From the § Department of Microbiology and the
Integrated Program in Cellular, Molecular, and
Biophysical Studies, Columbia University, College of Physicians and
Surgeons, New York, New York 10032
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ABSTRACT |
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The control of CD4 gene expression is
believed to be linked directly to the signaling events that mediate T
cell development and is directly dependent on the CD4
promoter. We have previously determined that this promoter contains
four factor-binding sites important for its function. One of these
sites, referred to as the P4 site, contains an Ets consensus
recognition sequence. Using functional and biochemical analyses, we
determine that Elf-1 binds to this site and specifically activates the
CD4 promoter, indicating that Elf-1 is playing an important
role in CD4 promoter function. In addition, a second
nuclear factor binds to this region. Although there are consensus
recognition sites for other factors, we demonstrate that none of these
factors binds to the P4 site, nor do other known members of the Ets
family. Thus, a novel transcription factor may bind to the
CD4 promoter and help mediate its function.
CD4 is a cell surface molecule that is essential for T cell
development and function (1-3). During antigen recognition, CD4 binds
to a nonpolymorphic region of the
MHC1 class II molecule,
thereby increasing the avidity of the T cell for its target. Both CD4
and CD8, a similar coreceptor molecule, also contribute to T cell
antigen receptor signaling by recruiting the tyrosine kinase
Lck to the T cell antigen receptor/CD3 complex and thus are
critical for the activation of the mature T cell (4). The decision to
express CD4 or CD8 on mature T cells correlates with both its MHC
restriction and its functional specialization and occurs during
development in the thymus (5, 6). Precursor T cells in the thymus
undergo a "double-positive" stage in the thymus in which the
thymocyte expresses CD4, CD8, and T cell antigen receptor on its
surface. The selection processes that determine both the antigen and
MHC class specificity of each T cell occurs at this stage (5, 7-11).
During this process, a decision is made to turn off either CD4 or CD8
so that the T cell will either become a
CD4+CD8 CD4 expression during development is controlled primarily by
transcriptional mechanisms (24). At least five transcriptional control
elements have been identified in the CD4 locus: a distal enhancer (25), a proximal enhancer (26, 27), the promoter (28-30), a
transcriptional silencer (24, 31, 32), and a novel thymocyte-specific
enhancer (33). Each of these elements contributes in different ways to
the specificity of CD4 gene expression. Thus, there are many
signaling events and complex interactions between transcription factors
that are necessary to mediate the complexities of tissue- and
developmental stage-specific CD4 expression. To address these issues,
we study the structural and factor binding requirements of each
regulatory element and their contribution to the control of
CD4 gene expression. We have determined that HES-1 and the
novel transcription factor SAF bind to functional sites in the
CD4 silencer and help mediate its function
(34).2 In addition, work by
Sawada and Littman (26, 27) has shown that a heterodimer of the
basic-helix-loop-helix proteins HEB and E12 bind to a functional site
in the proximal enhancer. However, all of these factors are expressed
in many different hematopoietic cells, and thus their expression
pattern cannot explain the specificity of CD4 gene expression.
To understand how subclass-specific expression of CD4 is controlled, it
is important to understand the functional interaction of the different
CD4 transcriptional control elements. The CD4 promoter is an important central regulatory element in the control of
CD4 gene expression; consequently, it is important to
understand the factors that bind to its functional sites and how they
interact with transcription factors that bind to the other elements. We have therefore conducted an extensive analysis of the CD4
promoter (28, 30). There are four factor-binding sites, denoted P1, P2,
P3, and P4, that are important for full promoter function (28). The
transcription factors c-Myb and Maz bind to sites P1 and P2,
respectively, implicating these factors in CD4 promoter function (30). The P4 region contains consensus initiator (36) and Ets
(37) sequences. Previous work by Klatzmann and colleagues demonstrated
that exogenously expressed Ets-1 is capable of binding and
transactivating the human CD4 promoter, consistent with
these observations (29). Here, we determine that the Ets consensus sequence in the P4 site is important for regulation of transcription of
the CD4 gene. Using functional and biochemical analyses, we determine that Elf-1 and not Ets-1 or Ets-2 binds to the Ets site and
mediates CD4 promoter function. In addition, we determine that a novel factor also binds the P4 site. Taken together, these data
support the hypothesis that Elf-1 acts as a positive regulator at the
CD4 promoter and, together with a novel factor, plays a complex role in the induction of CD4 gene expression.
Cell Transfection and Maintenance--
The
CD4+CD8 Generation of Promoter Mutations--
Site-directed mutagenesis
was used to construct all of the promoter mutations. Oligonucleotides
containing the mutations were obtained from Life Technologies, Inc. and
used for site-directed mutagenesis in a pKS vector as described (43).
Mutations were identified by DNA sequencing analysis or restriction
digests. Mutant promoters were cloned into the pGL2 vector (Promega),
which contains the luciferase gene. The promoter sequence and
orientation were confirmed by sequencing.
