From the Eppley Institute for Research in Cancer and
Allied Diseases and the ¶ Department of Pathology and
Microbiology, University of Nebraska Medical Center, Omaha, Nebraska
68198
Received for publication, July 26, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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Distal enhancers commonly regulate gene
expression. However, the mechanisms of transcriptional mediation by
distal enhancers remain largely unknown. To better understand distal
enhancer-mediated transcription, we examined the regulation of the
FGF-4 gene. The FGF-4 gene is regulated during
early development by a powerful distal enhancer located downstream of
the promoter in exon 3. Sox-2 and Oct-3 bind to the enhancer and are
required for the activation of the FGF-4 gene. Previously,
we implicated the co-activator p300 as a mediator of Sox-2/Oct-3
synergistic activation of a heterologous promoter, suggesting that p300
may play a role in mediating enhancer activation of the
FGF-4 gene. In this study, we provide both functional and
physical evidence that p300 plays an important role in the action of
the FGF-4 enhancer. Specifically, we show that E1a, but not
a mutant form of E1a that is unable to bind p300, inhibits enhancer
activation of the FGF-4 promoter. We also demonstrate that
Gal4/p300 fusion proteins can stimulate the FGF-4
promoter when bound to the FGF-4 enhancer.
Additionally, we present evidence that p300 mediation of the
FGF-4 enhancer requires acetyltransferase activity.
Importantly, we also show that Sox-2 and p300 are physically associated
with the endogenous FGF-4 enhancer but weakly associated
with the endogenous FGF-4 promoter. These results are
consistent with a model of transitory interaction between the distal
enhancer and the FGF-4 promoter. Our results also suggest
that intragenic distal enhancers may use mechanisms that differ from
extragenic distal enhancers.
Enhancers regulate the transcriptional activation of many, and
perhaps most, genes. It is thought that activators bound to an enhancer
located close to a promoter help recruit the preinitiation complex
(PIC)1 to the promoter (1).
This recruitment is likely to be mediated through the action of
co-activators (2). Much is known about how proximal enhancers (located
within 200-500 bp of a promoter) function (3); however, it is
unknown how activators bound to distal enhancer sequences, located
several kb away, are able to stimulate a promoter. Importantly, random
interaction between a promoter and an enhancer is likely to decrease
significantly when separated by distances greater than 200 bp (4).
Several models have been proposed to explain the mechanism(s) of distal enhancers. These models can be categorized as "contact" and
"noncontact" models. Contact models propose mechanisms of
enhancer action that result in a direct interaction between the
enhancer and promoter (1, 5-7), whereas noncontact models do not
propose direct enhancer-promoter interactions (8, 9).
To better understand the mechanisms of distal enhancers, we are
investigating the transcriptional regulation of the FGF-4 gene. The FGF-4 gene is regulated by a powerful distal
enhancer located 3 kb downstream of the promoter within the
untranslated region of the third exon (10-12). This gene serves as an
excellent model as many of the critical cis-regulatory
sites, and the factors that bind them have been identified in both the
promoter (12-17) and its distal enhancer (12, 17-21). Mechanisms for
enhancer action by distal enhancers located downstream of promoters
within the transcribed region are not readily explained or addressed by
current models of distal enhancer function.
Co-activators are believed to act as bridging molecules between
promoters and proximal enhancer regions, and their roles in the
activation of the proximal enhancer and promoter of the
interferon- Previously, we identified p300 as a co-activator that can mediate the
synergistic activation of transcription by the transcription factors
Sox-2 and Oct-3 (38). Importantly, FGF-4 expression requires
Sox-2 and Oct-3 binding to its distal enhancer (12, 20, 21), suggesting
a possible role for p300 in the regulation of the FGF-4
gene. However, the studies implicating p300 were performed using
heterologous promoter/reporter constructs in cells where Sox-2 and
Oct-3 are not expressed. To advance our understanding of the role of
p300 in the regulation of the FGF-4 gene and the mechanisms
of intragenic distal enhancers, we have employed F9 embryonal carcinoma
(EC) cells. These cells biochemically and morphologically resemble the
cells of the inner cell mass where Sox-2, Oct-3, and FGF-4 are
expressed (18, 39-41). In addition, transcription of the
FGF-4 gene decreases dramatically when EC cells undergo
differentiation (10, 12, 42). Hence, these cells provide an excellent
model for studying the regulation of FGF-4 expression. To
this end, several questions are addressed in this study. First, can
p300 mediate the action of the FGF-4 enhancer, and is its AT
activity necessary? Second, does p300 associate physically with the
endogenous FGF-4 enhancer and/or promoter? We demonstrate
that p300 can stimulate the FGF-4 promoter when tethered to
the enhancer, and full stimulation by p300 requires the
CCAAT box and GC boxes in the promoter. This
stimulation is dependent on the AT activity of p300. Importantly, we
also demonstrate that both Sox-2 and p300 physically associate with the
endogenous FGF-4 enhancer. Together, the findings described
in this study provide both functional and physical evidence that p300
can mediate the effect of a distal enhancer. In the case of the
FGF-4 gene, p300 may function both as an acetyltransferase
and as a transitory bridging factor able to promote enhancer-promoter interactions.
Cell Culture and Transient Transfections--
F9 embryonal
carcinoma (EC) cells were maintained in Dulbecco's modified
Eagle's medium (DMEM; Invitrogen) and 10% fetal bovine serum
(Hyclone) as described previously (12, 43). For the promoter/reporter
assays, F9 EC cells were seeded at 5 × 105 per 100-mm
dish in DMEM + 10% fetal bovine serum and transfected 24 h later
by the calcium phosphate precipitation method (12, 44). Cells were
incubated with the precipitate for 14-16 h, washed with DMEM, and
re-fed with DMEM + 10% fetal bovine serum. The following day, cells
were harvested, and cell extracts were prepared. Chloramphenicol
acetyltransferase (CAT) and Plasmids and Cloning--
The promoter/reporter constructs
kFGF427T+E (referred to as 427T+E) and
pCatSO3 have been described previously (12, 38, 45).
pBLCat6+E is based on a previous construct,
pBLCat6 (45), and contains the FGF-4 enhancer
from 427T+E ligated into the SacI site downstream
of the CAT gene. The remaining promoter/reporter constructs
are based on 427T+E. 427T+EnGSp was constructed
using the following primers in PCR with 427T+E to amplify
the entire plasmid but containing a Gal4 site in place of
the Sox-2 (HMG) and Oct-3
(POU) sites of the enhancer: upstream primer,
5'-ccgcggAGGACAGTCCACCGGGATAACTTAAAATACTATTCTG-3'; downstream primer, 5'-CTAAAGGAATGTGAAAGACA-3'.
