(Received for publication, September 16, 1994)
From the
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.
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) ()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- and -
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-
and -
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.
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 (G + C)
+ 2
(A + T) = T
), 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.
EMSA reactions were carried out in a final volume of 20 µl,
containing 1 µg of poly(dI-dC)poly(dI-dC), 2.5 mM MgCl
, 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
TBE (Tris-borate-EDTA).
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.
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.).
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- 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-, GAPDH, and
ribosomal S26 genes. Cells (2
10
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- 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.
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; E2 is the ets-1-binding
E2 element from
the TcR-
enhancer. Complexes were resolved by electrophoresis
through 5% polyacrylamide gel. The position of the Elf1
CD2E4
complex is indicated.
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 SOX4CD2E2 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 (CD2E2-3`), 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, SOX4
CD2E2 complexes
are clearly visible (lane 5). Within CD2E2, and overlapping
the region not present in CD2E2-
3`, is the sequence AACAATA (in
CD2E2-
3`, 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 (CD2E2-3`; 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.
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-(42, 43) ,
(7,
44, 45),
(46), and CD8
(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 CD8
gene were identified by DNase I footprinting. The
importance of the elements within the CD8
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-, 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 CD8
, 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.