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P300 Provides a Corepressor Function by Cooperating with YY1 and HDAC3 to Repress C-Myc

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P300 Provides a Corepressor Function by Cooperating with YY1 and HDAC3 to Repress C-Myc Oncogene (2008) 27, 5717–5728 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE p300 provides a corepressor function by cooperating with YY1 and HDAC3 to repress c-Myc N Sankar1, S Baluchamy1,3,4, R-K Kadeppagari1,3,5, G Singhal1,3, S Weitzman2 and B Thimmapaya1 1Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA and 2Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA We showed earlier that p300/CBP plays an important role Myc expression in normal cells is critical and is likely to in G1 progression by negatively regulating c-Myc and be mediated at least in part by the chromatin remodeling thereby preventing premature G1 exit. Here, we have complexes. The chromatin remodeling proteins p300 studied the mechanism by which p300 represses c-Myc and and CBP are two highly conserved nuclear phospho- show that in quiescent cells p300 cooperates with histone proteins that are recruited by a large number of deacetylase 3 (HDAC3) to repress transcription. p300 and transcription factors to activate transcription. These HDAC3 are recruited to the upstream YY1-binding site of proteins also contain intrinsic histone acetyl transferase the c-Myc promoter resulting in chromatin deacetylation activity that acetylates the nucleosomal histones and and repression of c-Myc transcription. Consistent with modifies the local chromatin structure to promote this, ablation of p300, YY1 or HDAC3 expression results transcription (Goodman and Smolik, 2000). Cell in chromatin acetylation and induction of c-Myc. These transformation by viral oncoproteins such as E1A is three proteins exist as a complex in vivo and form a dependent on its binding to and inactivating p300/CBP multiprotein complex with the YY1-binding site in vitro. (Frisch and Mymryk, 2002). p300 is mutated in several The C-terminal region of p300 is both necessary and forms of cancer suggesting a tumor suppressor role for sufficient for the repression of c-Myc. These and other this protein (Iyer et al., 2004). results suggest that in quiescent cells the C-terminal We previously showed that both p300 and CBP play region of p300 provides corepressor function and facili- an important role in quiescent cells by repressing Myc tates the recruitment of p300 and HDAC3 to the YY1- and preventing premature cell cycle G1 exit and that the binding site and represses the c-Myc promoter. This repressive activity of p300 is not dependent on its HAT corepressor function of p300 prevents the inappropriate activity (Baluchamy et al., 2003, 2005). We also showed induction of c-Myc and S phase. that E1A induces Myc in quiescent cells in a p300- Oncogene (2008) 27, 5717–5728; doi:10.1038/onc.2008.181; dependent manner (Kolli et al., 2001; Baluchamy et al., published online 9 June 2008 2007). In this paper, we present evidence that in quiescent cells, p300 cooperates with transcription Keywords: c-Myc; p300; gene expression; cell cycle; factor YY1 and histone deacetylase 3 (HDAC3) to YY1; HDAC3 repress the Myc promoter. p300 and HDAC3 associate with the Myc promoter upstream YY1-binding site resulting in the deacetylation of the Myc chromatin. YY1, p300 and HDAC3 bind to the Myc promoter YY1 Introduction site in vitro as a complex. The three proteins also form complexes both in vivo and in vitro. These and other The proto-oncogene c-Myc (referred to as Myc in this results suggest that the C-terminal region of p300 report) plays a pivotal role in the control of cell provides an essential corepressor function in recruiting proliferation and its expression is deregulated in most HDAC3 to the Myc promoter and keeping Myc in a forms of cancer (Dang, 1999). Stringent regulation of repressed state in growth-arrested cells. Correspondence: Dr B Thimmapaya, Department of Microbiology Results and Immunology, Northwestern University Medical School, 303 E. Chicago Avenue, olson 8452, Chicago, IL 60611, USA. E-mail: [email protected] Identification of p300 targets on human Myc promoter 3These authors contributed equally to this work. As p300 does not bind DNA directly, we reasoned that 4Current address: University of Illinois School of Medicine, Chicago, in quiescent cells a Myc promoter-specific transcription IL, USA. factor could recruit p300 and cooperate with deacety- 5Current address: LSU Health Sciences Center, New Orleans, LA, USA. lase(s) to repress the promoter. To identify the p300 Received 2 January 2008; revised 15 April 2008; accepted 1 May 2008; responsive elements, rat12 cells were transfected published online 9 June 2008 with Myc promoter–reporter constructs containing p300 is a corepressor of c-Myc N Sankar et al 5718 progressive deletions from the 50 end and