Polycomb inhibits 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 on Lys 27 () 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 compaction and other activities of PRC1 to identified autoregulatory loop, whose autoacetylation greatly en- H3K27me3-containing . 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 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 . 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 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 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 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. PRC1 subunits PC and access to the active site by and other substrates (30). PSC are present in fractions 8–11 and are cofractionated with CBP Autoacetylation of the AIL thus acts like a switch to potentiate in fractions 10 and 11 but not in fraction 8 (∼2 MDa) (Fig. 1C). robust acetylation activity. Furthermore, both PC and PSC could be coimmunoprecipitated Here, we show that PC binds directly to the CBP HAT domain via a major contact with the CBP AIL. We identify a short PC with CBP from fractions 10 and 11 (Fig. 1D), suggesting that PRC1 may be associated with CBP in a 1.5- to 1.8-MDa complex. motif with a Lys Arg Gly (KRG) core that is required for this GENETICS interaction and is conserved in all five mammalian PC orthologs We also examined the colocalization of PC and CBP on sali- (CBX2, CBX4, CBX6, CBX7, and CBX8). We show that PC vary gland polytene chromosomes by double immunostaining. competes with histone H3 for CBP binding and inhibits H3 CBP can be recruited to the insertion site of a transgene con- acetylation. PC binding to the AIL in vitro is impaired by CBP taining a PRE (35). More than one-third of identifiable PC- autoacetylation, and PC is preferentially associated with unacety- bound sites colocalized with CBP, and both are recruited to the lated CBP in vivo, further suggesting that PC binding negatively insertion site of a transgene containing a 670-bp Ubx PRE (Fig. regulates CBP HAT activity on chromatin. Knockdown of PC S1 A and B). This result is consistent with our previous obser- elevates the bulk H3K27ac level and the H3K27ac level at pro- vation from ChIP-chip data that PC and CBP co-occupy many moter regions of some genes targeted by both PC and CBP. genomic sites (36) with active or repressed marks (37). Overexpression of PC also reduces the elevated H3K27ac level We previously showed that CBP is physically associated with and the Polycomb phenotypes caused by TRX overexpression. Our several TrxG proteins in Drosophila embryos, including the H3K4 findings thus identify a previously unknown conserved activity of methyltransferase TRX (23), the H3K27 demethylase UTX, and PC in addition to the well-known binding of its chromodomain to the chromatin remodeler BRM (36). We found that PC was ab- H3K27me3-marked nucleosomes. We propose that PC inhibition of sent from IPs obtained with either anti-UTX or anti-BRM Abs CBP HAT activity plays a role in establishing and maintaining (Fig. 1E, lanes 4 and 5), suggesting that CBP/PC complexes are transcriptionally inactive chromatin states of PcG-regulated genes as distinct from CBP complexes containing UTX or BRM. However, well as modulating transcription of some active PcG-regulated genes. PC was coimmunoprecipitated with TRX (lane 6), suggesting that PC may be associated with a CBP/TRX complex (23, 35). Con- Results sistent with this possibility, PC and TRX co-occupy many PcG- PC Is Associated with Drosophila CBP in Vivo. To determine whether target genes genome-wide (21, 38). PC and CBP are physically associated in vivo, we performed (IP) from embryo nuclear extract (NE). PC Interacts with the CBP HAT Domain. We mapped the region of CBP was present in immunoprecipitates obtained with anti-PC CBP that interacts with PC by GST pull-down assays, initially by Abs (Fig. 1A, lane 3). In reciprocal IP with anti-CBP Abs, PC using a panel of GST-CBP fusion proteins to pull down endog- and the PRC1 subunit PSC [but not Sex comb on midleg (SCM)] enous PC from NE. The GST-CBP fusion proteins cover all of were coimmunoprecipitated with CBP (Fig. 1B, lane 3). In the the conserved domains of CBP: CH1 (residues 524–594); the residual unbound material, small but noticeable amounts of PC KIX domain (residues 938–1,018); CH3 (residues 2,333–2,470); and PSC were depleted by anti-CBP Abs (Fig. 1B, compare lane the catalytic core (residues 1,698–2,330), which includes the BD 5 with lane 6), suggesting that a portion of PC (and likely PRC1) (residues 1,699–1,806); the CH2 region (PHD and RING do- is stably associated with CBP in vivo. Interestingly, RPD3, which mains) (residues 1,807–1,940); and the HAT domain (residues associates with PRC1 and PRC2 and is required for deacetyla- 1,941–2,330) (Fig. 2D). GST-CBP1603-2678 (including catalytic tion of H3K27ac (8), coimmunoprecipitated not only with PC core + CH3) pulled down PC (Fig. 2A, lane 5), but CH1 and KIX (Fig. 1A, lane 3) as previously reported (34) but also with CBP did not (Fig. 2A, lanes 3 and 4). Further mapping of the PC-binding

Tie et al. PNAS | Published online January 22, 2016 | E745 Downloaded by guest on September 27, 2021 region within CBP revealed that PC interacts with the HAT domain (Fig. 2B, lane 5). To confirm this interaction in vivo, we cotransfected Dro- sophila S2 cells and performed IP. When FLAG-PC and FLAG- CBP-C (including catalytic core to C terminus) were coexpressed in S2 cells, both FLAG-PC and FLAG-CBP-C were immuno- precipitated from cell extracts with anti-PC Abs (Fig. 2C, Top, lane 5) but not with preimmune serum (Fig. 2C, Top, lane 4), indicating a specific association of PC with CBP-C in vivo. However, when FLAG-PC was coexpressed with FLAG-CBP- CΔ containing a deletion of BD, CH2, and a part of HAT (Fig. 2D), only FLAG-PC, but not CBP-CΔ, was immunoprecipitated with anti-PC (Fig. 2C, Bottom). All CBP constructs and PC in- teraction results are summarized in Fig. 2D.