Lowercase sequence denotes a SacII site used for
screening purposes, sequence in bold denotes the Gal4
binding site sequence, and the underlined sequence is wild-type
enhancer sequence. 427T SpDP-E is derived from
427T+E and contains mutations in both GC boxes of
the promoter but lacks the enhancer and was constructed by removing the
enhancer of 427T SpDP (15) by digestion and re-ligation of
the SacI sites flanking the enhancer. The enhancer
containing the Gal4 site was isolated from
427T+EnGSp by digestion with SacI and ligated
into the SacI sites of 427T SpDP-E and 427T
CCAATmut (containing a mutant CCAAT box and lacking the
enhancer) (46) to produce 427T GCmut+EnGSp and 427T
Cmut+EnGSp, respectively. A 2.4-kb BsaI fragment from
427T SpDP encompassing the GC box mutations of
the promoter was ligated to a 3.55-kb BsaI fragment isolated
from 427T Cmut+EnGSp to produce 427T
GC/Cmut+EnGSp. This construct contains mutations in the
GC boxes and CCAAT box of the promoter and an
enhancer with a Gal4 site in place of the HMG
and POU sites.
Expression plasmids utilized include the following: pCMV 12SE1a (E1a)
and pCMV 12SE1a Immunoprecipitation and Western Blotting--
F9 EC cells were
transfected with 1 µg of CMV Chromatin Immunoprecipitation (ChIP)--
ChIP was performed
essentially as described previously (50) on non-transfected or
pCMVFLAGSox-2-transfected F9 EC cells as indicated. Transfections were
performed using LipofectAMINE in conjunction with the PLUS reagent
(Invitrogen) (49). DNA-protein complexes were cross-linked by addition
of formaldehyde to a final concentration of 1% to the culture medium
for 10 min at room temperature. The cross-linking reaction was stopped
with addition of glycine (0.125 M final) for 5 min. Cells
were then washed and harvested by scraping into phosphate-buffered
saline plus 0.5 mM phenylmethylsulfonyl fluoride. The cells
were lysed in lysis buffer (5 mM PIPES, 85 mM
KCl, 0.5% IGEPAL CA-630, 0.5 mM phenylmethylsulfonyl
fluoride) plus various protease inhibitors, and nuclei were collected.
Nuclei were lysed in nuclei lysis buffer (50 mM Tris-Cl, 10 mM EDTA, 1% SDS, 0.5 mM phenylmethylsulfonyl
fluoride) plus various protease inhibitors and subjected to sonication
to produce DNA fragments of ~200 to 1500 bp in length. The samples
were subjected to centrifugation to pellet cell debris, and DNA-protein
complexes were precleared with protein A beads (Santa Cruz
Biotechnology, Inc.) for 1 h at 4 °C with rotation. Each sample
was then diluted with ChIP dilution buffer (0.01% SDS, 1% Triton
X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8, 167 mM NaCl), an aliquot was removed as "input" control, and the remaining sample was divided into two aliquots. M2
(anti-FLAG; Sigma) antibody or anti-p300 (N15; Santa Cruz
Biotechnology, Inc.), alone or conjugated to agarose beads, or
anti-p300 (C20; Santa Cruz Biotechnology, Inc.) was added to one
aliquot, and anti-Gal4 (DBD) (Santa Cruz Biotechnology, Inc.), alone or
conjugated to agarose beads, was added to the second aliquot as a
nonspecific antibody control. Samples were incubated with antibody
overnight with rotation at 4 °C. The following day, protein A beads
were added to the samples with antibody not conjugated to beads and incubated for 1 h at 4 °C with rotation to collect
DNA-protein-antibody complexes. Beads were washed, and DNA-protein
complexes were eluted by two successive incubations at 65 °C for 10 min with freshly prepared elution buffer (1% SDS, 50 mM
NaHCO3). NaCl to a final concentration of 0.3 M
and RNaseA was added to the eluted and input samples and incubated at
65 °C for 4-6 h to revert cross-links. Samples were precipitated
overnight at
The following primer pairs were used for the indicated regions
of the FGF-4 gene. FGF-4 promoter: FGFproU2,
5'-GTAAGGAAGGAGCACAGGAGAT-3' and FGFproL3,
5'-CAGACCTAAGTAGCGAGAGCAA-3'; FGF-4 enhancer: FGFenhUp, 5'-AGACTTCTGAGCAACCTCCCGAAT-3' and FGFenhDown,
5'-CAACTGTCTTCTCCCCAACACTCT-3'; exon 2 of FGF-4: FGFex2U2,
5'-CGCCTCCCCAGGTCTTCT-3' and FGFex2L, 5'-CAATCCCGCTAGTCTTGCTCAC-3'; 5'
region (3.6 kb upstream of the FGF-4 promoter): FGF5'U,
5'-ACCTCACCTGTGCCCTGTTGAAT-3' and FGF5'L, 5'-GTGCTCATCTCCCCAGACTGTTTCT-3'. PCR was performed on equivalent volumes of each sample and analyzed at multiple cycles. Quantification was performed using ImageQuant analysis software (Molecular Dynamics). Results are presented for one cycle within the linear range of amplification. PCR amplifications for each primer set were repeated at
least twice within each ChIP assay with similar results. All primers
were synthesized by the Eppley Cancer Institute Molecular Biology Core Facility.
E1a, but Not mE1a, Inhibits the Enhancer through the
HMG/POU Sites--
Co-activators, including p300, play
important roles in many enhancer-promoter interactions (3, 26, 27, 51,
52). Previous results indicate that p300 can mediate the synergistic action of Oct-3 and Sox-2 in HeLa cells (38). Individually, Sox-2 and
Oct-3 have been shown to stimulate expression of a heterologous promoter/reporter construct 3- to 5-fold. However, a synergistic stimulation is observed with both proteins (25- to 40-fold) (38, 53),
and a further stimulation is observed in the presence of added p300
(38). FGF-4 expression in F9 EC cells is dependent upon Oct-3 and Sox-2
binding to a distal enhancer (12, 20, 21). This enhancer is located
downstream of the FGF-4 promoter within the untranslated
region of the third exon of the FGF-4 gene. To better
understand the mechanism of the FGF-4 distal enhancer, we
examined whether p300 is involved in mediating its action. E1a and a
mutant E1a that is unable to bind p300 (mE1a) have been used previously
to implicate p300 as a possible co-activator of gene expression (52,
54-57). To test whether p300 may be involved in mediating the action
of the FGF-4 enhancer, we performed co-transfections with
various FGF-4 promoter/reporter constructs and an expression vector for either E1a or mE1a. Initially, the promoter/reporter construct 427T+E (Fig.