EMSAs--
EMSAs were conducted as described previously (24, 31,
32). The P4 site containing sequences from Shift-Western--
EMSA was performed essentially as above. The
binding reaction was scaled up 5-fold to contain 105 cpm of
P4 probe and 30-40 mg of total protein from S49, AKR, or D10 nuclear
extracts in 30-ml reaction volume. The reactions were then run at 250 V
on a 4% acrylamide gel at 4 °C for 4 h in glycine buffer (see
above). The gel was blotted simultaneously onto two different
membranes, nitrocellulose and DE81 paper, using a semidry transfer
apparatus as described (45). In short, the gel and all membranes were
placed in transfer buffer (39 mM glycine, 48 mM
Tris base, 20% methanol, 0.037% SDS) for 30 min. The nitrocellulose membrane was placed in direct contact with the gel followed by one
layer of 3MM paper and then the DE81 paper and transferred for 1 h
in a semidry transfer apparatus. The protein composition of the shift
complexes was analyzed by subjecting the nitrocellulose membrane to
Western blotting with the anti-Elf-1 antibody using the Roche Molecular
Biochemicals Western blotting kit (catalog no. 1520709) according to
the manufacturer's protocol. The DE81 paper was air dried and exposed
on film at Immunodepletion/Immunoprecipitation--
Protein
A-Sepharose CL-4B beads were purchased from Amersham Pharmacia
Biotech. Immunodepletion of S49, 293T and Elf-1-transfected 293T cell
nuclear extracts was carried out with the anti-Elf-1 antibody according
to the Amersham Pharmacia Biotech protocol. In short, the extracts were
preincubated with rabbit IgG (Santa Cruz Biotech, sc-2027) and 10 µl
of protein A-Sepharose beads for 2 h to eliminate nonspecific
binding. The beads were spun down, and the supernatant was then
incubated with the anti-Elf-1 antibody (sc-631X) for 2 h. Then 20 or 40 µl of protein A-Sepharose beads were added, and incubation
proceeded overnight with rotation. The beads were spun down and washed
three times with TSA solution (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 0.025% sodium azide), and then they were boiled
for 5 min at 100 °C in 2× SDS loading buffer (120 mM
Tris-HCl, pH 6.8, 4.6% SDS, 20% glycerol, and 10%
An Elf-1 Recognition Site in the P4 Region--
Using linker
scanning mutations and transient transfections, we have identified four
functional sites in the CD4 promoter, which we refer to as
P1, P2, P3, and P4 (30) (Fig.
1A). The P4 site encompasses
both the initiation point of transcription for the CD4 gene
and important 5' functional regions. An alignment of P4 with the
consensus binding sequences of several Ets family members suggested
that this site is a recognition site for an Ets family transcription
factor (Fig. 1, B and C). There are a large
number of transcription factors in the Ets family, all of which
recognize the sequence (A/G)(T/A)(G/C)(A/T)TCC(G/T)(G/C)Y with a
DNA-binding domain that shares a common structural motif. The core TCC
sequence is essential for Ets family factor DNA binding; specificity
between the different family members is determined by the surrounding
nucleotides (37, 46, 47). The P4 site contains an Ets core consensus
that most closely matches an Elf-1 recognition sequence and is almost
identical in sequence to the Elf-1 recognition site in the
CD4 locus distal enhancer, suggesting that the Ets family
factor binding to P4 may be Elf-1 (Fig. 1B). We conducted a
functional analysis to characterize P4 further (Fig.
2). A large mutation encompassing the Ets
consensus sequence results in a 75% decrease in CD4
promoter function as assayed by transient transfections in D10(CD4SP)
cells (Fig. 2A, pSS057 and pSS058, combined) (30). The
pSS057 mutation changes three bases in the 5' end of the P4 site,
whereas the pSS058 mutation changes two bases immediately downstream of
the pSS057 mutation. As can be seen in Fig. 2B, the pSS058
mutation caused an 80% decrease in promoter activity, whereas the
pSS057 mutation did not affect promoter function. We therefore conclude
that the 5' functional boundary of the P4 site is the G nucleotide at
the
To determine whether the consensus Ets site itself is important for
CD4 promoter function, we generated single site-specific mutations in each of the core nucleotides. As can be seen in Fig. 2A, mutations pSS055, pSS025, and pSS056 contain single-base
changes at each of the core TCC nucleotides. In addition, pSS054
contains a mutation that alters the two critical cytosines in the core. Each of these mutations decreases promoter function by at least 80%,
indicating that the Ets binding site is required for CD4 promoter function (Fig. 2B). Interestingly, mutation pSS031
also completely abrogates promoter activity. As discussed above,
sequences adjacent to the core TCC nucleotides affect the ability of
different members of the Ets family to bind to the consensus Ets
recognition sequence. Thus, the identity of the Ets family member
binding to a given site can be determined by studying the effects of
mutations in these regions on factor binding. Experiments by several
groups have demonstrated that Elf-1 is the sole member of the Ets
family that recognizes the core sequence TTCC but cannot bind to the sequence ATCC; all other members of the Ets family, including Ets-1,
Ets-2, Pu.1, PEA, Fli-1, Erg, and Elk-1, can bind to the ATCC core Ets
recognition sequence (46-50). This difference in DNA binding
specificity is due to a sequence differences in the Elf-1 DNA binding
domain in comparison with the other Ets family members (49, 50). The
pSS031 mutation is a point mutation that changes the first thymidine in
the core TTCC sequence in the P4 site to an adenine (Fig. 2); thus, our
functional data suggest that the factor binding to P4 and mediating
CD4 promoter function cannot recognize the ATCC variant of
the Ets consensus sequence. This observation is consistent with the
hypothesis that the endogenous factor mediating promoter function at
the P4 Ets site contains an Elf DNA-binding domain.
Elf-1 and Not Ets-1 or Ets-2 Binds Specifically to P4--
To
address this issue biochemically, we conducted a series of EMSAs to
determine whether Elf-1 binds the P4 site. Three major (A, B, and C)
and two minor protein-DNA complexes form when the P4 probe is used in
an EMSA reaction with nuclear extracts purified from CD4 SP
TH clones (Fig. 3A
and data not shown). Addition of excess nonradioactive P4
oligonucleotide inhibits formation of all complexes, whereas
nonspecific DNA did not, indicating that all of the factors that form
these complexes bind specifically to the P4 probe. Similarly, the
addition of large molar excesses of an oligonucleotide containing the
consensus Elf-1 recognition site from the CD4 distal
enhancer also inhibits complex formation. Addition of excess
nonradioactive oligonucleotide that contains a large mutation in the
Ets site (P4mETS; see Figs. 2 and 3A) does not inhibit the
formation of complexes A and B, suggesting that these two complexes
represent nuclear factors binding to a region surrounding the Ets
consensus site in P4. Formation of complex C is inhibited effectively
with the P4mETS oligonucleotide, suggesting that this complex
represents a factor binding to P4 outside of the consensus Ets
region.
