Coactivators p300 and PCAF physically and functionally interact with the foamy viral trans-activator
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Foamy virus Bel1/Tas trans-activators act as key regulators of gene expression and directly bind to Bel1 response elements (BRE) in both the internal and the 5'LTR promoters leading to strong transcriptional trans-activation. Cellular coactivators interacting with Bel1/Tas are unknown to date.
Transient expression assays, co-immunoprecipitation experiments, pull-down assays, and Western blot analysis were used to demonstrate that the coactivator p300 and histone acetyltransferase PCAF specifically interact with the retroviral trans-activator Bel1/Tas in vivo. Here we show that the Bel1/Tas-mediated trans-activation was enhanced by the coactivator p300, histone acetyltransferases PCAF and SRC-1 based on the crucial internal promoter BRE. The Bel1/Tas-interacting region was mapped to the C/H1 domain of p300 by co-immunoprecipitation and pull-down assays. In contrast, coactivator SRC-1 previously reported to bind to the C-terminal domain of p300 did not directly interact with the Bel1 protein but nevertheless enhanced Bel1/Tas-mediated trans-activation. Cotransfection of Bel1/Tas and p300C with an expression plasmid containing the C/H1domain partially inhibited the p300C-driven trans-activation.
Our data identify p300 and PCAF as functional partner molecules that directly interact with Bel1/Tas. Since the acetylation activities of the three coactivators reside in or bind to the C-terminal regions of p300, a C/H1 expression plasmid was used as inhibitor. This is the first report of a C/H1 domain-interacting retroviral trans-activator capable of partially blocking the strong Bel1/Tas-mediated activation of the C-terminal region of coactivator p300. The potential mechanisms and functional roles of the three histone and factor acetyltransferases p300, PCAF, and SRC-1 in Bel1/Tas-mediated trans-activation are discussed.
Keywords293T Cell P300 Domain Basal Transcriptional Machinery Transient Reporter Gene Reporter Gene Expression Assay
List of abbreviations used
2, and 3, Cys/His-rich domains of p300
long terminal repeat
Glutamine-rich domain of p300
steroid receptor coactivator
p300M, and p300C, amino-, middle and C-terminal regions of p300
primate foamy virus
internal promoter Bel1 response element
nuclear factor 1
Several models have been proposed to explain transcriptional activation. Coordinated recruitment of coactivators by diverse transcriptional activators to specific promoter target sites has been shown by a collective effort of many groups . According to the models, different coactivators either modify chromatin structure by altering the nucleosomal DNA thereby affecting its accessibility to DNA-binding proteins or, alternatively, form complexes with HAT activities that by acetylation of specific Lysines in histone N-terminal tails weaken interactions between DNA and the histone octamer. Moreover, the HAT activities of some coactivators acetylate non-histone substrates such as viral and cellular trans-activators, for instance p53 .
Diverse viral trans-activator proteins were found to interact with distinct domains of p300/CBP and PCAF. Prominent among them are the early adenoviral E1A antigen [10, 11], Epstein-Barr virus protein EBNA-2 , and human T-cell leukemia virus oncoprotein Tax [13, 14]. Several other modifications including methylation, phosphorylation, and ubiquitination lead to either diminished or increased DNA binding of the activators that, in turn, will result in either a repression or activation of gene expression. In addition, both coactivators were reported to interact with additional HAT enzymes, namely PCAF and SRC-1 [15, 16].
