(Received for publication, October 19, 1995; and in revised form, January 8, 1996)
From the
Human p300 protein is a cellular target of adenoviral E1A oncoprotein and a potential transcriptional coactivator. Both p300 and Rb family protein-binding regions of E1A are required for the repression of muscle gene expression, which is regulated by MyoD family transactivators. This implies that p300 is involved in MyoD-dependent transactivation. We show that the repression of MyoD-mediated E box (MyoD consensus) reporter activity by E1A is correlated with its interaction with p300, indicating that p300 participates in MyoD-dependent transactivation. In addition, p300 is able to interact both in vivo and in vitro with MyoD through a portion at the carboxyl-terminal cysteine/histidine-rich domain and associates with the components of the basal transcriptional complex through its two separate transactivation domains at the amino and carboxyl termini. Consistent with its role as a coactivator, p300 potentiates MyoD-activated transcription.
Human p300 protein was identified initially as a cellular target of adenoviral E1A oncoprotein(1, 2, 3) . The region of E1A interacting with p300 is distinct from the one that interacts with tumor suppressor retinoblastoma protein (Rb) and Rb family proteins. The interactions of E1A with both p300 and Rb family proteins are required for cellular transformation. p300 protein is expressed ubiquitously, and its sequence is conserved from nematode to human(4) , further implying the importance of its biological role.
The sequence of p300 reveals it to be a potential
transcriptional coactivator(5) . This potentiality is
consistent with the fact that p300 has transcriptional activity (6) and forms a complex with TBP ()in
vivo(7) . It was reported that human protein p300
potentiates the transactivation by the cAMP-responsive element-binding
protein (CREB), as does the homologous transcriptional coactivator,
mouse CREB-binding protein (CBP)(8) . However, the role and
significance of this p300 function in cellular control and E1A
transformation are unclear.
For transformation, E1A represses expression of muscle-specific genes and muscle differentiation(9) , which are regulated by the basic helix-loop-helix protein MyoD and other MyoD family transactivators (10, 11) . It was reported that this repression requires both regions of E1A to bind to p300 and Rb family proteins(11, 12, 13) . Thus, it is possible that p300 is involved in MyoD-dependent transactivation.
We used E1A as an investigative tool to test this hypothesis. We studied the interaction between p300 and MyoD proteins and demonstrated that p300 potentiates MyoD-dependent transactivation. To examine the transcription potential of p300, we also identified the regions of the p300 protein containing transcriptional activities and their direct interactions with the components of the basal transcriptional complex.
Figure 1:
p300 plays a
positive role in MyoD-mediated transactivation. A, U-2 OS
human osteosarcoma cells were transiently transfected with 4 µg of
p4RTKCAT, 0 µg (lanes 1 and 5) or 1, 4, and 8
µg (lanes 2-4) of pCMVMyoD, and 4 µg of
pCMV-p300 (lane 5), followed by CAT assays as described
under ``Materials and Methods.'' A representative experiment
with its percent conversion is shown. Means of percent conversion from
three independent experiments ± S.D. from lanes 1-5 are 1 ± 0, 50 ± 5.1, 74 ± 12.5, 53 ±
8.1, and 4 ± 1.4. B, U-2 OS cells were transfected with
4 µg of p4RTKCAT, 4 µg of pCMVMyoD, 0, 0.5, 2, and 4 µg of
pCMV12S (lanes 1-4), and 1, 2, and 4 µg of dl
2-36 (lanes 5-7), followed by CAT assays. A
representative experiment with its percent conversion is shown. Means
of percent conversion from three independent experiments ± S.D.
from lanes 1-7 are 62 ± 4.2, 52 ± 4.1, 5
± 2, 1 ± 0, 33 ± 1.9, 30 ± 3.5, and 26
± 1.4. C, U-2 OS cells were transfected with 4 µg
of p4RTKCAT, 4 µg of pCMVMyoD, 0 (lanes 1, 3, and 5) or 4 (lanes 2, 4, and 6) µg of
pCMV12S, and either blank (lanes 1 and 2), 4 µg
of pCMV
-p300 (lanes 3 and 4), or 4 µg of
p300 mutant vector 1-1737 (encoding a p300 fragment of amino
acids 1-1737) (lanes 5 and 6), followed by CAT
assays. ``Transactivation'' refers to the relative CAT
activity (mean with S.D., n = 3) compared with the
activity in the absence of MyoD.
