements within the CD8alpha enhancer has been demonstrated by mutation (47) . Protected elements containing no binding site consensus are represented with a ?. Underlined motifs have been shown to bind purified or recombinant protein.



When peripheral T cells are activated either by polyclonal mitogen or specific antigen/MHC complexes, the level of CD2 at the cell surface increases by 3-6-fold(36) . This increase has been postulated to act as a mechanism by which the initial antigen driven activation signal is amplified for optimal T cell activation. An important question concerning this regulation of CD2 expression is whether the increase in cell surface expression on T cell activation is regulated at the transcriptional level. The presence of a sequence within the CD2E4 element that is similar to the NF-AT site within the IL-2 enhancer suggests that this may be a target of TcR-mediated T cell activation. The NF-AT site of the IL-2 gene enhancer has been shown to bind inducible fos/jun complexes together with a constitutively expressed or preexisting lymphoid specific component, NF-ATc or NF-ATp, that is translocated to the nucleus upon T cell activation(29, 30, 31) . The formation of this complex is necessary for transcription of the IL-2 gene and the subsequent proliferation of the T cell. We show here that the NF-AT-like sequence within CD2E4 is unable to act as an inducible element. Thus, no activation of transcription from CAT reporter constructs containing the CD2 enhancer was observed in EL4 cells, which can be induced to up-regulate IL-2 expression. Consistent with this observation, antigen stimulation of the human T helper clone HA1.7 results in an increase in surface CD2 expression without affecting the level of CD2 mRNA. Taken together these results suggest that any increases in CD2 surface expression are not due to increased transcription of the CD2 gene or increased CD2 mRNA stability, but to post-translational mechanisms.

We have shown that the CD2E4 element binds the Elf1 transcription factor. Elf1 is a member of the ets family of transcription factors, the expression of which is confined primarily to T cells and was first identified as a factor that is able to bind to the NF-AT site of the IL-2 enhancer(38) . However, the relevance of Elf1 to the regulation of IL-2 transcription is unclear. It is expressed constitutively in the nuclei of T cells, whereas the factors responsible for the NF-AT-mediated induction of IL-2 expression are not present in nuclei of uninduced T cells. Furthermore, two different cytoplasmic components of the NF-AT complex have recently been cloned and neither show any homology to Elf1. Thus, the CD2E4 element may represent a more likely target for binding of Elf1 in vivo. Recently, expression of the human IL-3 and GM-CSF genes has also been shown to require Elf1 binding sites(39, 40) . However, in both these cases, the Elf1 binding sites are adjacent to AP-1 sites, and both Elf1 and AP-1 sites are required for T cell-specific gene expression and inducibility. In the absence of an AP-1 site, an Elf1 binding site (as in CD2E4) may be required only for the T cell specificity of gene expression.

Comparison of the CD2E1, -E3, -E4, and -E5 sequences with known transcription factor binding motifs reveals that each of these elements shows homology to the canonical HMG site bound by the T cell-specific transcription factors, TCF1 and TCF1-alpha, and the sex-determining gene, SRY(8, 22, 23) . However, none of these sites is a perfect match with the consensus binding motif, and none was able to bind the HMG transcription factor SOX4 in vitro. The CD2E2 element also contains an HMG-like binding sequence, AACAATA, although this differs significantly from the normal HMG site (WWCAAAG). However, bacterially expressed SOX4 was able to bind to CD2E2 in vitro and, when co-expressed in Jurkat or K562 cells, activated transcription from a synthetic enhancer comprising three tandem copies of the CD2E2 element. SOX4 was originally identified as a transcriptional activator that was capable of binding and transactivating a motif (AACAAAG) found in several T cell-specific enhancers(37) . We have identified a variant, but related, site to which SOX4 is able to bind and via which it can activate transcription. It will be of interest to determine which of these types of sites (WWCAAAG or AACAAT) is most relevant for SOX4 binding rather than binding of other HMG proteins. The consensus binding site for SRY has recently been determined as AACAAW and it has been suggested that subgroups of HMG family proteins will preferentially bind different A/T-rich DNA sequences(41) . The results presented here support this notion, that the SRY/SOX proteins preferentially recognize a DNA sequence different from that recognized by the TCF/LEF subgroup of HMG proteins.

The core of the CD2 enhancer has been shown to bind at least two major classes of transcription factors (see Fig. 9), the SOX and ets families. Each of these families contain members that have been shown to be important for the transcription of other T cell-specific genes. Thus, HMG, ets, and GATA sites are present within all five of the enhancers of T cell specific genes shown in Fig. 9(7, 42, 43, 44, 45, 46, 47) . CREs are present in all but the TcR- enhancer and LyF-1 sites in both CD2 and CD8alpha, but not in any of the TcR gene enhancers. The binding of factors to these elements within the CD2 enhancer may, therefore, explain its tissue specificity. In this context, the lymphoid specific factor SOX4 was shown to act as a transactivator in this system. In contrast, we obtained no evidence that the NF-AT complex, which plays a key role in the induction of a number of cytokine genes upon antigen stimulation, had any functional effect upon the CD2 enhancer. It will be of interest to determine whether these transcription factor families play any role in the LCR activity of the CD2 enhancer in addition to determining the tissue specificity of expression.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Columbia University, Dept. of Microbiology, 701 W. 168th St., New York, NY 10032.

To whom correspondence should be addressed. Tel.: 44-71-269-3069; Fax: 44-71-269-3479.

(^1)
The abbreviations used are: LCR, locus control region; TcR, T cell receptor; HMG, high mobility group; EMSA, electrophoretic mobility shift analysis; GST, glutathione S-transferase; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; PMA, phorbol 12-myristate 13-acetate; SEB, staphylococcal enterotoxin B; TNF, tissue necrosis factor; TCF, T cell transcription factor; IL, interleukin; CAT, chloramphenicol acetyltransferase; kb, kilobase(s); CRE, cyclic AMP response element.


ACKNOWLEDGEMENTS

We thank Dr. J. M. Leiden for the generous gift of Elf1 cDNA and Drs. J. R. Lamb and R. O'Hehir for providing the HA1.7 T cell clone.


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