PC Binds Directly to the AIL Within the CBP HAT Domain. We used recombinant PC and GST-CBP purified from Escherichia coli to determine whether PC interacts directly with CBP in vitro. We found that PC was pulled down by GST-CBP fusion proteins containing the entire catalytic core or just the HAT domain (Fig. 3A, Top, lanes 3 and 6), but not by GST-CBP fusion proteins containing just the BD and CH2 domains (Fig. 3A, Top, lanes 4 and 5), indicating direct interaction between PC and CBP HAT. By further examining N- and C-terminal halves of the HAT domain, we found that HAT-C (CBP2132–2330), which includes the ∼70-residue AIL, retained strong PC-binding ability, whereas HAT-N (CBP1941–2138) mediates a weaker interaction with PC (Fig. 3A, Top, lanes 7 and 8).

Fig. 3. PC75–228 binds directly to CBP HAT and AIL. (A and B) Purified GST- CBP fragments (Bottom, Coomassie blue staining; constructs are shown in C) and purified PC (full-length and fragments) were used in pull-down assays. PC was detected by WB with anti-His Ab. (C) Schematic summary of GST-CBP constructs tested and results of PC binding. (Top) Conserved chromodomain (CD) and conserved C-terminal motif (c-box) are indicated in black in the schematic of PC. Note that CD-deleted PC and PC75–228 are sufficient for interaction of CBP HAT-C1 (residues 2,132–2,247, including AIL).

To map the region of PC that is responsible for its direct binding to CBP, we generated recombinant PC fragments and tested their binding to GST-CBP fusions to catalytic core fragments. PC1–86, containing the chromodomain (residues 25–74), did not bind to CBP, whereas the complementary PC75–390 missing the chromo- domain retained binding to GST-CBP HAT (Fig. 3A, panels 2 and 3). PC75–228, missing both the chromodomain and the conserved C-terminal RING/SCE-binding motif (39, 40), is sufficient for the PC interaction with CBP HAT, whereas PC222–390 only binds weakly (Fig. 3A, panels 4 and 5). In tests of subfragments of HAT-C, HAT-C1 (CBP2132–2247, containing the AIL) is sufficient for strong PC binding (Fig. 3B, lane 4), whereas HAT-C2 (CBP2132–2193, which lacks the AIL) binds weakly to full-length PC but not to PC75–228 (Fig. 3B, panels 1 and 2, lane 5). Furthermore, GST-AIL (CBP2175–2247) also pulled down PC (full-length) and PC75–228 (Fig. 3B, lane 6), although their signals are not as strong as HAT-C1 (Fig. 3B, Fig. 2. CBP HAT domain interacts with PC. (A and B) Purified GST-CBP lane 4). PC222–390 barely binds to the AIL but binds the region fragments (Bottom, Coomassie blue staining; constructs are shown in D) and preceding the AIL (HAT-C1 and HAT-C2) (Fig. 3B, panel 3). embryo NE were used in pull-down assays. PC, PSC, and SU(Z)12 were ana- Deletion of two poly-His stretches in the central region of PC did lyzed by WBs. (C) Transiently coexpressed FLAG-PC and FLAG-CBP-C or FLAG- Δ not affect its binding (Fig. 3B, panel 4). All tested GST-CBP PC and FLAG-CBP-C (deletion of the central region of CBP-C) in S2 cells constructs and PC-binding results are summarized in Fig. 3C.We were immunoprecipitated with anti-PC Abs (lane 5) and analyzed by WBs with anti-FLAG mAbs. Preimmune serum (lane 4) was used as a negative conclude that the AIL-containing HAT-C1 is sufficient for a control. Unbound proteins are shown in lanes 2 and 3. (D) Schematic sum- robust PC interaction. PC75–228 binds directly to the CBP AIL, mary of GST-CBP and FLAG-CBP constructs and results of PC binding to CBP. and PC222–390 binds weakly to the region preceding the AIL.