In this system E1A 12S repressed the MyoD-dependent E box activity in a dose-dependent manner (Fig. 1B, lanes 1-4). When we used an E1A 12S mutant bearing an amino-terminal deletion (dl 2-36) that loses its ability to bind to p300, but not to Rb family proteins(18, 24, 25) , it repressed the reporter activity much less than the wild type (Fig. 1B, lanes 5-7), indicating that this repression by E1A may require its binding to p300.
To confirm this possibility, a p300
expression construct (pCMV-p300) was cotransfected with E1A
vector. Ectopic p300 largely released the E1A repression (Fig. 1C, lane 4 compared with lane 2). When
the wild type p300 construct was replaced by a p300 mutant 1-1737
that removes the E1A-binding domain and carboxyl-terminal sequence, E1A
did not repress any reporter activity (Fig. 1C, lane
6). Thus the E1A repression of reporter activity is associated
with its ability to bind to p300, indicating that p300 is involved in
MyoD-dependent transactivation. The stimulation of reporter activity by
E1A in the presence of p300 mutant (Fig. 1C, lane 6)
may be associated with E1A's ability to perturb p300 function
indirectly, as shown by Arany et al.(6) . In addition,
p300 mutant 1-1737 that still contained transcriptional activity,
but at a reduced level (data not shown), affected reporter activity in
the absence of E1A in the system (Fig. 1C, lane 5).
This result also suggests that p300 plays a role in MyoD-dependent
transactivation.
Figure 2: p300 associates MyoD in vivo. Immunoblot with an anti-MyoD antibody (Santa Cruz) following immunoprecipitation of whole cell extracts prepared from C2C12 myoblast cells in lysis buffer containing either 250 mM salt (lanes 2 and 3) or 150 mM salt (lanes 1, 4, and 5) with an anti-p300 polyclonal antiserum (5 µl). NRbS, controls for immunoprecipitation with normal rabbit serum. WCL, whole cell extract without immunoprecipitation.
Figure 3: p300 specifically binds MyoD in vitro at the carboxyl-terminal cysteine/histidine-rich domain. A, GSTMyoD, GSTTBP, and GSTTFIIB fusion proteins and GST alone were expressed from pGEX-MyoD, pGEX-hTBP, pGST-IIB, and pGEX-2T (Pharmacia) vectors in E. coli, purified by being immobilized on glutathione-agarose beads (Pharmacia), and analyzed on 15% SDS-polyacrylamide gel. LMW, low molecular weight markers (Bio-Rad: 112, 84, 53.2, 34.9, and 28.7 kDa). B and C, the purified bacterial GST-MyoD fusion protein or GST alone was incubated with roughly equal molar amount of labeled p300, either wild type (WT) in B or fragments indicated by amino acids in C, as described under ``Materials and Methods.'' The binding assay in C was carried out in a higher stringency condition with 250 mM NaCl and 0.5% Nonidet P-40 in binding buffer. Input refers to in vitro translation products used in the binding assays. Roughly equal volume amount of each in vitro translation product was used in the ``Input'' panel in C.
Through deletion analysis, the portion of p300 containing the affinity to MyoD was detected in fragment 1514-1922 (Fig. 3C, lane 1) that covers the third cysteine/histidine-rich conserved domain (C/H3). The fragment 1737-2414, that contains a carboxyl portion of the C/H3 domain, was still able to bind to MyoD (lane 2), while the fragment 1945-2414, the most carboxyl-terminal region, was not (lane 3). These interactions between p300 and MyoD at the C/H3 region were specific, because no interactions were detected for GST alone and for the control protein (lane 6). This MyoD-binding portion of p300 is separate from the amino-terminal portion (1-663) that binds to CREB protein(8) . Although the fragment 1-596 bound to GSTMyoD slightly (lane 5), a weak interaction between this amino-terminal fragment and GST alone was detected. The results obtained from the GST binding assays were repeatable when the same stringency conditions were used for binding and washing. The results were also essentially the same when the assays were carried out in lower stringency conditions (data not shown).