The apparently nonpathogenic primate foamy viruses (PFV) show a wide host range and tissue tropism and have been developed into vectors that efficiently transduce SCID-repopulating cells . The PFV Bel1/Tas protein has been characterized as a transcriptional trans-activator of the acidic class and is known to directly interact with its responsive elements (BRE) [18, 19]. Bel1/Tas is a nuclear protein and acts as the key regulator absolutely required for virus replication. The minimal Bel1-specific DNA target site is 27 base-pairs long and located within the internal promoter (IP.BRE) upstream of the second cap site that is part and parcel of the second PFV transcription unit . Additional Bel1/Tas DNA target sites in the LTR region of the PFV DNA genome were not analyzed in this study. The acidic trans-activation domain (TAD) was mapped to the C-terminus of Bel1 with little if any protein homology to other FV Bel1/Tas proteins from different species. Previously, we identified the nuclear factor 1 (NF1) as a repressor of Bel1/Tas-mediated trans-activation . This repression was due to the fact that the specific family members NF1-C and -X interacted with parts of the IP.BRE and its flanking sequences. Since the NF1-mediated repression of the promoter of mouse mammary tumor virus was abrogated by distinct coactivators , we investigated which of the known coactivators and HAT proteins were capable of interacting with the PFV Bel1/Tas activator in the context of the IP.BRE promoter that is absolutely required for virus replication . Here we report that the Bel1/Tas DNA binding protein functionally interacted with p300 and with the well-known HAT factor PCAF. In addition, SRC-1 enhanced Bel1/Tas trans-activation. This is the first time that these cellular coactivators have been shown to interact with the Bel/Tas1 trans-activator protein. Furthermore, Bel1/Tas binding to the C/H1 domain of p300 and coactivator-driven trans-activation seem to follow a unique pathway.
Coactivator p300 enhances Bel1/Tas-mediated activation
Bel1/Tas interacts with p300 in vivo
Mapping of the p300-Bel1/Tas interaction domain
To summarize this part, our data show that p300 physically interacted with Bel1/Tas in vivo, and that the C/H1 domain of p300 was responsible for this interaction at least in vitro.
Effect of the Bel1-C/H1 domain on Bel1/Tas-mediated activation by p300C
PCAF interacts with Bel1/Tas
To assess whether SRC-1a was able to physically interact with the Bel1/Tas protein, immunoprecipitations and Western blot analysis were carried out under different conditions. The specificity of SRC-1a was ascertained by using monoclonal antibody directed against SRC-1a in control reaction. However, evidence for an interaction between the SRC-1a protein and Bel1/Tas was not obtained.
It was previously reported that Bel1/Tas is capable of inducing the expression of many cellular genes . While it is known that Bel1/Tas binds directly and to a large number of DNA target sites [18, 19, 20, 21], the mechanism of activation and the identity of the cellular partner molecules of Bel1/Tas remained unknown. As a first step, we have sought to identify the cellular proteins that interact with the PFV retroviral trans-activator and mediate its activating potential. The data presented here show that the coactivators p300 and HAT PCAF physically bound Bel1/Tas in vitro and both enhanced Bel1/Tas-mediated activation whereas SRC-1 enhanced with Bel1/Tas activation without direct binding. According to our data, Bel1/Tas specifically interacted with the C/H1 domain of p300, although we cannot rule out binding to other p300 domains with much lower affinity not detectable under the rather harsh conditions of co-immunoprecipitation used here.
When the levels of the relative luciferase activity of p300FL and its three shortened versions are compared, it is noteworthy that p300C reached the highest level of enhancement of Bel1/Tas-mediated activation (Fig. 2). Besides its intrinsic HAT activity, p300C contains both the intact C/H3 and Q domains that interact with the HAT enzymes PCAF and SRC-1, respectively [10, 24, 25]. These three HAT enzymes are likely responsible for the large enhancement of the observed trans-activation either directly by acetylation of Bel1/Tas or indirectly by histone acetylation, or both. In contrast to p300C, p300N and p300M do not possess any HAT activities nor do they bind to HAT-containing interaction partners. On the other hand, it is well known that p300 and its three subregions bind a plethora of various partner molecules leading to either activation or repression of transcription. Since the high level of p300C-mediated activation was partially inhibited in cotransfections with C/H1 and pbel1s, competition for Bel1/Tas between the C/H1 and the p300C-terminal interaction partners PCAF and SRC-1 cannot be ruled out so that both direct and indirect mechanisms might be responsible for the relative increase in Bel1/Tas-mediated activation by p300C. When Bel1/Tas binds to the C/H1 domain, the degree of Bel1/Tas acetylation may be much lower, since the protein surface of Bel1/Tas may be occluded and, hence less accessible. Some residual Bel1 acetylation might still occur by endogenous p300 and PCAF. Other factors might play additional roles. The ability of full-length p300 to trans-activate Bel1/Tas was relatively low for two reasons. First, the transfection efficiency of the full-length p300 is very low because of its large plasmid size, and the concentration of endogenous p300 is limiting. Secondly, the activation loop of p300 HAT is not fully activated by auto-acetylation as required for full trans-activation .