As discussed above, the pSS031 mutation, representing a change in the
core Ets recognition site from TTCC to ATCC, can be recognized by all
members of the Ets family except Elf-1 (49, 50). This change results in
the loss of Elf-1 binding and subsequent transcriptional control
element function. Our functional data presented above suggest that the
factor binding to P4 cannot recognize the ATCC variant of the Ets
consensus site, implying that the P4-binding factor has an Elf DNA
binding domain. Thus, should endogenous Elf-1 bind to P4, we predict
that a mutant oligonucleotide with a single base change that alters the
TTCC to ATCC should neither be able to inhibit complex formation with
the unmutated P4 probe in an EMSA experiment nor be able to form
complexes A and B when used as a probe. We tested these predictions
directly using additional EMSA experiments. As can be seen in Fig.
3A, with large molar excesses of the 0031 oligonucleotide we
could successfully inhibit the formation of complexes C, but not
complexes A and B, satisfying our first prediction. Fig. 3B
demonstrates that the 031 probe, like the P4 probe, is able to form
complex C, but unlike the P4 probe, it is unable to form complexes A
and B when incubated with D10(CD4SP) cell extract. As a control, we showed that an oligo with an extensive mutation of the Ets core consensus, P4mEts, also fails in complex formation and so does oligo
058, which has a similar mutation to 031 (see Fig. 2A). In
addition, we demonstrated that the complexes formed by the 031 probe
are specific, because they can be competed away with the P4mEts and 031 oligos, but not with the linker oligo (Fig. 3B). Thus, we
satisfy our second prediction and demonstrate that a single base pair
change, previously proven to abrogate Elf-1 complex formation (49),
abrogates formation of complexes A and B.
To demonstrate conclusively that Elf-1 is binding to P4, we conducted
EMSA supershift experiments with an antiserum specific for Elf-1 (Fig.
4). We could completely ablate complex B
with the addition of an anti Elf-1 antiserum and detected a novel
slower-mobility complex (Fig. 4A). We could detect no
changes when we incubated the EMSA reaction with similar amounts of
normal rabbit serum. In contrast, we could not supershift any of the
P4-binding complexes using an antiserum generated against the Ets-1 and
Ets-2 proteins (Fig. 4A and data not shown). We obtained
similar results using nuclear extracts from CD8 SP, DP, and DN T cells,
indicating that Elf-1 is expressed in all of these cells types and is
capable of binding to the P4 site of the CD4 promoter (Fig.
4B). Thus, the EMSA and supershift data are consistent with
the functional data and suggest that Elf-1 is binding to the
CD4 promoter and contributing to CD4 promoter
function.
The Complex A Does Not Contain Elf-1--
Interestingly, we could
not supershift complex A (Figs. 3 and 4). It is possible that this
complex represents Elf-1 either in a different conformation or bound in
a multifactor complex, the formation of which blocks the epitope
recognized by the antiserum. Alternative, this complex could represent
a novel transcription factor. To distinguish between these
possibilities, we conducted multiple biochemical experiments using the
anti-Elf-1 antibody to characterize the complex A. Should Elf-1 be
present in complex A and the antiserum-reactive epitopes be blocked by
protein conformation or by a bound cofactor, we would predict that
denaturing the complex would expose the epitope and thus enable the
antiserum to identify the Elf-1 component. To test this, we conducted a
large-scale EMSA reaction with the P4 probe, resolved the reaction on a
nondenaturing polyacrylamide gel as for a normal EMSA experiment, and
simultaneously transferred the protein products in the gel under
denaturing conditions to nitrocellulose and the radioactive DNA probe
to DE81 paper. The nitrocellulose membrane was then probed with the
anti-Elf-1 antisera, and the DE81 paper was exposed to x-ray film.
Thus, two superimposable images were formed; proteins identified by Western blotting can be assigned to complexes formed in the EMSA experiment by aligning the images. Should complex A contain Elf-1, the
epitope of which is blocked by protein conformation or by a bound
cofactor, we would predict that we would be able to detect Elf-1 in
complex A in this experiment because the protein is denatured during
the transfer to the membrane. As seen in Figs. 4 and
5, using nuclear extracts from D10(CD4
SP), AKR1G1(DP), and S49(DN) T cells, we could detect multiple
complexes binding to the P4 probe (Fig. 5, EMSA panels, A, left
lane; B and C, left and middle lanes). As
expected, we can detect Elf-1 binding to the probe (Fig. 5,
It is possible that the sensitivity of the shift-Western assay is
insufficient to detect Elf-1 in the A complex. To address this, we
immunodepleted the nuclear extracts with the anti-Elf-1 antisera and
used these extracts in EMSA reactions with the P4 probe. Although
complex A is not recognized by the antisera, should it contain Elf-1 in
a different conformation, complete immunodepletion of all Elf-1 in the
cell may shift the overall equilibrium of the conformation or binding
reaction, thus leading to a diminution of intensity of complex A on
EMSA. We immunoprecipitated Elf-1 out of S49(DN) extracts and tested
the efficiency of the immunodepletion by Western analysis (Fig.