1A), containing the
FGF-4 enhancer placed downstream of the CAT
reporter gene driven by 427 bp of the FGF-4 promoter, was
co-transfected with an expression vector for E1a or mE1a into F9 EC
cells. E1a caused a dose-dependent decrease in
transcriptional activity, whereas mE1a had little or no effect (Fig.
1B). The lack of effect by mE1a, which is 35 amino acids smaller than E1a, was not because of a lack of expression as both E1a
and mE1a are expressed at similar levels in F9 EC cells (Fig. 1C). Similar to previous observations with the same antibody
(47), multiple bands were detected for both E1a and mE1a. It has been suggested that the multiple species are because of post-translational modifications (58). To determine whether E1a was acting through the
FGF-4 enhancer, we utilized a promoter/reporter construct (TKCAT+E) in which the FGF-4 promoter in
427T+E was replaced with the thymidine kinase
(TK) promoter (Fig.
2A). Again, a
dose-dependent decrease in transcriptional activity was
observed in the presence of E1a but not with mE1a (Fig. 2B).
To further define the region in the enhancer affected by E1a, the
promoter/reporter construct pCatSO3 was co-transfected with
expression vectors for E1a or mE1a into F9 EC cells. pCatSO3
contains six copies of the Sox-2 (HMG) and
Oct-3 (POU) binding cassette (as it appears in
the FGF-4 enhancer) upstream of the SV40 promoter
driving the CAT reporter gene (Fig.
3A). E1a, but not mE1a,
inhibited the Sox-2/Oct-3 activation of the SV40 promoter
(Fig. 3B). As a control, E1a and mE1a expression vectors
were co-transfected with the TKCAT and SV40
parental plasmids to verify that E1a and mE1a do not affect the basal
activity of the TK and SV40 promoters (data not
shown). Together these results suggest that p300 can mediate the action
of the FGF-4 enhancer, as well as transcriptional activation
by Sox-2/Oct-3.
p300 Stimulates the FGF-4 Promoter When Tethered to the
Enhancer--
Previously, we demonstrated that the C-terminal region
of Sox-2 is able to stimulate a heterologous promoter/reporter
construct in HeLa cells identifying the C-terminal region as the
transactivation domain (TAD) (38). The Sox-2 TAD is inhibited by
E1a but not mE1a, and p300 recovers the E1a inhibition (38). Similarly, E1a, but not mE1a, inhibits Oct-3 stimulation of promoter/reporter constructs in HeLa
cells.2 This suggested
that Sox-2 and Oct-3 activate the FGF-4 promoter by
interacting with p300. To test this possibility functionally, we
replaced the Sox-2 and Oct-3 binding sites of the
enhancer in 427T+E with a binding site for the yeast
transcription factor Gal4 (427T+EnGSp; see Fig.
4A). This promoter/reporter
construct was transfected into F9 EC cells with an expression vector
for a fusion protein, Gal4/ Sox-2206-319,
containing the Gal4 DBD and the transactivation domain of Sox-2. These
transfections resulted in ~30-fold stimulation of the
FGF-4 promoter whereas co-transfections with expression
vectors for Gal4 alone or Gal4/Sox-2 constructs containing further
deletions of the C-terminal end of Sox-2 failed to significantly
stimulate the FGF-4 promoter (data not shown). These results
indicate that the C-terminal region of Sox-2 functions as a TAD in the
context of a natural promoter in cells where it is expressed. More
importantly, these results, together with the E1a results above and
earlier observations that p300 is able to mediate the TAD of Sox-2
(38), further suggest a role for p300 as a co-activator of Sox-2/Oct-3
action from the FGF-4 enhancer.
If p300 mediates the action of Sox-2/Oct-3 from the enhancer then
tethering p300 directly to the enhancer should result in a stimulation
of the FGF-4 promoter, bypassing the necessity of Sox-2/Oct-3 binding. Indeed, bypass experiments have implicated CBP, a
homolog of p300, in the mediation of gene expression (27, 51, 59).
Additionally, by directly targeting a factor to a gene, problems
inherent in overexpression studies can be avoided. To test whether p300
is able to stimulate the FGF-4 promoter from the enhancer,
427T+EnGSp was transfected into F9 EC cells with expression
vectors for fusion proteins containing the DBD of Gal4 and aa 1-2414
(full-length) or aa 964-1922 of p300 (Gal4/p300 1-2414 or Gal4/p300
964-1922, respectively). Both fusion proteins stimulated the
FGF-4 promoter from the FGF-4 enhancer (Fig.
4B). The differences in stimulation of 427T+EnGSp
by full-length p300, aa 1-2414, (15-fold), and amino acids 964-1922
of p300 (over 100-fold) may be because of differences in expression
levels (data not shown). As a control, F9 EC cells were co-transfected
with the Gal4/p300 expression vectors with the promoter/reporter
construct 427T+EnHPmut, which contains mutated
HMG and POU sites and no Gal4 binding
site (Fig. 4A). Relative to 427T+EnGSp, a small
stimulation of 427T+EnHPmut was observed with both Gal4/p300
vectors (Fig. 4B). This stimulation is likely to be because
of p300 interaction(s) with the FGF-4 promoter.
Interestingly, Gal4/CBP fusion proteins also stimulated
427T+EnGSp (data not shown). In direct contrast, Gal4/Tip60,
although expressed at levels comparable with Gal4/p300 964-1922,
failed to stimulate 427T+EnGSp (data not shown). Tip60 is a
co-activator with AT activity (60). Thus, not all co-activators with AT
activity can mediate the action of the FGF-4 enhancer.
Importantly, these results indicate that p300/CBP can stimulate the
FGF-4 promoter when tethered to the FGF-4
enhancer downstream of the promoter, suggesting that Sox-2/Oct-3
binding may promote the association of p300 and the FGF-4
enhancer. Of particular interest is the observation that the fusion
protein containing aa 964-1922 of p300, which includes the bromodomain
and AT domain, was itself able to simulate the FGF-4
promoter when tethered to the enhancer. E1a is known to inhibit p300 by
inhibiting its AT activity (61). Taken together with the E1a results,
these observations suggest a possible role for the AT activity of p300
in the mediation of the FGF-4 enhancer. This possibility is
addressed experimentally below.