Since the relative activation by SRC-1 was not as high as that of p300C, we consider the HAT activity of PCAF as one of the major players of Bel1/Tas-mediated trans-activation. This result is supported by the observed enhancement of Bel1/Tas activation after cotransfection with PCAF that resulted in higher levels of Bel1 acetylation thereby leading to increased binding to the IP.BRE [J. Bodem, personal communication]. Thus, the observed high level of enhancement of p300C might be due to the synergistic effects brought about by formation of ternary p300C-PCAF-Bel1 and binary p300C-SRC complexes, respectively. In these multimeric protein complexes, Bel1/Tas binds p300C indirectly through PCAF. Consistent with the HAT activities of PCAF and p300, we detected acetylated Bel1/Tas in pbel1s-transfected 293T cells using monoclonal antibody against acetyl-Lysine after immunoblotting (our unpublished data). It is intriguing that the distribution of the closely spaced Lysines of Bel1/Tas apparently mimics the correspondingly spaced Lysines in histones. This observation is further complicated by our observation that cotransfection with higher levels of PCAF led to a reduced stability of Bel1/Tas (Fig. 9). We assume that PCAF acetylates or even hyper-acetylates the Bel1/Tas protein at closely spaced Lysines in analogy to other activators reported previously . The decreased stability of acetylated Bel1/Tas might indicate that modified Bel1/Tas is less stable than the unmodified form. This observation adds an additional layer of combinatorial regulation to Bel1/Tas-mediated trans-activation.
It is intriguing that some viral trans-activators interact with more than a single p300 domain [10, 14]. However, Bel1/Tas might recruit a second interacting region of p300 through binding PCAF (Fig. 10) that is known to interact with a p300 domain different from the C/H1 domain (Fig. 1) . The complex nature of p300-Bel1/Tas interactions reported here might serve to strengthen the overall binding affinity between Bel1/Tas and the PCAF interaction domain of p300 within a larger transcriptional complex . PCAF is known to specifically acetylate distinct Lysine residues of a subset of core histones and thereby regulate the transcriptional activity of many genes depending on the genetic context. It is well documented that acetylation, methylation and other covalent histone modifications are essential signals for the regulation of transcription .
It remains to be seen whether the stronger level of enhancement of p300C is a special if not unique feature of Bel1/Tas activation and due to over-expression or to repressive effects of other p300-interacting protein factors that cannot bind to the truncated p300 protein. Alternatively, many other factors were reported to bind to the C-terminal domains of p300 that also encompass general transcription factors TBP and TFIIB proteins that might also be responsible for the enhancement observed here [1, 2, 3].
In search of viral and cellular activators that are comparable with the ability of Bel1/Tas to interact with the C/H1 domain of p300, we found one case. A report indicates that EBNA-2 protein shares many features with Bel1/Tas that include the C-terminal acidic activation domain as well as the abilities to bind both the C/H1 domain and PCAF . There remain two differences, however. First, EBNA-2 binds to both the C/H1 and the C/H3 domain, and, secondly, PCAF does not coactivate EBNA-2 in strong contrast to Bel1/Tas .