6A). The presence of Elf-1 is
easily detectable in the immunoprecipitate (Fig. 6A,
Finally, we transfected 293T cells with an Elf-1 expression vector,
purified nuclear extracts, and used these extracts in EMSAs. The 293T
cell line does not express endogenous Elf-1 (Fig. 6A);
should complex A represent Elf-1 in a different protein conformation or
Elf-1 bound to a cofactor, we would predict that we would be able to
detect both complexes on EMSA in the transfected 293T cells but not the
untransfected 293T cells. As can be seen in Fig. 6A, using
Western analyses, we could detect two novel protein species at the
appropriate molecular weight for Elf-1 in the transfected 293T cells
but not the untransfected cells. Immunodepletion of these extracts led
to the complete depletion of both of these protein species, suggesting
that both represent Elf-1 (Fig. 6A). The reason for the
difference between the two species is unknown, but these species may
represent different stages of phosphorylation of Elf-1. EMSA with
nuclear extracts purified from the Elf-1-transfected 293T cells
revealed a single new complex that comigrates with the Elf-1 complex in
the T cell nuclear extracts; however, we could not detect complex A
(Fig. 6B). This novel complex in the nuclear extracts from
Elf-1-transfected 293T cells could be detected on the Western blot of a
shift-Western experiment (data not shown) and disappeared upon
immunodepletion with anti-Elf-1 antibody (Fig. 6A). Thus, we
can conclude that the full-length Elf-1 product binds specifically to
the Ets sequence of the P4 probe and that complex A does not form in
the presence of Elf-1 in 293T cells. Taken together, the above
experiments support the hypothesis that complex A does not contain
Elf-1.
Characterization of the Second P4-binding Factor--
Although
complex A does not contain Elf-1, it is possible that this complex
represents one of the other two members of the Elf-1 family (51-53).
One of these factors, MEF, is myeloid-specific; as complex A is clearly
present in all T cells, MEF is unlikely to be a component of this
complex (53). The second member of the Elf-1 family, NERF (51), has a
DNA-binding domain that is almost identical to that of Elf-1 and has a
broad tissue distribution; it is therefore possible that the Elf-1-like
factor binding to P4 in our EMSA experiments is NERF. To address this
issue directly, we conducted additional biochemical studies using an
antiserum generated against human NERF. We first determined the
expression pattern of NERF using Western analysis on human and murine
cell lines. We could detect NERF in HELA and 293T cells, but not in Jurkat or Namalwa cells (Fig.
7A), suggesting that NERF is
not expressed in the lymphoid lineage. In addition, 293T cells
expressed NERF but did not form either complex A or B in our EMSA
experiments (Fig. 6B). We could detect Elf-1 in all
lymphocyte lines, as well as in Hela, but not the 293T cells,
consistent with the expression pattern of Elf-1 (Fig. 7B).
These data suggest that NERF is not likely to be factor that we detect
binding to P4 as complex A. Interestingly, we could not detect NERF
expression in any of the murine cell lines, including the nonlymphoid
L929 cell line. It is possible that the anti-NERF antibody does not
recognize NERF of murine origin; alternatively, a murine homologue of
NERF may not exist. However, our data presented above from the human
cell lines exclude NERF as a possible P4-binding protein. Another
possibility is that this second factor could be recognizing a different
recognition sequence in the P4 region. An extensive data bank search
showed that there were weak consensus Ikaros and TFII-I recognition
sites within the P4 region (35, 64). However, using EMSA competition as
well as antibody supershift/ablation experiments, we were unable to
demonstrate that these factors are binding to the P4 probe, suggesting
that complex A is not likely to be either of these factors (data not
shown).
To characterize the second P4-binding factor further, we conducted UV
cross-link experiments to estimate its molecular weight. A preparative
EMSA reaction was resolved on a nondenaturing polyacrylamide gel and
exposed to ultraviolet light. The two P4-binding complexes were then
excised and eluted from the gel, resolved on a denaturing polyacrylamide gel, and visualized using autoradiography. As can be
seen in Fig. 8, the molecular mass of
complex A is approximately 75 kDa (Fig. 8, left lane);
subtracting the molecular mass of the DNA probe, we obtained an
apparent molecular mass of 60 kDa for the DNA-binding component of
factor A. All of the members of the Ets and Elf transcription factor
families, including MEF and NERF, size much larger on an SDS-PAGE gel
(51, 53); thus, our data support the above conclusions that the second
P4-binding factor is not a known member of these families. The
lower P4 protein-DNA complex is approximately 110 kDa (Fig. 8,
right lane), giving an apparent molecular mass for the lower
P4-binding factor of 95 kDa, which is the size of Elf-1 when analyzed
on an SDS-PAGE gel. Thus these results are consistent with our antibody
experiments and support our hypothesis that the factor forming complex
B contains Elf-1.
Elf-1 Activates the CD4 Promoter--
Our data suggest that Elf-1
binds specifically to the CD4 promoter at P4. Thus, we
predicted that we would be able to activate transcription from a
reporter construct containing the CD4 promoter in a
CD4+ T cell by overexpressing Elf-1. To test this, we
conducted cotransfection experiments (Fig.
9). In this experiment, we transfected
the pSS006 reporter construct into different T cell lines with
increasing amounts of an expression construct containing the murine
Elf-1 gene under the transcriptional control of the CMV LTR. The pSS006 construct contains the intact Elf-1 Binds to the P4 Site and Activates CD4 Promoter
Function The observation that Elf-1 is important in CD4 gene
expression is especially interesting in light of recent work on the
role of the Ets transcription factors in lymphocyte development (47). The control of CD4 gene expression is intimately linked to
many different aspects of T cell development and function, and thus the
factors that bind to the CD4 transcriptional control
elements and mediate their function are believed to be linked to these processes. Elf-1 may be playing multiple roles in the control of
CD4 gene expression during these events. It is interesting to speculate that Elf-1 may be playing a role in linking signaling from
the T cell antigen receptor to the CD4 gene during T cell development via the mitogen-activated protein kinase (MAPK) pathway, which has been described as being important in thymic selection (54,
55). In Drosophila, several Ets transcription factors are
targets of the MAPK pathway (see below for discussion); by analogy,
Elf-1 may be a target of the mammalian MAPK pathway. One attractive
hypothesis is that signaling from the MAPK pathway affects Elf-1
function, thus inducing or repressing the expression of CD4.