E746 | www.pnas.org/cgi/doi/10.1073/pnas.1515465113 Tie et al. Downloaded by guest on September 27, 2021 Autoacetylation of CBP Impairs Its Interaction with PC. The AIL KRG core of PC75–228 with AAA abrogates its strong binding PNAS PLUS becomes highly acetylated by autoacetylation (32). We mutated a to GST-CBP HAT domain (Fig. S2D, panel 1, lane 3). cluster of three highly conserved Lys residues in the Drosophila We also conducted GST pull-down assays with the mouse PC CBP AIL (indicated in boldface: KKxxKxK 2226) that are auto- orthologs mCBX6, mCBX7, and mCBX8, and found that all of acetylated in mammalian p300 (32) to Ala or Gln (3K/A and 3K/Q them bind to the CBP HAT domain (Fig. S2E,lanes3–5). Fur- in Fig. 4C) and tested them in GST pull-downs. Compared with the thermore, the KRG/AAA mutation in mCBX7 and mCBX8 se- wild-type (wt) GST-CBP catalytic core (Fig. 4A,lane3),the verely impaired their binding to the CBP HAT (Fig. S2E, lanes 8 presence of the 3K/A mutation in the AIL slightly weakened and 9); CBX6 containing a deletion that removes its central region, the interaction with full-length PC (Fig. 4A, lane 4), whereas the but not its KRG motif, retained its binding ability, and mCBX8 3K/Q mutation, which mimics Lys acetylation, greatly impaired containing a deletion of its C terminus, but not its KRG motif, also PC binding (Fig. 4A, lane 5), suggesting that acetylation of one or retained its binding ability (Fig. S2E, lanes 6 and 7), further sug- more of these Lys may impair the interaction of the AIL with gestingthatKRGmotifiscritical for CBP HAT binding. The results full-length PC. However, both the 3K/A and 3K/Q mutations of these binding assays are summarized in Fig. S2F. We conclude almost abolished the interaction between GST-CBP catalytic that PC binding to the CBP HAT domain is conserved in mammals, core and PC75–228 (Fig. 4A, panel 2, lanes 4 and 5), suggesting strongly suggesting that it is functionally important in vivo. that one or more of these Lys are also particularly important for the interaction of the CBP AIL with this central region of PC PC Inhibits CBP HAT Activity in Vitro. The conserved interaction that binds most strongly to the AIL. This result also suggests that between PC and the CBP HAT domain and the preferential as- the weaker effect of the 3K/A mutation on binding of the CBP sociation of PC with unacetylated CBP in vivo suggested that PC core to full-length PC is due to additional CBP core contacts may have an effect on CBP HAT activity. We first tested whether with PC sequences that lie outside of the PC75–228 polypeptide. PC affects the interaction between the HAT domain and its his- The GST-CBP catalytic core becomes partially autoacetylated tone H3 substrate in vitro in a GST-CBP pull-down assay. As when expressed in E. coli, and the presence of the 3K/A mutation shown in Fig. 5A, PC binding to GST-CBP1603–2678 (lanes 3–5) resulted in reduced autoacetylation in E. coli (Fig. 4B, Middle, was inversely proportional to the amount of core histones added, compare lane 5 with lane 3), suggesting that one or more of these suggesting that PC competes with histone H3 for binding to the Lys residues are also autoacetylation sites in Drosophila CBP. To CBP HAT domain. Using an in vitro HAT assay with purified test whether autoacetylation affects the binding of CBP to PC, GST-CBP1603–2678 and recombinant histone H3 as a substrate, the wt and 3K/A mutant GST-CBP catalytic core proteins were we detected decreased yields of H3K27ac and H3K18ac when both further autoacetylated in vitro by incubating them with increasing amounts of PC were added to the reaction (Fig. 5B, acetyl-CoA (Fig. 4B, Middle, compare lane 4 with lane 3 and lane lanes 3–5). This result suggests that PC inhibits H3 acetylation by 6 with lane 5). This hyperacetylation impaired PC binding in CBP. PC had a similar inhibitory effect when using purified GST- pull-down assays (Fig. 4B, Top, compare lane 4 with lane 3 and CBP1689–2330 (CBP