Of note, an additional HAT enzyme, GCN5, did not interact with Bel1/Tas when tested in reporter genes assays indicating that only distinct HAT sets such as those identified in this report specifically interact with Bel1/Tas in trans-activation. The precise roles of the HAT activities of PCAF, p300, and SRC-1 during Bel1/Tas-mediated trans-activation remain to be addressed in future studies.
Coactivators PCAF and p300 were identified to physically and functionally interact with the spumaviral Bel1/Tas trans-activator. Coactivator SRC-1 was found to strongly enhance Bel1/Tas-mediated trans-activation. The C/H1 domain of p300 was responsible for binding the retroviral activator and found to partially inhibit the p300-driven trans-activation.
Mouse monoclonal antibodies directed against the FLAG epitopes of p300FL, p300N, p300M, p300C, and PCAF were purchased from Sigma, rabbit polyclonal antibodies against the N-terminal p300FL, the C-terminal p300C, and SRC-1a from Santa Cruz Biotechnology. The polyclonal serum direct against Bel1/Tas was used as described previously . Typically, 5 μl of each antiserum was used for each immunoprecipitation. Monoclonal antibody against acetyl-Lysine was purchased from Sigma.
Plasmids, cells, transfections, and metabolic labeling
Plasmids pUC18, pCMVβ-gal, pbel1s , pGL3-pro-IP. BRE (-1 to -192), pCI-FLAG-p300FL, pCI-FLAG-p300N, pCI-FLAG-p300M, pCI-FLAG-p300C , and pCI-FLAG-PCAF  were separately, or in the combinations indicated, transfected into 293T cells using Lipofectamine 2000 (Invitrogen). In general, unless otherwise indicated, 0.1 – 10 μg plasmid DNA were transfected into 293T cells grown in Petri dishes with a diameter of six cm. Full-length pCI-FLAG-300FL plasmids and three different shortened versions (Fig. 1, constructs no. 1 to 4) were grown in E. coli, DH5α cells . The PFV internal promoter was constructed by PCR-mediated amplification of the defined promoter fragments as reported previously [19, 21]. Recombinant clone p300N-C/H1-ΔKIX* (construct no. 5, Fig. 1), was constructed by first generating two PCR products with pCI-p300FL as template using the sense (s) and antisense (as) primers s1: 5'-CTTATGGTTCACCATATACTCAGAATCC-3', as1: AAACTGGAACCATGCCTGCATTTCTCTTATCACC-3', s2: 5'-GAAATGCAGGCATGGTTCCAGTTTCCAT-3', and as2: 5'-GGAAGGAACTGGCCCTGGTTGGAAGGCTGTTG-3' to amplify the sequence from nucleotide 755 to 1275 and nucleotides 1984 to 2297 fused in-frame. The resulting DNA product of 833 nucleotides was cloned into the pCR2.1 Topo vector and designated as pCR2.1-ΔKIX. An SphI-NotI DNA fragment obtained from pCR2.1-ΔKIX and was inserted into pCI-p300N that had been predigested with Sph I and NotI. The borders of construct no. 5 are shown in Fig. 1 and the expressed recombinant protein had the expected size. p300-C/H1-ΔKIX (no. 6) was constructed by inserting the SphI/NotI DNA fragment from pGEX-C/H1 (35) into pCI-p300N digested with SphI/NotI for expression of residues 1 to 424 of p300. Construct no. 7 (Fig. 1) was constructed by digesting pCI-p300N with SphI and re-ligating the larger fragment for the expression of residues 1 through 347 of p300. Finally, p300ΔN-ΔC/H1-ΔKIX was constructed from pCI-p300N by digesting with MunI and re-ligated to express residues 1 to 196 of p300. Bacterial plasmids coding for glutathion-S-transferase (GST) fusion proteins pGST-C/H1 (328–424) and pGST-KIX (436–661) (Fig. 1) were constructed from pGEX-6p-2GST-p300  and grown in E. coli, BL21 cells. pCR3.1-FLAG-SRC-1a was grown in E. coli, JM109 cells . Human 293T or HeLa cells were cultivated in DMEM medium supplemented with 1% penicillin and streptomycin, 1% Glutamine and 10 % fetal calf serum.
