The amino-terminal C/H1 domain of CREB binding protein mediates zta transcriptional activation of latent Epstein-Barr virus.
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Latent Epstein-Barr virus (EBV) is maintained as a nucleosome-covered episome that can be transcriptionally activated by overexpression of the viral immediate-early protein, Zta. We show here that reactivation of latent EBV by Zta can be significantly enhanced by coexpression of the cellular coactivators CREB binding protein (CBP) and p300. A stable complex containing both Zta and CBP could be isolated from lytically stimulated, but not latently infected RAJI nuclear extracts. Zta-mediated viral reactivation and transcriptional activation were both significantly inhibited by coexpression of the E1A 12S protein but not by an N-terminal deletion mutation of E1A (E1ADelta2-36), which fails to bind CBP. Zta bound directly to two related cysteine- and histidine-rich domains of CBP, referred to as C/H1 and C/H3. These domains both interacted specifically with the transcriptional activation domain of Zta in an electrophoretic mobility shift assay. Interestingly, we found that the C/H3 domain was a potent dominant negative inhibitor of Zta transcriptional activation function. In contrast, an amino-terminal fragment containing the C/H1 domain was sufficient for coactivation of Zta transcription and viral reactivation function. Thus, CBP can stimulate the transcription of latent EBV in a histone acetyltransferase-independent manner mediated by the CBP amino-terminal C/H1-containing domain. We propose that CBP may regulate aspects of EBV latency and reactivation by integrating cellular signals mediated by competitive interactions between C/H1, C/H3, and the Zta activation domain. (
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The histone acetylase PCAF is a phorbol-ester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness.
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Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes. To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells. The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes. By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins. This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities. Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes. Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-alpha-stimulated gene expression in U937 cells. Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-alpha-inducible promoter activity. These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness. (
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Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A.
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Histone acetyltransferases (HAT) play a critical role in transcriptional control by relieving repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for inhibition of p300-dependent transcription by Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein. (
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Transcriptional regulation of the mouse ferritin H gene. Involvement of p300/CBP adaptor proteins in FER-1 enhancer activity.
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We previously identified a major enhancer of the mouse ferritin H gene (FER-1) that is central to repression of the ferritin H gene by the adenovirus E1A oncogene (Tsuji, Y., Akebi, N., Lam, T. K., Nakabeppu, Y., Torti, S. V., and Torti, F. M. (1995) Mol. Cell. Biol. 15, 5152-5164). To dissect the molecular mechanism of transcriptional regulation of ferritin H, E1A mutants were tested for their ability to repress FER-1 enhancer activity using cotransfection with ferritin H-chloramphenicol acetyltransferase (CAT) reporter constructs. Here we report that p300/CBP transcriptional adaptor proteins are involved in the regulation of ferritin H transcription through the FER-1 enhancer element. Thus, E1A mutants that failed to bind p300/CBP lost the ability to repress FER-1, whereas mutants of E1A that abrogated its interaction with Rb, p107, or p130 were fully functional in transcriptional repression. Transfection with E1A did not affect endogenous p300/CBP levels, suggesting that repression of FER-1 by E1A is not due to repression of p300/CBP synthesis, but to E1A and p300/CBP interaction. In addition, we have demonstrated that transfection of a p300 expression plasmid significantly activated ferritin H-CAT containing the FER-1 enhancer, but had a marginal effect on ferritin H-CAT with FER-1 deleted. Furthermore, both wild-type p300 and a p300 mutant that failed to bind E1A but retained an adaptor function restored FER-1 enhancer activity repressed by E1A. Sodium butyrate, an inhibitor of histone deacetylase, mimicked p300/CBP function in activation of ferritin H-CAT and elevation of endogenous ferritin H mRNA, suggesting that the histone acetyltransferase activity of p300/CBP or its associated proteins may contribute to the activation of ferritin H transcription. Recruitment of these broadly active transcriptional adaptor proteins for ferritin H synthesis may represent an important mechanism by which changes in iron metabolism are coordinated with other cellular responses mediated by p300/CBP. (
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Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300.
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The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 were found to act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors. (
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Murine double minute (MDM2) blocks p53-coactivator interaction, a new mechanism for inhibition of p53-dependent gene expression.
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The ability of the p53 tumor suppressor to induce cell cycle arrest and cell death is closely regulated under normal conditions. The transcriptional activity of p53 is negatively controlled by murine double minute (MDM2). p53 requires the coactivator CREB-binding protein (CBP), or its structural homolog, p300, to stimulate transcription of responsive genes. Here we find that the transactivation domain of p53 selectively interacts with the N- and C-terminal regions of CBP/p300. A mutant CBP lacking the N terminus failed to stimulate p53-dependent transactivation. In both p53 null Saos2 cells, and in UV-irradiated MCF7 cells, we observed that MDM2 associates with the N-terminal region of CBP/p300. Because p53 interacts with both MDM2 and CBP/p300 through its trans-activation domain, we examined the role of MDM2 in p53-coactivator interactions. MDM2 blocked CBP/p300 recruitment in vitro and inhibited the interaction of the transactivating region of p53 with both the N- or C-terminal regions of CBP/p300 in a mammalian two-hybrid assay. These observations suggest that MDM2 may be inhibiting p53 trans-activation by shielding its activation domain from the coactivators, a new mechanism for the inhibition of p53-dependent gene expression. (
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Cooperation of Sp1 and p300 in the induction of the CDK inhibitor p21WAF1/CIP1 during NGF-mediated neuronal differentiation.
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Addition of nerve growth factor (NGF) to PC12 cells promotes neuronal differentiation while inhibiting cell proliferation. In order to understand how NGF exerts its antimitogenic effect during differentiation, we have studied the mechanism by which this factor activates the promoter of the CDK inhibitor p21W4F1/CIP1. The minimal region of the p21 promoter required for the NGF-induction was mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. This GC-rich region was shown to interact specifically with the transcription factor Sp1 and the related protein Sp3, in either exponentially-growing or NGF-treated PC12 cells. The addition of NGF resulted in an accumulation of the transcriptional co-activator p300 in complexes associated with the NGF-responsive region. Transcriptional activity of Sp1, Sp3 and p300 was specifically induced by NGF in a Gal4-fusion assay, indicating that induction of p21 during neuronal differentiation may involve regulation of the activity of these factors by NGF. Furthermore, p300 was able to act as a co-activator for Sp1-mediated transcriptional activation in PC12 cells, suggesting that p300 and Sp1 may cooperate in activating p21 transcription during the withdrawal of neuronal precursors from the cell cycle. This hypothesis is supported by experiments showing that p300 and Sp1 form complexes in PC12 cells. (
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Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation.
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During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle. (
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