Figure 4: p300 contains two separate transactivation domains. Diagrams showing p300 and its deletions fused to Gal4 DNA-binding domain. The striped box refers to the Gal4 DNA-binding domain. The filled box represents the conserved regions of p300. The number in the diagram indicates the amino acid positions of p300. The cysteine/histidine-rich regions are indicated as C/H1-3. B, bromodomain. TA, regions containing transcriptional activity. The values in the Transactivation column are the relative CAT activity (mean ± S.D., n = 3) compared with the activity of Gal4 1-147 alone. For CAT assays, U-2 OS cells were transiently transfected with 4 µg of pG5e1bCAT and 4 µg of either Gal4 1-147, Gal4p300, or one of the p300 deletion vectors, as described under ``Materials and Methods.''
An additional question is whether the amino- and carboxyl-terminal transactivation domains of p300 interact with the basal transcription complex directly. Previous reports showed that p300 formed a complex with TBP in vivo(7) , and CBP interacted with TFIIB at the conserved C/H3 region(1680-1812)(23) . By using in vitro binding assay with TBP and TFIIB fused to GST, we detected a specific affinity between the carboxyl-terminal transactivation domain of p300 (1737-2414) containing a C/H3 region and either TBP or TFIIB (Fig. 5A). The amino-terminal portion of p300 (1-743), inducing a high level of transactivation, also bound to TBP but not to TFIIB (Fig. 5B).
Figure 5: p300 communicates with the components of the basal transcriptional machinery through its transactivation domains. A and B, the purified bacterial fusion proteins GSTTBP and GSTTFIIB and GST alone (Fig. 3A) were incubated with labeled p300 fragments indicated by amino acids and analyzed for binding, as described under ``Materials and Methods.'' The binding assays were performed in the binding buffer containing 100 mM NaCl and 0.1% Nonidet P-40. Input refers to in vitro translation products used in the binding assays.
Figure 6:
p300 potentiates MyoD-activated gene
expression. A, F9 teratocarcinoma cells were cotransfected
with p4RTKCAT (4 µg) and other vectors as indicated, followed by
CAT assays as described under ``Materials and Methods.'' Transactivation refers to the relative CAT activity (mean with
S.D., n = 3) compared with activity in absence of
ectopic MyoD and p300. B, U-2 OS cells were cotransfected with
4 µg of p4RTKCAT, 2 µg of pCMVMyoD, and 0, 2, and 8 µg of
either pCMV-p300 (p300 WT) or one of two p300 deletion
mutants as indicated, followed by CAT assays as described under
``Materials and Methods.'' Transactivation represents the relative CAT activity (mean with S.D., n = 3) compared with the activity in the absence of ectopic
p300. C, Western blot analysis of U-2 OS cells transfected
with 4 µg of pCMVMyoD and 0 (lane 2) or 8 µg of either
p300 mutant dl 242-1737 (lane 3) or 1545-1922 (lane
4). Control cells received no transfection (lane
1).
To confirm the role of p300 in MyoD-dependent transactivation, we selected p300 mutants that interfered the transcription activity of endogenous p300 protein in transfected U-2 OS cells. As shown in Fig. 6B, two mutants markedly affected the MyoD-dependent transactivation, one (dl 242-1737) positively (lanes 6 and 7) and the other (1514-1922) negatively (lanes 4 and 5). We believe that the effects of these p300 mutants are related to their disturbing the endogenous p300 function, because they did not show relevant effects on the activity of the promoter driving the ectopic MyoD expression (data not shown) or the MyoD level in the transfected cells (Fig. 6C, lanes 3 and 4).
A new type of transcriptional regulator has been classified recently as the transcriptional coactivator that communicates between transactivators binding on enhancer DNA and the basal transcriptional complex formed on the promoter near the transcription initiation site (26) . This communication is required for transcriptional activation by the transactivators. p300 protein contains a bromodomain that is a hallmark of certain transcriptional adapter proteins(5) . A sequence at the C/H3 domain of p300 has homology to the yeast ADA2 protein(27) , a putative transcriptional coactivator(28) . These p300 properties suggest that p300 is a potential transcription coactivator. This potentiality is supported by evidence demonstrating that p300, like its homologous protein, can interact with the transactivator CREB and potentiate its transactivation(8) , that a fusion of E2 DNA-binding domain to p300 transactivates an E2-binding site reporter(6) , and that p300 can form a complex in vivo with TBP, a component of the basal transcriptional complex(7) .
