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Polycomb Inhibits Histone Acetylation by CBP by Binding Directly to Its Catalytic Domain

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Polycomb Inhibits Histone Acetylation by CBP by Binding Directly to Its Catalytic Domain Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain Feng Tiea,1, Rakhee Banerjeea, Chen Fua, Carl A. Strattona,2, Ming Fangb, and Peter J. Hartea,1 aDepartment of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106; and bInstitute of Life Sciences and State Ministry of Education Key Laboratory of Developmental Genes and Human Diseases, Southeast University, Nanjing, China 210096 Edited by Jasper Rine, University of California, Berkeley, CA, and approved November 23, 2015 (received for review August 5, 2015) Drosophila Polycomb (PC), a subunit of Polycomb repressive com- have been shown to mediate ubiquitylation of histone H2AK118 plex 1 (PRC1), is well known for its role in maintaining repression of (K119 in mammals). This modification has been reported to be the homeotic genes and many others and for its binding to trime- linked to Polycomb silencing in mammalian ES cells (14, 15) but thylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. is dispensable for silencing in Drosophila (16) and for mouse Here, we identify a novel activity of PC: inhibition of the histone embryogenesis (13, 17). The PC subunit contains a conserved acetylation activity of CREB-binding protein (CBP). We show that PC N-terminal chromodomain (18) that binds specifically to the and its mammalian CBX orthologs interact directly with the histone H3K27me3 mark deposited by PRC2 (19, 20), thereby targeting acetyltransferase (HAT) domain of CBP, binding to the previously the chromatin compaction and other activities of PRC1 to identified autoregulatory loop, whose autoacetylation greatly en- H3K27me3-containing nucleosomes. Consistent with this observa- hances HAT activity. We identify a conserved PC motif adjacent to tion, the genome-wide binding pattern of PRC1 is highly correlated the chromodomain required for CBP binding and show that PC bind- with inactive genes marked by H3K27me3. However, several recent ing inhibits acetylation of histone H3. CBP autoacetylation impairs studies have revealed that PRC1 is also bound at promoters of PC binding in vitro, and PC is preferentially associated with unace- many active genes that contain little or no H3K27me3 (21, 22), tylated CBP in vivo. PC knockdown elevates the acetylated H3K27 suggesting that PRC1 can be targeted independent of the PC (H3K27ac) level globally and at promoter regions of some genes chromodomain and may also negatively modulate transcription of that are bound by both PC and CBP. Conversely, PC overexpression active genes. decreases the H3K27ac level in vivo and also suppresses CBP-depen- The repressive effects of PRC1 and PRC2 are antagonized by dent Polycomb phenotypes caused by overexpression of Trithorax, TrxG proteins, which include histone modifying and chromatin an antagonist of Polycomb silencing. We find that PC is physically remodeling enzymes. Prominent among the antagonistic activities associated with the initiating form of RNA polymerase II (Pol II) and associated with TrxG proteins is H3K27 acetylation, which is cata- that many promoters co-occupied by PC and CBP are associated with lyzed by the acetyltransferase CREB-binding protein (CBP) in paused Pol II, suggesting that PC may play a role in Pol II pausing. Drosophila and by the closely related CBP and p300 proteins in These results suggest that PC/PRC1 inhibition of CBP HAT activity mammals (8). We previously found that some H3K27ac is de- plays a role in regulating transcription of both repressed and active pendent on the Trithorax protein (TRX) (8), a well-known antag- PC-regulated genes. onist of Polycomb silencing, and reflects a direct interaction of TRX with CBP (23). Moreover, both the elevated H3K27ac level and Polycomb | CBP | acetylation | histone H3K27 | Drosophila impaired Polycomb silencing phenotypes caused by overexpression of TRX in vivo are suppressed by reducing the CBP level (23). he Polycomb group (PcG) and Trithorax group (TrxG) pro- CBP and p300 play important roles as transcriptional coac- Tteins are well known for their mutually antagonistic roles in tivators. Their histone acetyltransferase (HAT) activity is required maintaining, respectively, stable heritable repression and activa- tion of genes that specify the different cell identities comprising Significance the body plans of multicellular organisms. Two principal types of PcG-containing complexes, termed Polycomb repressive complex The Polycomb protein (PC) is well known for its role in tran- 1 (PRC1) and PRC2, have been identified in Drosophila and in scriptional silencing and binding to trimethylated histone H3 mammals (1). PRC1 and PRC2 are recruited to their target genes Lys27 (H3K27me3). We report here that PC inhibits the histone by specialized “Polycomb response elements” (PREs) in Dro- acetyltransferase (HAT) activity of CREB-binding protein (CBP). sophila (2, 3) and by unmethylated CpG islands in mammals (4). PC interacts directly with the CBP HAT