Along these lines, it is interesting to note that the overexpression of
Elf-1 cannot transactivate the CD4 promoter in
CD4 Other Factors Bind to the P4 Region--
Interestingly, we have
also shown that a novel factor also binds to the P4 region. The role of
this novel protein in the control of CD4 gene expression is
unknown. It is possible that this novel protein mediates the effect of
Elf-1 on CD4 promoter function. There are several examples
in which two factors bind to the same site and have different effects
on the control of gene expression during development. The proper
development of the R7 photoreceptor in Drosophila
melanogaster requires signaling from the transmembrane receptor
protein tyrosine kinase Sevenless (Sev) (56). Signals through Sev are
transmitted through the MEK-1 and MAPK homologues DSOR1 and Rolled to
two transcription factors: Pointed P2 and Yan (57). Both Pointed and
Yan are homologues of mammalian Ets/Elf family transcription factors
and play important roles in the control of developmental gene
expression. Pointed is a positive transcription factor, whereas Yan
represses transcription; whether or not the target gene is transcribed
is dependent on which of the two factors predominates. It is
interesting to speculate that Elf-1 and the novel factor are
functioning in a similar manner at the CD4 promoter. However, additional experiments are necessary to demonstrate this point conclusively.
We can also identify other protein-DNA complexes in our EMSA
experiments with the P4 probe (Fig. 3). The P4 site is only two bases
5' of the initiation site of transcription of the CD4 gene containing a consensus initiator (Inr) sequence. The close proximity of
the Elf-1 and Inr sites in the CD4 promoter may indicate
that Elf-1 and/or the Elf-like factor interact(s) directly with
Inr-binding proteins to mediate promoter function. Ets proteins have
been demonstrated to be weak activators by themselves, and their
diversity of cell type expression is believed to be mediated at least
in part through the association with cofactors. For example, Ets-1 binds to a functional site in the T cell antigen receptor The Elf-1-containing P4-binding Complex Also Binds to the CD4 Locus
Distal Enhancer--
We have previously shown that the distal enhancer
contains multiple factor-binding sites, including an Elf-1 recognition
site. Interestingly, the Elf-1 probe from the CD4 locus
distal enhancer is capable of competing away formation of all of the
CD4 promoter P4-binding complexes (Fig. 3), indicating that
the P4-binding factors have the same sequence specificity as the distal
enhancer-binding factors. In addition, EMSAs with the Elf-1 site from
the distal enhancer as a radioactive probe reveal the same complexes as
EMSAs with the P4 probe (data not shown). These data indicate that
similar Elf-1-Inr protein complexes may be binding both the promoter
and the distal enhancer. Most promoter-binding factors can function equally well as enhancer-binding factors; therefore, the presence of
similar complexes binding to both an enhancer and a promoter of a gene
is not unprecedented. However, these experiments predict that the
specificity of function of the promoter and the distal enhancer would
be very similar. Our previous experiments, as well as those published
here, support this hypothesis (25, 28). The similarity of function of
these two elements is most likely the result of similar transcription
factor complexes binding to critical functional sites in both elements.
Although the distal enhancer was first described as functioning on the
CD4 promoter in transient transfection studies, its functional relevance for in vivo CD4 gene
expression has not been demonstrated (25). Transgenic experiments
demonstrate that the CD4 distal enhancer is not necessary
for the correct developmental expression of the CD4 gene and
indeed cannot enhance the CD4 promoter in vivo,
providing evidence that it is irrelevant for the control of
CD4 gene
expression.3 It is possible
that the distal enhancer may be a control element for a different gene
located in the CD4 locus. There are at least two genes
located in the CD4 locus adjacent to the distal enhancer: LAG-3, a gene with structural similarity to CD4 located
immediately downstream of the distal enhancer; and the parathymosin
gene (59, 60). It is possible that the CD4 locus distal
enhancer serves as a regulatory element for one of these other genes
rather than the CD4 gene. Should this be the case, the
requirement for similar Elf-1 protein complexes binding to these two
control elements indicates that the expression of these genes may be
coordinately controlled. This is at least partly the case for the
LAG-3 gene, which is expressed at high levels in activated T
cells (61-63). However, to address this issue in detail, it will be
necessary to generate mice that are transgenic for constructs that
contain different combinations of the CD4 promoter and
enhancer elements to determine how these elements function
independently of each other. We are currently conducting experiments to
test these hypotheses further.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
helper T cell (TH) that
recognizes antigen bound to MHC class II or a
CD4
CD8+ cytotoxic T cell (TC)
that recognizes antigen bound to MHC class I (12-16). Thus the control
of expression of the CD4 gene is directly linked to T cell
development (17-23).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
TH2 clone D10 and the
CD4
CD8+ TC clones L3 and B18 (38)
were maintained in Click's medium (EHAA) supplemented with 10% fetal
calf serum, penicillin/streptomycin (0.1 mg/ml), L-glutamine (2 mM), 2-mercaptoethanol (50 µM), and interleukin-2 (10-30 U/ml). AKR1G1 (a double
positive lymphoma), S49 (a double negative lymphoma), and 293T cells
were maintained in the same medium without interleukin-2. The cells
clones were stimulated every 2 weeks with antigen as described (28).