We demonstrated that p300 is a transcriptional coactivator for MyoD. The analysis with E1A and the potentiation of MyoD-dependent transactivation by p300 indicate that p300 participates in transactivation mediated by the interaction between the E box enhancer and MyoD. The interactions between MyoD and p300 proteins, and between p300 and either TBP or TFIIB, suggest that p300 can communicate directly between the E box-binding MyoD and the basal transcriptional complex during transactivation. This feature of p300 defines it as a transcriptional coactivator for MyoD.
The association between p300 and MyoD was detected only in a low stringency salt condition in vivo. This result is consistent with the fact that interactions of transcription factors are generally weak and instant, and it is difficult to detect them in vivo except by use of a two-hybrid system. The association between MyoD and Rb was detected also in a similar low stringency salt condition(29) . This condition is created by a lysis buffer containing a 150 mM rather than a 250 mM salt concentration and is closer to the physiological salt condition in the cell.
A transcription factor communicates directly or indirectly with the basal complex through its transactivation domain. The transactivation activities of p300 were detected in separate amino- and carboxyl-terminal regions. The interactions between the amino- and carboxyl-terminal regions of p300 and either TBP or TFIIB in vitro indicate that p300 can associate with the basal complex directly. However, these results cannot rule out an indirect interaction of p300 with the basal transcriptional complex occurring simultaneously, as was reported by Abraham et al.(7) when they found that the interaction between p300 and TBP may be mediated by two 64- and 59-kDa polypeptides. It is also possible that p300 interacts with and is mediated by other transcriptional factors during transactivation(30, 31) .
The dominant negative and
dominant positive mutants of p300 were shown to affect endogenous p300
function in MyoD-dependent transactivation of the E box reporter in
this study. Because the dominant negative mutant 1514-1922 is
able to bind to MyoD but has no transcriptional activity, its negative
role may be associated with sequestering MyoD from the wild type p300
in cells. The dominant positive mutant dl 242-1737, while it has a high
transactivation ability, it lacks a large middle portion, including the
bromodomain and the second cysteine/histidine-rich region. Results of
other Gal4 reporter assays imply a negative role for this middle
portion of p300 in transactivation, ()and thus the mutant dl
242-1737 could bypass repressor involvement. It also contains the
MyoD-binding domain, it is able to interact with MyoD (data not shown),
and it could therefore deliver its higher transactivation potential to
the E box via MyoD. These data further support the contention that p300
serves as a coactivator for the myogenic transactivator MyoD.
All
MyoD family transactivators can transactivate the E box enhancer. Under
the condition of muscle differentiation, MyoD induces the expression of
myogenin. In order to distinguish the E box activity mediated by MyoD
from that mediated by other myogenic transactivators, we carried out
experiments in non-muscle cells under nondifferentiation conditions.
However, we discovered that the dominant positive and negative mutants
of p300 affected E box reporter activity during MyoD-induced
differentiation in a way similar to that detected in nondifferentiation
conditions. The effect of p300 mutants on E box activity
without ectopic MyoD was observed also in C2C12 myoblast and human
rhabdomyosarcoma cells under low serum differentiation
conditions.
It appears that p300 modulates transactivation
by MyoD, and possibly by other MyoD family transactivators, under
myogenic differentiation. Consistent with this, we observed that the
p300 mutant 1-1737 bypassed E1A repression of myogenin-induced E
box reporter activity,
indicating a potential role for p300
in myogenin-dependent transactivation.
The function of p300 as a coactivator for MyoD, and possibly for other MyoD family transactivators, suggests one of the mechanisms by which it can be involved in the regulation of muscle differentiation. This mechanism probably plays an important role because, for efficient repression of muscle-specific gene expression and differentiation, E1A oncoprotein must have the ability to attack both p300 and Rb family proteins. This does not rule out the possibility that the repression of the muscle-specific gene by E1A also is correlated with its direct interaction with myogenin(32) , which could be another pathway for E1A to inhibit the myogenic process. It has been shown that the p300 protein is involved, during keratinocyte differentiation, in the induction of the cyclin-dependent kinase inhibitor p21 (33) , which is regulated by MyoD and which is required for permanent cell cycle withdrawal during muscle differentiation(17, 34) . This result further supports the proposition that p300 participates in myogenesis through modulating MyoD-dependent transcription.