domain, binding to its The discovery of enzyme activities associated with PRC2 and autoregulatory loop, whose autoacetylation greatly enhances PRC1 has provided important insights into their functions. PRC2 enzyme activity. PC binding inhibits histone H3 acetylation. trimethylates histone H3 on Lys27 (H3K27me3), and the genome- Interestingly, CBP autoacetylation impairs PC binding in vitro, wide distribution of its H3K27me3 product is highly correlated and PC is preferentially associated with unacetylated CBP in with transcriptionally silent genes (5). Moreover, Drosophila har- vivo. Altering PC levels in vivo alters the acetylated H3K27 boring a histone H3K27R or H3K27A point mutation fails to (H3K27ac) level in a predictable manner. PC inhibition of CBP silence PcG target genes, indicating that this modification is HAT activity at enhancers and promoters with paused RNA essential for silencing (6, 7). The repressive effect of H3K27 polymerase II may affect regulation of both repressed and methylation by PRC2 is thought to be due, in part, to direct active genes. blocking of H3K27 acetylation (H3K27ac) (8), a mark of active enhancers and promoters, because methyl- and acetyl modifi- Author contributions: F.T. and P.J.H. designed research; F.T., R.B., and C.A.S. performed cations of the Lys e-amino group are mutually exclusive. research; F.T., C.F., M.F., and P.J.H. analyzed data; and F.T. and P.J.H. wrote the paper. Biochemical studies have shown that PRC1, composed of core The authors declare no conflict of interest. subunits Polycomb (PC), PH, PSC, and RING/Sex combs extra This article is a PNAS Direct Submission. (SCE), can exert a repressive effect on transcription from 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. chromatin templates in vitro by inhibiting nucleosome remodeling 2Present address: Production Department, Cell Signaling Technology, Beverly, MA 01915. (9, 10) and transcription initiation (11) and by promoting chro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. matin compaction (12, 13). The RING and PSC subunits of PRC1 1073/pnas.1515465113/-/DCSupplemental. E744–E753 | PNAS | Published online January 22, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1515465113 Downloaded by guest on September 27, 2021 for CBP/p300-dependent transcription from chromatin in vitro PNAS PLUS and in vivo (24, 25). They are recruited by hundreds of different transcription factors (26, 27) to specific genomic sites, notably enhancers, where they acetylate multiple Lys residues in their histone and nonhistone substrates, including many transcription factors (28). Sequence-specific targeting of p300 catalytic domain to specific enhancers and promoters, using a Cas9-p300 fusion protein, is sufficient to induce H3K27 acetylation and robust transcription of the associated genes (29). CBP and p300 are large modular proteins with multiple con- served domains, including KIX, Cys/His-rich region 1 (CH1), bromodomain (BD), CH2 (composed of a discontinuous PHD finger domain interrupted by a RING finger domain), HAT, and CH3 domains. Their tandemly arranged BD, PHD, RING, and Fig. 1. PC is physically associated with Drosophila CBP. (A and B) Immuno- HAT domains are required for robust acetyltransferase activity blots of CBP and PRC1 subunits after IP from embryo NEs with anti-PC and in vitro and in vivo. A recent p300 crystal structure (30) revealed anti-CBP Abs (lane 3). NEs were treated with ethidium bromide (EtBr) to eliminate DNA-mediated protein associations. An asterisk next to the RPD3 that these domains form a single compact structural module “ ” blot in A indicates the heavy chain of IgG. (C) Fractionation of NE on a referred to as the catalytic core. The RING domain is posi- Superose 6 (size exclusion) column. Proteins in fractions were analyzed by tioned over the substrate-binding pocket of the HAT domain, Western blots (WBs). The size standards and fraction numbers are indicated with which it makes multiple contacts, partially occluding it, at the top. (D) IP from fractions 10, 11, 12, 14, and 16 in C with anti-CBP Abs. suggesting that the RING domain may play a role in regulating Mock IP from NE without Abs serves as a negative control (lane C). (E)PCis substrate binding and/or HAT activity of the native enzyme (30). also associated with TRX, but not with UTX or BRM. IPs were performed with The p300 HAT activity toward its histone and nonhistone anti-CBP (positive control), anti-UTX, anti-BRM, and anti-TRX Abs (lanes 3–6). substrates is activated by autoacetylation and inactivated by Preimmune serum (lane 2 in A, B, and E) serves as a negative control. deacetylation by SIRT2 (31). Autoacetylation occurs in trans,pre- dominantly on multiple conserved Lys residues within a flexible (Fig. 1B, lane 3), suggesting that a small portion of RPD3 may be “autoinhibitory loop” (AIL) (also known as an autoacetylation loop) associated with the CBP/PC complex. and greatly stimulates HAT activity (32). It induces conformational To confirm the association of PC and CBP further, we frac- changes within the HAT domain (33) that are thought to allow tionated NEs on a size exclusion column.
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