maintained del-6 is repressed as efficiently as del-1 plasmid contain- under serum-free medium for 48 h, then serum stimu- ing 2130 bp promoter sequences (Figure 1b) and p300 lated for 2 h and the luciferase activity was quantified. fail to repress when the YY1-binding site in del-6 is Note that in this report, rat12 cells were used for mutated (Figure 1c). transient assays, whereas human breast epithelial cells (MCF10A) were used to study the endogenous Myc Recruitment of p300 and HDAC3 to the YY1 site, promoter. In our earlier studies, we used both MCF10A transcription initiation and coding regions and their and rat12 cells to show that the endogenous Myc effect on chromatin acetylation promoter is repressed by p300 (Baluchamy et al., 2003). Normally, the endogenous Myc promoter is minimally However, transfection efficiency of MCF10A cells is active transcriptionally and it is transiently induced after very poor and thus MCF10A cells could not be used for serum stimulation. We previously showed that the best transient transfection assays. Data shown in Figure 1b way to study the repression of the endogenous Myc indicate that a deletion mutant containing 647 bp promoter by p300 is to overexpress p300 in quiescent upstream from the cap site (del-6) was efficiently cells then induce Myc by serum stimulation (Baluchamy repressed by p300 (the numbering is relative to the et al., 2003). Thus, we overexpressed FLAG p300 in major P2 promoter start site; see Figure 1a; Zou et al., quiescent human MCF10A cells, serum stimulated for 1997). Although truncation of another 300 bp of the 2 h to induce Myc and then performed ChIP assays with promoter resulted in a significant loss of basal activity, primer pairs that encompass the upstream 647 bp of the this mutant also lost its response to p300 indicating that promoter sequences (Figure 2a shows the time course). transcription factors binding to sequences between À647 p300 is also reported to be associated with the and À347 bp contribute to the repression of Myc by transcriptional initiation and elongation complexes p300 (del-7; Figure 1b). Previous studies in rat and raising the possibility that in p300 overexpressing cells, mouse B cells have identified functional YY1 and increased levels of p300 may also be found in transcrip- Blimp1 sites abutting each other in the region between tional initiation and coding regions (Black et al., 2006; À350 and À450 bp of the Myc promoter (Figure 1a and Guermah et al., 2006). Therefore, we also scanned three d; Zou et al., 1997). Mutagenesis of the putative Blimp1 randomly chosen regions downstream of the RNA start site in the context of del-6 promoter did not affect site for p300 occupancy. A western blot shown in repression by p300, whereas mutation of the YY1 site Figure 2c indicated that only p300 levels increased in resulted in the complete loss of repression (plasmid p300 overexpressing cells. The occupancy of p300, YY1, YY1M, Figure 1c). Electrophoretic mobility shift assays HDAC1, -2 and -3 was quantified using real-time PCR (EMSA) using nuclear extracts prepared from human with the primer pairs shown in Figure 2b. Changes in MCF10A cells and an YY1-specific antibody showed the levels of occupancy of these proteins in different that human YY1 protein binds to this sequence (super- regions of the promoter in p300 overexpressing cells as shifted band shown by an open arrow in Figure 1e). compared to that of control samples are shown as a bar Note that in the absence of p300 overexpression the diagram in Figure 2d (see legend to Figure 2d for further reporter activity of YY1M construct is similar to that of details). These data show that the occupancy of YY1 the WTplasmid even though repression in YY1M and p300 was enriched by about 10- and 5-fold, construct is relieved (Figure 1c). We do not have a clear respectively, in the YY1-binding region (region II). A explanation for this at present. We assayed these 20-fold increase in the occupancy of HDAC3 but not plasmids 2 h after serum stimulation and it is possible HDAC1 or 2 was also noted in region II. HDAC1 and 2 that when stimulated with serum, in the absence of p300 occupancy levels did not change in any of the regions overexpression, the YY1-mediated repression of Myc is tested, and in fact, they were slightly reduced in several relieved in the WTplasmid and thus both plasmids show regions (Figure 2d). There was no increase in YY1 similar levels of reporter activity. Nonetheless, under occupancy levels in any other regions of the promoter. these assay conditions, when p300 is overexpressed, There was also no change in p300 or HDAC3 occupancy Figure 1 Identification of the p300-responsive sequences on the human Myc promoter, and the YY1-binding site as a p300 target. (a) Map of the human Myc promoter and positions of YY1 and Blimp1 sites relative to the P2 promoter transcription start site.
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