Plasmid p300FL was transfected into 293T cells and metabolically labeled with L-[35S]-Methionine plus L-[35S]-Cysteine (spec. act. of 37 TBq/mM, PRO-MIX, Amersham) for 6 hr. Cells were harvested and used for immunoprecipitation as described above. The precipitates were analyzed by SDS-PAGE on 12% gels, dried, and exposed on KODAK Biomax MR1 films.
Luc reporter gene expression assays
Plasmid pCMV-βgal directing β-galactosidase expression from the CMV-IE promoter was used for normalization of transfection efficiency. Luc reporter gene assays were performed and quantified as described  using a Luminoskan TL Plus luminometer (Labsystems, Frankfurt, FRG). pUC18 vector plasmid DNA was used as carrier DNA to equalize the DNA concentration of each transfection. Cells were harvested 18 h after transfection. The results of luc assays were based upon at least triplicate experiments on multiple independent occasions. Expression levels were monitored by Western blot analysis.
Immunoprecipitation was performed as described previously with minor modifications . Lysates of subconfluent cotransfected layer of 293T cells were prepared by first washing the cells with PBS, and subsequently lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl-fluoride containing 1% (v/v) Triton X-100. To inhibit unspecific protease activity, protease inhibitors (Biomol) were added to the lysis buffer. Lysates were precleared with protein A-SepharoseCL-4B (Amersham Bioscience AB, Uppsala). Co-immunoprecipitation of p300FL, p300N, p300M, p300C, SRC-1, and PCAF were performed with rabbit anti Bel1 antiserum . The immune precipitates were retrieved with protein A-SepharoseCL-4B (Pharmacia) and eluted by boiling. To detect the specific precipitate, immunoblotting was performed with specific antibodies against expressed p300, shortened p300 versions, and SRC-1. For the detection of expressed PCAF, Western blot analysis with monoclonal anti-FLAG antibody was carried out. The immunoprecipitates were washed three times with the lysis buffer and analyzed by immunoblotting on 12% gels for expressed p300FL and on 14% gels for shortened p300 forms, PCAF, and SRC-1a. The experiments were repeated at least three times, specially the immunoprecipitations of lysates of p300C-transfected cells.
GST pull-down assays
The recombinant GST-C/H1 and GST-KIX proteins were expressed in E. coli BL21/DE3 cells after transformation with the corresponding plasmids. Expressed proteins were purified by binding to glutathione Sepharose 4B resin. Each of the GST-fusion proteins bound to glutathione Sepharose 4B were mixed with lysates obtained from pbel1s-transfected 293T cells. After incubation at 4°C overnight in binding buffer  and extensive washing with the binding buffer, bound proteins were eluted, separated by SDS-PAGE, and visualized by staining with Coomassie Brilliant Blue according to Ariumi  or detected by Western blot analysis.
Cells were harvested two days after transfection by lysis in 1% SDS and the protein concentration was determined using the DC protein assay (BioRad). Identical amounts of proteins were separated by SDS-PAGE on 12% gels, blotted, reacted with monoclonal serum directed against the FLAG epitope of the four different FLAG-tagged p300 (Sigma), or polyclonal serum direct against Bel1 , and detected by enhanced chemoluminescence.
We thank Kunitada Shimotohno for critically reading the manuscript and helpful comments and our colleagues for generously providing plasmids and reagents: Shelley Berger, Don Chen, Ella Englander, Richard Gronostajski, Tasuku Honjo, Steve McMahon, Bert O'Malley, Marc Montminy, Sharon Roth, and Kunitada Shimotohno. We are grateful to Jennifer Reed for critically reading our paper and to Gholamreza Darai and Harald zur Hausen for support. The Fond der Chemischen Industrie supported this work.
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