The GC Boxes and CCAAT Box of the Promoter Are Required for Optimal
Stimulation by p300--
Three cis-regulatory sites have
been identified in the FGF-4 promoter (12, 14). Two
GC boxes, which bind Sp1 and Sp3 in vitro (17),
reduce expression of 427T+E by ~25% when mutated (15),
and a CCAAT box, which binds NF-Y both in
vitro and in vivo (13, 14, 16), results in an ~50%
reduction of 427T+E activity when mutated
(14).3 Both Sp1 and NF-Y have
been shown to physically and functionally interact with and be
acetylated by p300 (33, 62-64), suggesting that p300 may interact with
these factors when bound to the FGF-4 promoter.
To test whether regulatory elements in the promoter are necessary for
the stimulation by enhancer-tethered p300, mutations were introduced in
the promoter within the context of 427T+EnGSp. 427T
GCmut+EnGSp contains mutations in both GC boxes, which
inactivate their function (15). 427T Cmut+EnGSp contains a
mutation in the CCAAT box, which inactivates its function
(46). 427T GC/Cmut+EnGSp contains the same
mutations in both the GC boxes and the CCAAT box
(Fig. 5A). When transfected
into F9 EC cells alone, these constructs exhibited little activity
compared with the wild-type construct 427T+E (Fig.
5B). This was expected, because the Sox-2 and
Oct-3 binding sites in the enhancer were disrupted by
replacement with a Gal4 binding site. When Gal4/p300 1-2414
was co-transfected with the constructs containing mutations in either
the two GC boxes or the CCAAT box, a small
decrease in stimulation (~25-30%) was observed when compared with
the stimulation of 427T+EnGSp containing a wild-type
promoter (Fig. 5B). Co-transfection of Gal4/p300 1-2414
with the construct containing all three sites mutated resulted in a
50% reduction in stimulation. These results argue that p300 requires
interactions with NF-Y and the factors binding the GC boxes
(Sp1/Sp3) for optimal stimulation. A complete reduction in stimulation
of the mutated promoter was not observed. This suggests p300 has
another function in addition to interacting with factors that bind to
the GC boxes and CCAAT box of the promoter. Interestingly, a similar decrease in activity of the promoter/reporter construct containing all three promoter sites mutated was observed when
co-transfected in conjunction with the Gal4/p300 fusion protein containing the bromodomain and AT domain, Gal4/p300 964-1922 (data not
shown).
p300 AT Activity Is Necessary for FGF-4 Expression--
Based on
the observations above and on prior observations of the necessity of AT
activity in the regulation of other genes by p300 (23, 31-35, 65), we
investigated the role of AT activity in the p300 stimulation of the
FGF-4 promoter. Utilizing the 427T+EnGSp promoter/reporter construct, transfections were performed with Gal4/p300 constructs harboring several aa changes in the AT domain. These mutations have been shown to reduce the AT activity of p300 to
<1% of wild-type (35). Gal4/p300 ATmut and Gal4/p300 964-1922 ATmut
vectors express fusion proteins containing the Gal4 DBD and either
full-length p300 or aa 964-1922 of p300 with the aa changes in the AT
domain mentioned above. Compared with Gal4/p300 964-1922, which shows
50-200-fold stimulation of the FGF-4 promoter, the AT
mutant fails to stimulate the FGF-4 promoter. To ensure that
the failure to stimulate was not because of a lack of expression, nuclear extracts from cells transfected with the fusion proteins were
analyzed for expression. A 2- to 4-fold lower expression of the AT
mutant was observed (Fig. 6B).
However, the differences in expression cannot account for the
50-200-fold difference in the ability of these proteins to stimulate
the FGF-4 promoter. Similarly, Gal4/p300 1-2414 ATmut
failed to stimulate 427T+EnGSp (data not shown).
To further test the necessity of the AT activity of p300 in the
mediation of the FGF-4 enhancer, F9 EC cells were
co-transfected with the promoter/reporter construct 427T+E,
which does not contain a Gal4 binding site, with expression
vectors for Gal4/p300 (1-2414 or 964-1922) AT mutants. Interestingly,
both the Gal4/p300 1-2414 AT mutant and the Gal4/p300 964-1922 AT
mutant inhibited the expression of 427T+E in a
dose-dependent manner (data not shown). These results suggest that the AT mutants are acting as dominant-negatives to directly inhibit the FGF-4 promoter. Together, these results
and those described in Fig. 6 argue that the AT activity of p300 is required for stimulation of the FGF-4 promoter.
p300 and Sox-2 Are Both Associated with the Endogenous FGF-4
Enhancer--
The above observations are indicative of a mechanism
whereby Sox-2/Oct-3 binding to the enhancer acts to recruit p300,
possibly through interaction with the TAD of Sox-2 (38). p300 could
then interact with and acetylate transcription factors and/or histones associated with the FGF-4 promoter. If this is correct then
p300 would be expected to be associated with the FGF-4
enhancer and possibly the FGF-4 promoter. To address this
possibility, ChIP analyses were performed using antibodies that
recognize p300. Specifically, F9 EC cells were treated with
formaldehyde to cross-link DNA-protein complexes, nuclear extract was
prepared, and DNA was sheared. Samples were split into two aliquots.
One aliquot was immunoprecipitated with an antibody that recognizes the
N terminus of p300, and the other aliquot was immunoprecipitated with a
nonspecific antibody to serve as a control. Primers were used to
specifically amplify regions of the enhancer, the promoter, or exon 2 of the FGF-4 gene (Fig.
7A). In each case, the same
DNA preparations of immunoprecipitated chromatin fragments were used in
the PCR reactions. We observed a 9-fold enrichment of the endogenous
FGF-4 enhancer in DNA-protein complexes immunoprecipitated
with antibodies that recognize the N terminus of p300 (Fig.