Nuclear extracts were prepared using a modified Dignam protocol (39) or
by the Schrieber protocol (40). Transient transfections were performed between day 4 and day 6 after stimulation as described (28). In short,
two plasmids were introduced together into cells by the DEAE-dextran
method; the first was an experimental plasmid with the test
CD4 promoter cloned upstream of the luciferase gene in pGL2,
and the second was the transfection control plasmid pRL-TK (Promega)
containing the Renilla luciferase gene under the control of
the tk promoter. Cells were harvested after 48 h, and extracts were prepared for the Dual Luciferase assay as recommended by the
manufacturer (Promega). All data points are corrected for transfection
efficiency and are presented as a percentage of the wild type
CD4 promoter activity. At least five independent
transfections were performed for each construct, and the percentages
were averaged as described (30). When different amounts of the
pRcCMV-mElf-1 plasmid (41) were cotransfected for the transactivation
experiments, the DNA amount was kept constant by the addition of an
irrelevant plasmid (pKS). The fold activation was calculated relative
to the CD4 wild type promoter. 293T cells were transfected
with the Elf-1 expression vector by the calcium phosphate precipitation method (42). Forty-eight hours later, the cells were harvested, and
nuclear protein extracts were made for use in EMSA as described below.
20 to +4 of the murine CD4 promoter was subcloned and used as a radioactive probe
(2-5 × 104 cpm/reaction) for EMSA with equal protein
amounts of nuclear extracts from D10, L3, S49, and AKR1G1 cell lines.
The reaction also included 1 µg of dIdC as a nonspecific competitor,
1 mM spermidine, and reaction buffer (10 mM
HEPES, pH 7.9, 50 mM NaCl, 5 mM Tris-HCl, pH
7.5, 25 mM EDTA, 1 mM dithiothreitol, and 10%
glycerol). For competitions, a 50- or 100-fold molar excess of
nonradioactive oligonucleotides was used. Competitors included P4, the
mutant P4 site pSS031, P4mEts (Fig. 2), the distal enhancer Elf-1
binding site 5'-CTTGGGGGCAGGACTTCCTGTTTCT-3' (DE-Elf), and the pKS
linker between the XbaI and XhoI restriction
enzyme sites as a nonspecific oligonucleotide. After 15 min of
incubation at room temperature, the reactions were resolved on a
nondenaturing 4% polyacrylamide gel and run at 150 V for 3 h at
room temperature in glycine buffer (190 mM glycine, 25 mM Tris-HCl, pH 8.5, 1 mM EDTA). Anti-Elf-1 antibody (sc-631X), anti-NERF antibody (sc-6829X), and rabbit IgG
(sc-2027) were obtained from Santa Cruz Biotech. Supershifts were
performed by adding 1 µg of antibody to the EMSA reaction and
incubating on ice for 15 min before loading on gels. Gels were dried
and exposed on x-ray film overnight at
70 °C with an intensifying
screen. For the fractionation of the EMSA complexes on the SDS-PAGE
gel, EMSA binding reactions were resolved on a nondenaturing acrylamide
gel as described above. The gel was then exposed to UV irradiation as
described, the appropriate complexes were visualized by
autoradiography, excised from the gel, boiled for 5 min in SDS sample
buffer, placed into the well of a 10% SDS-PAGE gel, and subjected to
electrophoresis (44).
70 °C with an intensifying screen. All radioactivity
was captured on the DE81 paper, thus providing an image of all the
complexes formed that was superimposable with the Western blot. No
radioactivity could be detected on the nitrocellulose membrane.
-mercaptoethanol). This constitutes the IP panel in Fig.
6A. The supernatant was spun one more time to remove any
remaining beads, and aliquots were boiled in 2× SDS loading buffer for
5 min. This constitutes the post-IP supernatant panel in Fig.
6A. The boiled fractions were analyzed on an 8% SDS gel and
transferred overnight to a nitrocellulose membrane using the Bio-Rad
wet transfer system, and then a Western blot with anti-Elf-1 antibody
was performed to demonstrate the level of Elf-1 depletion from the extracts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
9 position, and thus the sequences in the P4 site that are
important for promoter function include the consensus Ets recognition
site.
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Fig. 1.
The P4 site of the CD4
promoter contains an Ets consensus sequence. A,
schematic representation of the minimal CD4 promoter and its
cis- and trans-regulatory elements; see text for details. The sequence
around the P4 site is shown in detail, with the Ets core consensus
sequence underlined. Consensus binding sequences for several
Ets family members (C) are aligned with the broad family
consensus sequence, the CD4 distal enhancer Elf-1 binding
sequence and the CD4 promoter P4 site (B). The
Ets core consensus is outlined. The sequence degeneracy
around the core consensus determines the specificity of binding
in vivo for different members of the family.
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Fig. 2.
Functional analysis of the P4 site.
A, the sequence of the P4 probe, representing the
CD4 promoter between 16 and +6, is shown and aligned with
the Ets consensus sequence. The transcription start site is indicated
by an arrow. The pSS series indicates mutations in the P4
site that were generated in the context of the entire minimal promoter
(
101 to +71). P4mETS is an oligonucleotide, synthesized to contain a
mutant Elf-1 site at the P4 site. DE-Elf is an oligonucleotide that
spans the Elf site of the CD4 distal enhancer. B,
functional activity of the mutant CD4 promoter in the
CD4+CD8
TH cell clone D10.
Transfection efficiency was controlled as described under "Materials
and Methods." The activity of the mutant promoters is described as a
percentage of the wild type promoter activity, which is taken to be
100%. Raw luciferase units for the unmutated promoter were 2-4 × 104 light units. The data are representative of 7-10
independent transfections.
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Fig. 3.