7B). In contrast, the promoter and exon 2 region of the
FGF-4 gene show approximately a 2- to 4-fold and less than a
2-fold enrichment, respectively, in DNA-protein complexes
immunoprecipitated with antibodies that recognize p300, compared with
DNA-protein complexes immunoprecipitated with a nonspecific antibody
(Fig. 7B). In the case of exon 2, PCR with exon 2 primers
was allowed to continue until amplification was observed. This
illustrates that no significant enrichment of exon 2 fragments occurred
with p300 antibodies. Moreover, the experiment with the antibody that
recognizes the N terminus of p300 was performed twice, and similar
results were obtained. To confirm these findings, this experiment was
repeated with a second p300 antibody that recognizes its C terminus. In
this experiment, an ~7-fold enrichment of the endogenous
FGF-4 enhancer and no enrichment of exon 2 were observed
(data not shown). The enrichment of the enhancer region but lack of
enrichment of the exon 2 region with p300 antibodies demonstrates that
p300 is associated specifically with the enhancer region of the
FGF-4 gene. However, the association of p300 with the
FGF-4 promoter appears to be weak. Results with the
427T+EnHPmut, which show that Gal4/p300 can stimulate the promoter when the enhancer is nonfunctional (Fig. 4B), argue
that p300 may be interacting with the promoter, as well as the
enhancer. Hence, if p300 associates with the FGF-4 promoter,
it appears to do so transiently. Alternatively, the weak association of
p300 with the promoter may be a result of poor cross-linking because of
an indirect interaction between p300 and the FGF-4 promoter.
If the interaction between the enhancer and promoter is transitory,
then Sox-2 would also be expected to associate with the enhancer and
transiently with the promoter. To test this possibility, we performed
ChIP assays with an antibody that recognizes Sox-2. A Sox-2 antibody
sensitive enough for ChIP is not available commercially. Therefore, a
vector expressing FLAG-tagged Sox-2 (FSox-2) was transfected into F9 EC
cells, and the M2 (anti-FLAG) antibody was utilized. A significant
enrichment of the endogenous FGF-4 enhancer was observed
with the M2 antibody compared with a nonspecific antibody (Fig.
7C). No enrichment of a 5' region (3.6 kb upstream of the
FGF-4 promoter) was observed, indicating that the enrichment of the enhancer is specific. Again, similar results were obtained when
this experiment was repeated. Thus, these results formally demonstrate
that Sox-2 binds to the endogenous FGF-4 enhancer. As with
p300, only a slight enrichment (~1.6-fold) of the promoter region was
observed (Fig. 7C). The weak association of FSox-2 with the
promoter is consistent with either a transitory enhancer-promoter interaction or is indicative of a mechanism by which p300 recruitment to the enhancer merely initiates a series of events that do not involve
a direct interaction between the enhancer and promoter.
p300 and its homolog CBP interact with a wide array of proteins,
implicating them in the regulation of many genes (24). Previous studies
in this laboratory (38) demonstrated that p300 can mediate Sox-2/Oct-3
synergistic activation, specifically through the TAD of Sox-2, as well
as through Oct-3.4 Based on
the studies reported here, p300 appears to play a major role in
mediating the enhancer activation of the FGF-4 promoter. We
demonstrate that E1a, but not a mutant form of E1a that is unable to
bind p300, blocks the function of the FGF-4 enhancer. This
inhibition appears to be mediated primarily through the
Sox-2/Oct-3 binding sites of the enhancer. We also
demonstrate that p300/CBP, when tethered to the enhancer, can stimulate
the FGF-4 promoter from a distance. Previously, p300/CBP was
shown to stimulate the Previous studies suggested that Sox-2 binds to the FGF-4
enhancer (20, 21). However, the studies described in this report provide formal proof for Sox-2 association with the endogenous FGF-4 enhancer. Importantly, our studies also provide direct
evidence of p300 association with a downstream, intragenic distal
enhancer in the endogenous FGF-4 gene. This directly
implicates p300 in the mediation of enhancer regulation of the
FGF-4 gene. Association of p300 and Sox-2, albeit a weak
association especially of Sox-2, with the FGF-4 promoter was
also observed. Interestingly, although some Gal4/CBP fusion proteins
were able to stimulate the FGF-4 promoter when tethered to
the enhancer, CBP was not observed to be associated with either the
FGF-4 enhancer or promoter in ChIP assays with two different
CBP antibodies (data not shown). However, it is possible that the
epitopes of CBP recognized by the antibodies used were masked.
Mechanistically, p300 could mediate the effects of the enhancer by a
noncontact or indirect mechanism that would involve p300 recruitment to
the enhancer and the initiation of a series of events leading to
activation of the promoter. Indirect mechanisms of enhancer action have
been proposed in two different models, a DNA tracking model (9) and a
linking model (8). The DNA tracking model proposes movement of
co-factors recruited to the enhancer along the chromatin. Because
Gal4/p300 can stimulate the promoter when tethered to the enhancer, we
believe that p300 is unlikely to be mediating the action of the
enhancer by a tracking mechanism. Because of the intragenic location of
the enhancer, we believe a linking mechanism is also unlikely. The
linking model (8) proposes that facilitator proteins (66) bind to the
intervening sequences beginning at the enhancer and then spread toward
the promoter to propagate a signal. Binding of proteins to the
intervening (transcribed) sequences between the enhancer and promoter
of the FGF-4 gene could impede transcription. Alternatively,
p300 may be mediating the effects of the enhancer by a stable direct
enhancer-promoter interaction that is undetectable using the ChIP assay
or through a direct interaction that is transitory. Our functional and
physical data are consistent with a contact model involving a
sustained, direct interaction between the enhancer and promoter, as
proposed in the facilitated tracking (7) and looping (5, 6) models.
In the facilitated tracking model, enhancer-bound factors and
associated co-activators track along the DNA in small steps, resulting
in the formation of intermediate loops until the promoter is reached,
and a stable loop is formed. One of the earliest models proposed to
explain enhancer function, and the one for which the most evidence
exists, is a looping model (5, 6, 67-72). In this model, chromatin
between the enhancer and promoter forms a loop, allowing the
enhancer-bound activators to contact factors bound at the promoter and
recruit the PIC. Based on our ChIP analyses, if a direct
enhancer-promoter interaction occurs, p300 is either not closely
associated with the promoter or mediates a transitory interaction.
Based on the data presented here demonstrating p300 is strongly
associated with the endogenous FGF-4 enhancer and weakly
with the FGF-4 promoter and that enhancer-tethered p300 can
stimulate the promoter from a distance, we currently favor a model
involving a direct but transitory interaction between the
FGF-4 enhancer and promoter. Evidence for transitory
interactions, referred to as a "hit and run" mechanism, has been
demonstrated in several cases (73-75). For example, p300 associates
transiently at a single time point, and CBP associates in a cyclic
nature with the CATD promoter following estrogen stimulation
(73). Factors bound to the distal enhancer of the glnAp2
gene interact with the holoenzyme bound to the promoter, transcription
is initiated, and the enhancer-promoter interaction is then
destabilized (74). In addition, the glucocorticoid receptor was
observed to continuously exchange between binding to regulatory sites
and the unbound state (75). In the bound state, the glucocorticoid
receptor is thought to recruit secondary factors such as chromatin
remodeling factors, before dissociating from the chromatin. Based on
the results presented here, we propose the following model for
p300-mediated distal enhancer regulation of the FGF-4 gene
(Fig. 8). Cooperative binding of Sox-2
and Oct-3 results in p300 associating with the enhancer. p300 interacts transiently with NF-Y and/or the factors binding to the GC
boxes of the promoter bridging the enhancer and promoter. In this
regard, p300 has been shown to interact with Sp1 (63) and NF-Y (33). p300 then acetylates proteins in the FGF-4 promoter in
preparation for remodeling of chromatin and subsequent recruitment of
other complexes such as chromatin remodeling complexes (76), mediator (77, 78), and the PIC. Recruitment of these secondary complexes would
result in a dissociation of p300 and the FGF-4 enhancer from
the promoter. An association of p300 with the enhancer would persist in
anticipation of the next round of transcription.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene have been characterized extensively (22,
23). One such co-activator is p300 and its homolog, CBP. p300/CBP
interact with a variety of transcription factors and proteins of the
PIC (reviewed in Refs. 24 and 25). p300/CBP is able to mediate the
transcriptional activation of a variety of promoters (3, 26, 27) and
has been found to be associated directly with several promoters (22, 28-30). Often, p300/CBP-mediated transcription is dependent upon its
acetyltransferase (AT) activity (23, 31-35). p300/CBP also has been
shown to stimulate several promoters when tethered to distal sequences
(27, 36), and it has been demonstrated to associate endogenously with
the LCR of the
-globin gene (28) and the distal enhancer
of the PSA gene (37). Both the
-globin LCR and
the PSA enhancer are located in extragenic regions upstream of the promoters they regulate. In contrast to promoters and proximal enhancers (3, 24-26, 29, 30), the roles of p300/CBP in the function of
distal enhancers are poorly understood.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activities were
determined as reported previously (21). Molar amounts of DNA were kept
constant with the addition of null vectors. All transfections were
normalized by addition of 1 µg of CMV
-galactosidase (Clontech). Transfections were performed in
duplicate or triplicate, and representative transfections are shown.
2-36 (mE1a), a mutant E1a unable to bind p300/CBP,
(47); Gal4/p300 1-2414 and Gal4/p300 964-1922 (48); Gal4/Tip60, which
was received from John Lough (Medical College of Wisconsin); and
pCMVFLAGSox-2 (38). Gal4/p300 expression vectors, which lack AT
activity, Gal4/p300 ATmut, and Gal4/p300 964-1922 ATmut, were
constructed as follows. The plasmid pBSp300 AT2 was obtained from Lee
Kraus. This construct contains full-length p300 with six aa changes in
the AT domain that have been shown to reduce the AT activity to <1%
of wild-type (35). The p300 AT2 mutant in pBSp300 AT2 was
amplified by PCR with the upper primer
5'-TATAgtcgacGATGGCCGAGAATGTGGTG-3' and the lower primer 5'-ACTAAAGGGAACAAAAGCTGGG-3'. The upper primer contains a
SalI site (lowercase), and the lower primer is specific for
sequence within the pBS vector downstream of the inserted p300
sequence. Following amplification, the fragment was digested with
SalI and NheI and ligated into the
SalI and XbaI sites of CMVGal4 (38). The
resulting expression vector, Gal4/p300 ATmut, contains an in-frame
fusion between the Gal4 DNA binding domain (DBD) and p300 AT2. The
vector Gal4/p300 964-1922 ATmut expresses a fusion between the Gal4
DBD and aa 964-1922 of p300 lacking AT activity. This construct was
produced by ligating a PshAI-BstEII fragment from pBSp300
AT2, encompassing the six amino acid changes in the AT domain, into the
PshAI and BstEII sites in Gal4/964-1922. All engineered constructs were confirmed by sequencing. All primers were
synthesized, and sequencing was performed by the Eppley Cancer Institute Molecular Biology Core Facility.
-galactosidase and 8 µg of the
vectors expressing Gal4/p300 964-1922, Gal4/p300 964-1922 ATmut, E1a,
or mE1a using LipofectAMINE PLUS (Invitrogen) as described by Nowling
et al. (49). Nuclear extracts were prepared using the NE-PER
nuclear and cytoplasmic extraction Kit (Pierce) according to
manufacturer's directions. Immunoprecipitation (IP) of the Gal4/p300
fusion proteins or E1a proteins from nuclear extracts was accomplished
by addition of Gal4 (DBD) antibody conjugated to agarose beads (Santa
Cruz Biotechnology, Inc.) or E1a antibody (anti-E1a clone M73; Upstate
Biotechnology, Inc.), respectively. Following an overnight incubation
at 4 °C, protein G beads (Santa Cruz Biotechnology, Inc.) were added
to the anti-E1a samples and incubated for 1 h at 4 °C, and
beads were collected and washed. Proteins were eluted by addition of
sample buffer (62 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10%
glycerol, 0.1% w/v bromphenol blue) plus 10% 1 M
dithiothreitol and boiling. Eluted proteins were run on a denaturing
Tris-glycine gel (Invitrogen) and transferred to polyvinylidene
fluoride membrane (Immobilon-P; Millipore). Membranes containing Gal4
proteins were probed with Gal4 (DBD) antibody (Santa Cruz
Biotechnology, Inc.), and membranes containing E1a proteins were probed
with the same E1a antibody used in the IP. Proteins were detected by
enhanced chemifluorescence (Amersham Biosciences) as described
previously (38). Membranes were scanned on a Storm PhosphorImager
(Molecular Dynamics), and bands were quantified using the ImageQuant
analysis software (Molecular Dynamics) where indicated.
-Galactosidase activities were determined (see above) using 5 µl
of the cytoplasmic fraction of the extract. Transfection of the
wild-type Gal4/p300 construct was 2-3-fold more efficient than the AT
mutant construct. All IPs and Western analyses were performed at least
three times with representative blots shown.