EMSA of the P4 site. A, EMSA
using D10 (CD4+CD8 ) nuclear extracts and the
P4 probe. Nonradioactive competitors are indicated above the lanes and
are used in 50-200-fold molar excess. P4mETS has the Ets
core consensus mutated, DE is the DE-Elf, L is
pKS linker sequence, and 031 is an oligonucleotide
containing the pSS031 T to A mutation (see Fig. 2A for
sequences). Arrows indicate the Elf-1 sequence-specific
complexes (A and B). The thin arrow
indicates a third major specific complex binding to P4 outside of the
Ets consensus (C). Three minor specific complexes were also
present (see text for details). B, EMSA using D10(CD4SP)
cell extracts and four different probes, as indicated above the lanes.
Probe sequences are shown in Fig. 2A. Cold competition of
the complexes, formed with the 031 probe, are shown; conditions are as
in Fig. 3A.
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Fig. 4.
Endogenous Elf-1 binds to the P4 region.
Supershifts using D10 extracts (A) and L3, AKR1G1, and S49
extracts (B) with the P4 probe are shown. Antibodies used
were polyclonal antisera and are indicated above the lanes. The
anti-Ets-1/2 antibody recognizes both Ets-1 and Ets-2. The anti-Elf-1
antibody is Elf-1-specific. Rabbit IgG is used as preimmune control.
For A, two different amounts of each antibody were used;
3× indicates 3-fold more antibody used. Arrows
indicate native and supershifted Elf-1 complexes.
-Elf-1 blot panels). This band does not appear in the lanes containing EMSA reactions supershifted with the anti-Elf-1 antisera, because its mobility has been slowed significantly by the
anti-Elf-1 antibody. These data strongly support our previous data,
suggesting that this complex represents endogenous Elf-1 binding to P4.
Interestingly, we were not able to detect Elf-1 in complex A on Western
analysis under any conditions (Fig. 5,
-Elf-1 blot panels;
arrow labeled A ). This is especially clear in the
supershift lanes, in which signal from the Elf-1 complex cannot obscure
a fainter signal from the A complex. These data are also consistent
with our EMSA-supershift data described above and suggest that complex
A does not contain Elf-1.
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Fig. 5.
The upper complex does not contain
Elf-1. EMSA binding reactions using the P4 probe and D10
(A), AKR1G1 (B), or S49 (C) nuclear
extracts were resolved on 4% native polyacrylamide gels, and then each
gel was simultaneously blotted onto nitrocellulose membrane
(right panels) and DE81 paper (left panels) as
described under "Materials and Methods." During the transfer, the
DNA-protein complexes were dissociated, such that the proteins remain
bound to the nitrocellulose membrane, whereas the radioactively labeled
probe migrated through the nitrocellulose membrane and was retained on
the DE81 membrane. Thus, any protein detected on the membrane by
Western blot (right panels) could be subsequently identified
as a member of a particular complex by alignment with the image from
the DE81 paper (left panels). The amount of extract used and
the presence or absence of anti-Elf-1 antibody in the binding reaction
is indicated above each lane. Western analysis was done with anti-Elf-1
antibody (right panels). Arrows indicate the
position of the Elf-1 complex and complex A.
-Elf-1
IP), although it is not detectable in the supernatant as compared
with an equal amount of untreated extract (Fig. 6A, post IP
sup.). We could detect no differences between immunodepleted and
untreated extracts in EMSA experiments with non-Elf site-containing
probes, indicating that the depleted extracts are essentially intact.
As shown in Fig. 6B, after the immunodepletion of S49
nuclear extracts with anti-Elf-1 antibody, the Elf-1 complex did not
form, whereas all other complexes remained unaffected. Because complex
A remained unaffected by immunodepletion with anti-Elf-1 antibody, this
experiment further supports the notion that complex A does not contain
Elf-1.
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Fig. 6.
Immunodepletion of Elf-1 abrogates complex
formation. A, efficiency of the immunodepletion of
Elf-1 from nuclear extracts. Immunodepleted S49-, 293T-, and
Elf-1-transfected 293T nuclear extracts and immunoprecipitated Elf-1
from the same extracts were run on an 8% SDS-PAGE gel and analyzed by
Western blotting with the anti-Elf-1 antibody. The position of the
Elf-1 protein is indicated by an arrow. The extracts are
indicated above each lane. The second of the two S49 lanes represents
treatment with twice as much antibody. The left panel shows
the immunoprecipitated Elf-1 protein from all extracts, analyzed by
Western blot. The right panel shows the SDS-PAGE analysis of
the post-IP supernatants (indicated by sup after the cell
line name). As a control, nonimmunodepleted extracts from S49(DN) and
293T cells are run on the same blot. Equal amounts of total protein was
loaded on each lane as estimated by the Bradford assay and confirmed by
Ponceau S staining of the membrane. B, EMSA of
immunodepleted and untreated extracts from S49, 293T, and
Elf-1-transfected 293T cells with the radioactively labeled P4 probe.
Extracts and their treatment are indicated above each lane. The
triangles indicate increasing amount of extract added for the 293T- and
Elf-1-transfected 293 lanes; for the S49 lanes, it represents
increasing amount of antibody used for the immunodepletion. Complexes A
and B are indicated by filled and empty arrows,
respectively.
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Fig. 7.
Expression pattern of Elf-1 and NERF.
Western blot with anti-Elf-1 antibody (A) and anti-NERF
antibody (B). The cell lines used are indicated above the
lanes. The relative migration of the molecular mass markers is
indicated on the side (in kDa). Labeled arrows point to
Elf-1 and NERF, detected on the blots by their respective
antibodies.
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Fig. 8.
Determining the molecular mass of the
P4-binding complexes. Preparative EMSA binding reactions were
resolved on a nondenaturing PAGE gel; the gel was exposed to UV
radiation, as described under "Materials and Methods"; complex A,
the Elf-1 complex, and the intervening space were excised; and the
protein-DNA products were eluted and resolved on a denaturing PAGE gel.
The migration position of the complexes is indicated both on the
SDS-PAGE and EMSA gels. Molecular mass markers are indicated.
101 minimal CD4 promoter
driving the firefly luciferase gene; should Elf-1 bind to the P4 site, we would predict that we would be able to transactivate the
CD4 promoter by overexpressing Elf-1. As a specificity
control, we conducted similar experiments using the pSS031 and pSS054
reporter constructs, which contains a site-specific mutations in the
Elf-1 recognition site (Fig. 2). As can be seen in Fig. 9, we detected a dose-dependent increase in CD4 promoter
function in the CD4+CD8+ T cells using the
pSS006 construct and increasing amounts of the CMV-Elf-1 expression
plasmid; addition of comparable amounts of CMV plasmid alone does not
lead to promoter induction. This induction is also dependent upon the
presence of the Elf-1 recognition site in P4; addition of increasing
amounts of CMV-Elf-1 plasmid to the pSS054 transfection leads to only a
slight increase in promoter activity. These data indicate that Elf-1 is
capable of binding to P4 and inducing promoter function, supporting the
hypothesis that endogenous Elf-1 is in fact mediating CD4
gene expression in vivo at the promoter. To test whether
Elf-1 can transactivate the CD4 promoter in T cells that do
not express CD4, we conducted transactivation experiments
with the CMV-Elf-1 expression and the pSS006 reporter vectors in the L3
and B18 CD8 SP TC clones (Fig. 9). Interestingly, although
we could transactivate the CD4 promoter in the
CD4+CD8+ T cell line, we could not do so in
either CD8 SP TC clone, even with the addition of large
amounts of CMV-Elf-1 expression plasmid. We obtained similar results
with the CD4
CD8
T cell line S49 (data not
shown). These data suggest that Elf-1 is not capable of transactivating
the CD4 promoter in cell types that do not express CD4.
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Fig. 9.
Transactivation of the CD4 promoter by
Elf-1. Five µg of the minimal CD4 promoter luciferase
reporter plasmid or its mutant version pSS054 were cotransfected with
increasing amounts of pRcCMVmElf-1 as indicated. The total DNA
transfected was kept constant at 20 µg by addition of pKS plasmid
where necessary. Wild type promoter activity is taken to be 1, as a
basis for comparison. Transactivation is represented as relative
activity, compared with the wild type promoter. The data are
representative of five independent transfections for each of the cell
lines used. The developmental stage of each cell line is
indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Our data indicate that endogenous Elf-1, a member of the
Ets family, binds to the P4 functional site of the murine
CD4 promoter and activates its function. We draw this
conclusion on the basis of results we have obtained from seven
different experimental approaches. First, we demonstrate that a subset
of the T cell nuclear factors binding to the P4 region recognize the
Ets consensus sequence (Fig. 3). Second, we took advantage of the fact
that Elf-1, alone of all the Ets family members, cannot bind to the Ets
core sequence variant ATCC. Using biochemical experiments we
demonstrate that the nuclear factors binding to P4 also cannot recognize a ATCC variant of the P4 sequence (Figs. 2 and 3). In addition, the single base mutation of the P4 site to the ATCC sequence
abrogates CD4 promoter function (Fig. 2). Third, we were able to supershift a major P4-binding complex using antisera directed against Elf-1, but not with normal rabbit serum or antisera directed against Ets-1, Ets-2, or the other Ets family members, indicating that
it is unlikely that these other Ets family members are binding to P4
(Fig. 4 and data not shown). Fourth, we could also detect the presence
of Elf-1 in a major P4 protein-DNA complex by shift-Western experiments
(Fig. 5). Fifth, Elf-1-depleted T cell extracts failed to form the
specific P4 complex on EMSA (Fig. 6). Sixth, nuclear extracts purified
from 293T cells transfected with an Elf-1 expression construct formed a
novel complex with the P4 probe on EMSA that can be depleted with the
anti Elf-1 antisera; this complex migrated in the exact same manner as
the putative Elf-1-containing complex in T cell nuclear extracts (Fig.
6). Finally, we could transactivate the CD4 promoter by
overexpressing Elf-1 in a CD4+ T cell line; this
transactivation is dependent on the intact Elf-1 site (Fig. 9).
T cells but can do so in CD4+ T cells,
indicating that there may be functional differences in Elf-1 between T
cells of different developmental phenotypes.
chain gene enhancer and is believed to function only in conjunction with
LEF-1, the recognition site of which is directly adjacent to the Ets
consensus (58). As the initiator sequence is immediately 3' of the
Elf-1 recognition site, it is possible that an initiator factor may be
interacting with Elf-1 at different stages of development to convey
promoter specificity.
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ACKNOWLEDGEMENTS |
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We thank Drs. Martine Roussel and Frank Fitch for the CMV-Elf-1 expression plasmid and the B18 TC clone, respectively. We also thank Drs. Kathryn Calame, Steve Greenberg, Alessandra Pernis, and Chris Schindler for helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1 AI34925 and by a grant from the Irma T. Hirschl-Monique Weill Caulier Charitable Trust (to G. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Microbiology, Columbia University, College of Physicians and Surgeons, 701 W. 168th St., New York, NY 10032. Tel.: 212-305-2743; Fax: 212-305-8013; E-mail: siu{at}cusiu3.cpmc.columbia.edu.
2 W. W. S. Kim and G. Siu, submitted for publication.
3 M. Adlam, R. D. Allen, and G. Siu, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MHC, major histocompatibility complex; MAPK, mitogen-activated protein kinase; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation.
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REFERENCES |
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