20 °C by addition of 2.5 volumes of 100% ethanol,
resuspended in proteinase K buffer (10 mM Tris-Cl, pH 7.5, 5 mM EDTA, 0.25% SDS), and treated with proteinase K for
2 h at 45 °C. DNA was purified using the Geneclean turbo kit (Q
Biogene) following the manufacturer's directions. ChIP experiments
were repeated at least twice with similar results.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of E1a on enhancer-mediated
stimulation of the FGF-4 promoter. A,
physical map of FGF-4 promoter/reporter construct
427T+E, containing 427 bp of the FGF-4 promoter
driving the CAT reporter gene. The FGF-4 enhancer
(316 bp) is located downstream of the CAT gene to
recapitulate its endogenous placement. GC and GT,
Sp1/Sp3 binding sites; CCAAT, CCAAT box; TATA,
TATA box; HMG, Sox-2 binding site; POU, Oct-3
binding site. B, transfection of F9 EC cells with 15 µg of
427T+E alone or with increasing amounts of a CMV expression
vector for 12S E1a (E1a) or 12S E1a 2-36
(mE1a), as indicated. A constant amount of DNA was achieved
by addition of the null CMV vector pCMV5. Results are presented as CAT
expression relative to the expression of 427T+E alone, which
was set to 1. The mE1a vector expresses a mutant E1a unable to interact
with p300/CBP. This experiment was repeated twice with similar results.
C, protein expression in F9 EC cells. E1a proteins were
immunoprecipitated from nuclear extracts and subjected to Western blot
analysis as indicated under "Experimental Procedures." *, E1a
protein; #, mE1a protein. Approximate molecular mass is indicated on
the left.
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Fig. 2.
Effects of E1a on enhancer-mediated
stimulation of the TK promoter. A,
physical map of TKCat+E promoter/reporter construct
containing the TK promoter driving the CAT gene
mediated by the FGF-4 enhancer placed downstream of the
CAT gene. GT, Sp1/Sp3 binding site;
HMG, Sox-2 binding site; POU, Oct-3 binding site.
B, transfection of F9 EC cells with 7.5 µg of
TKCat, TKCat+E alone, or TKCat+E and
increasing amounts of CMV expression vectors for 12S E1a
(E1a) or 12S E1a 2-36 (mE1a), as indicated. A
constant amount of DNA was achieved by addition of the null CMV vector
pCMV5. Results are presented as CAT expression relative to the
expression of the TKCat parental vector, which was set to 1. The mE1a vector expresses a mutant E1a unable to interact with
p300/CBP. This experiment was repeated twice with similar
results.
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Fig. 3.
Effects of E1a on Sox-2/Oct-3 activation of
the SV40 promoter. A, physical map of
pCatSO3 promoter/reporter construct containing six copies of
the Sox-2 (HMG) and Oct-3
(POU) binding cassette (as it appears in the
FGF-4 enhancer) upstream of the SV40 promoter
driving CAT gene expression. B, transfection of
F9 EC cells with 10 µg of pCatSO3 alone or with increasing
amounts of CMV vectors expressing 12S E1a (E1a) or 12S
E1a 2-36 (mE1a), as indicated. A constant amount of DNA
was achieved by addition of the null CMV vector pCMV5. Results are
presented as CAT expression relative to the expression of
pCatSO3 alone, which was set to 1. The mE1a vector expresses
a mutant E1a unable to interact with p300/CBP. This experiment was
repeated twice with similar results.
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Fig. 4.
Stimulation of the FGF-4
promoter by Gal4/p300 fusion proteins. A,
physical map of 427T+EnGSp and 427T+EnHPmut
promoter/reporter constructs. Both constructs are driven by the
wild-type FGF-4 promoter. The FGF-4 enhancer in
427T+EnGSp contains a single Gal4 binding site
(GAL4) in place of the Sox-2 (HMG) and
Oct-3 (POU) binding sites. The enhancer in
427T+EnHPmut contains mutated HMG and
POU sites. GC and GT, Sp1/Sp3 binding
sites; CCAAT, CCAAT box; TATA, TATA box.
B, transfection of F9 EC cells with 15 µg of
427T+EnGSp or 427T+EnHPmut alone or with 5 µg
of a vector expressing Gal4/p300 1-2414 (1-2414) or
Gal4/p300 964-1922 (964-1922). Results are presented as
relative CAT expression over the expression of 427T+EnGSp
(solid bars) or 427T+EnHPmut (open
bars) alone, which were set to 1. Gal4/p300 1-2414 and Gal4/p300
964-1922 are vectors expressing fusions between the DNA binding domain
of Gal4 and full-length p300 (aa 1-2414) and the bromodomain and AT
domain of p300 (aa 964-1922), respectively. This experiment was
repeated at least twice with similar results.
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Fig. 5.
Effects of mutations in the GC
boxes (GC; Sp1/Sp3 binding sites) and the
CCAAT box (CCAAT) on the stimulation
of the FGF-4 promoter by Gal4/p300 fusion
proteins. A, physical map of the promoter/reporter
constructs containing various mutations in the promoter as described
under "Experimental Procedures." GT, Sp1/Sp3 binding
site; TATA, TATA box; HMG, Sox-2 binding site;
POU, Oct-3 binding site; GAL4, Gal4 binding site.
B, transfection into F9 EC cells with 15 µg of the various
promoter/reporter constructs depicted in A alone or with 5 µg of Gal4/p300 1-2414, a vector expressing a fusion of the Gal4 DNA
binding domain and full-length p300. A constant amount of DNA was
achieved by addition of the null CMV vector pCMV5. Results are
presented as CAT expression relative to expression of
427T+E, which is set to 1. Gal4/p300 1-2414 is a vector
expressing a fusion between the Gal4 DNA binding domain and full-length
p300. This experiment was repeated at least twice with similar
results.
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Fig. 6.
AT activity is necessary for p300 stimulation
of the FGF-4 promoter. A, transfection
of F9 EC cells with 15 µg of 427T+EnGSp and increasing
amounts of an expression vector for Gal4/p300 964-1922
(964-1922) or Gal4/p300 964-1922 ATmut (964-1922
ATmut), as indicated. Results are presented as CAT expression
relative to the expression of 427T+EnGSp alone, which is set
to 1. B, expression of Gal4 fusion proteins in transfected
F9 EC cells. Nuclear extracts from transfected cells were
immunoprecipitated with anti-Gal4 (DBD)-conjugated agarose beads and
subjected to Western blot analysis with a Gal4 (DBD) antibody.
Gal4/p300 964-1922 is an expression vector for a fusion between the
Gal4 DNA binding domain and aa 964-1922 of p300, which includes the
bromodomain and AT domain. Gal4/p300 964-1922 ATmut is identical to
Gal4/p300 964-1922 but lacks AT activity. This experiment was repeated
at least twice with similar results.
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Fig. 7.
Association of p300 and Sox-2 with the
endogenous FGF-4 gene. A, physical map
of the endogenous FGF-4 gene (not drawn to scale). The
promoter region between 427 and +1 contains two GC boxes
(Sp1/Sp3 binding site; GC), a CCAAT box
(CCAAT), and a TATA box (TATA).
FGF-4 contains three exons (shaded boxes, labeled
1, 2, and 3). The 316-bp
FGF-4 enhancer (+2777 to +3093) is located within the 3' UTR
(+1555 to + 3854) of the third exon and contains a Sox-2
(HMG) binding site, an Oct-3 (POU)
binding site, and a GT box (Sp1, Sp3 binding site;
GT). Regions amplified in B and C are
indicated below the physical map. B, ChIP assays
were performed on nontransfected F9 EC cells. DNA-protein complexes
were cross-linked, chromatin was sonicated, and p300-associated DNA
fragments were immunoprecipitated with an antibody to the amino
terminus of p300 (p300). A nonspecific antibody (NS) was
used as a negative control. Fragments were amplified using primers for
the enhancer, promoter, or exon 2 regions of the FGF-4 gene
within the same sample, as indicated and quantified. C, ChIP
assays were performed on F9 EC cells transfected with an expression
vector for FLAG-tagged Sox-2 (FSox-2). DNA-protein complexes
were cross-linked, chromatin was sonicated, and FSox-2-associated DNA
fragments were immunoprecipitated with a FLAG (M2) antibody.
A nonspecific antibody (NS) was used as a negative control.
Fragments were amplified using primers for the enhancer, promoter, or
exon 2 regions of the FGF-4 gene within the same sample, as
indicated, and quantified. ChIP experiments and amplifications within
each ChIP experiment were repeated at least twice with similar
results.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin promoter when tethered to
an upstream LCR (27) and a synthetic promoter from a downstream distal
position (36). In this study, we demonstrate that p300 can stimulate a
natural promoter from a downstream distal position. Importantly, we
provide evidence that acetylation plays a significant role in the
transcriptional regulation of the FGF-4 gene, as is the case
with other genes (31, 37). In this regard, p300 lacking AT activity
fails to stimulate the FGF-4 promoter when tethered to the
FGF-4 enhancer but acts as a dominant-negative to decrease
transcription from the FGF-4 promoter. Together, these
results argue strongly that p300 and its intrinsic AT activity are
important in mediating enhancer-regulated transcription of the
FGF-4 gene.
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Fig. 8.
Model for p300-mediated mechanism of
FGF-4 enhancer action. A, before
transcription of the FGF-4 gene, DNA binding transcription
factors bind to their cognitive sites. B, p300 interacts
with Sox-2 and Oct-3 at the enhancer and transiently with transcription
factors binding the promoter, thus bridging the enhancer and promoter.
p300 then acetylates proteins associated with the promoter.
C, secondary factors then interact with the promoter. p300
and the enhancer dissociate from the promoter, and p300 remains
associated with the enhancer.
Recent studies on the PSA gene have shed some light on mechanisms of distal enhancer transcriptional control. The PSA gene promoter is regulated by an enhancer located 4 kb upstream. Shang et al. (37) observed RNA PolII, p300/CBP, and histone acetylation associated with both the PSA promoter and enhancer in vivo but not with intervening sequences. These results are strongly indicative of a direct interaction between the enhancer and promoter through a stable looping mechanism involving p300/CBP. Similarly, we observe a strong association of p300 with the distal enhancer of the FGF-4 gene, supporting a role for distal enhancer mediation by p300/CBP. In contrast, only a weak association of p300 with the promoter is observed. Therefore, if direct interaction occurs, FGF-4 enhancer-promoter interaction is transitory, and a stable loop does not form. In this regard, the location of the PSA and FGF-4 distal enhancers are different, extragenic and intragenic, respectively. Hence, our results suggest different mechanisms may be used to mediate extragenic enhancers, such as that of the PSA gene, and intragenic enhancers located in introns and exons, such as that of the FGF-4 gene. Intragenic enhancers may employ a hit and run mechanism to avoid interference with transcription of the gene that could result from the formation of a stable interaction between the enhancer and promoter.
In conclusion, our findings provide both functional and physical
evidence that p300 can mediate the effects of the FGF-4
enhancer. Given the recent findings described in two other studies, one involving an LCR and one involving an upstream distal
enhancer (28, 37), our work adds further evidence that p300 and CBP can
mediate the effects of regulatory sequences located at considerable distances from the promoters that they control. However, our studies differ from a previous report (37) dealing with the PSA
gene. For this gene, recent studies suggest a mechanism involving
stable enhancer-promoter interactions for extragenic distal enhancers. In contrast, our findings are suggestive of a mechanism involving transient enhancer-promoter interactions for intragenic distal enhancers. Hence, we suggest that different mechanisms are likely to
mediate the effects of different classes of distal regulatory sequences. However, p300/CBP may be a common co-activator involved in
mediating their influence on promoter activation.
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ACKNOWLEDGEMENTS |
---|
We thank Antonio Giordano for plasmids Gal4/p300 1-2414 and Gal4/p300 964-1922, John Lough for plasmid Gal4/Tip60, and Lee Kraus for plasmid pBSp300 AT2. Hua Xiao and Mark Groudine are thanked for reading the manuscript and making helpful comments.
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FOOTNOTES |
---|
* This work was supported in part by NCI, National Institutes of Health Grant CA 74771. Core facilities of the University of Nebraska Medical Center, Eppley Cancer Institute used in the course of this work were supported in part by NCI Laboratory Cancer Research Center Support Grant CA 36727.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.
§ Supported in part by NCI, National Institutes of Health Training Grant CA 00476.
To whom correspondence should be addressed: Eppley Inst. for
Research in Cancer and Allied Diseases, University of Nebraska Medical
Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Tel.:
402-559-6338; Fax: 402-559-4651; E-mail: arizzino@unmc.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M207567200
2 L. Johnson and A. Rizzino, unpublished results.
3 L. Johnson and A. Rizzino, unpublished results.
4 L. Johnson and A. Rizzino, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: PIC, preinitiation complex; FGF-4, fibroblast growth factor-4; AT, acetyltransferase; LCR, locus control region; CBP, cAMP-response element-binding protein-binding protein; EC, embryonal carcinoma; ChIP, chromatin immunoprecipitation; PSA, prostate-specific antigen; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; aa, amino acid; DBD, DNA binding domain; IP, immunoprecipitation; TK, thymidine kinase; TAD, transactivation domain.
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