core) in the HAT assay (Fig. S3A,lanes3 lane 6 with lane 5), suggesting that PC binds preferentially to the and 4) and recombinant human nucleosomes as substrates (Fig. hypo- or unacetylated CBP HAT in vivo. To test this possibility, S3A,lanes12–17). Furthermore, PC75–228 also inhibited H3 we compared the acetylation status of the CBP present in anti- acetylation by CBP (Fig. S3A,lanes6–8), although to a somewhat GENETICS PC immunoprecipitates from embryo NE with the acetylation lesser extent than full-length PC, indicating that inhibition does status of the CBP remaining in the residual unbound soluble not require the PC chromodomain or the conserved C-terminal fraction after anti-PC IP, which was then recovered by anti-CBP motif (c-box) that binds the RING subunit of PRC1 (39, 40). IP (Fig. 4D). CBP, acetylated CBP, and PSC were determined by To determine whether the inhibitory effect of PC on CBP Western blots (Fig. 4E). Both CBP and PSC were readily de- HAT activity has been evolutionarily conserved, we tested tected in anti-PC immunoprecipitates (Fig. 4E, lane 1), but whether human PC2 (CBX4) also inhibits CBP HAT activity. acetylated CBP was not (Fig. 4E, Middle, lane 1). Acetylated Preincubating CBP with purified human CBX4 before addition CBP, assayed with a “pan–acetyl-Lys” Ab, was only detected in of acetyl-CoA to an in vitro HAT assay severely impaired acet- the unbound fraction (i.e., not associated with PC) (Fig. 4E, ylation of free histone H3 and recombinant human nucleosomes lanes 2 and 3), indicating that PC is preferentially associated with (Fig. S3B, Left and Right, lanes 3–5). This result suggests that unacetylated or hypoacetylated CBP in vivo (Fig. 4F). inhibition of CBP HAT activity by PC is conserved in mammals. CBP HAT activity is greatly enhanced by autoacetylation, PC Binding to the CBP HAT Domain Is Conserved in Mammals. To predominantly on multiple Lys in the AIL (32). We generated a determine whether there is a conserved CBP-binding motif in PC GST-CBP catalytic core containing a deletion of just the AIL and its mammalian orthologs, we aligned their sequences and (ΔK2184-G2242 or ΔAIL) and compared its HAT activity with found a short conserved region just past the chromodomain. This the HAT activity of the wt construct. As shown in Fig. 5C, al- region is rich in basic amino acid residues, has a KRG core (Fig. though wt CBP core exhibited strong autoacetylation and H3 S2F), and is present only in PC orthologs (CBX2, CBX4, CBX6, acetylation activities (Fig. 5C, Left, lane 2), CBP core ΔAIL CBX7, and CBX8) and not in the related HP1 class of chro- exhibited undetectable autoacetylation and very weak H3 acet- modomain proteins (CBX1, CBX3, and CXB5). We performed ylation (Fig. 5C, Left, lane 3). Preincubation of wt CBP core with GST pull-down assays using purified CBX4 (also known as hu- PC before addition of acetyl-CoA resulted in strong inhibition of man PC2). GST-CBX4 (full-length) and a CBX4 with a deletion H3 acetylation, reducing it to a level similar to the level observed of the N-terminal 20 residues (ΔN20), including part of its with the CBP core ΔAIL in the absence of PC (Fig. 5C, Bottom chromodomain, pulled down CBP HAT (Fig. S2A, lanes 3 and Left, compare lane 4 with lane 3). Similar preincubation of the 4), whereas deletion of the N-terminal 78 residues (ΔN78) or CBP core ΔAIL with PC had little or no additional effect on its substitution of KRG73 with three Ala’s (AAA) severely impaired weak H3 acetylation activity (Fig. 5C, Bottom Left, lane 5). These CBP HAT binding (Fig. S2A, lanes 5 and 6). In reciprocal pull- results, together with the direct binding of PC to the unacety- down assays, GST-CBP core and GST-CBP-C1 pulled down lated AIL, support the conclusion that the AIL mediates the in- CBX4 and its chromodomain-deleted form CBX4ΔN66 (Fig. S2 hibition of CBP HAT activity by PC. They also strongly suggest B and C, lane 3). Furthermore, the GST-CBP core containing that the acetylated AIL is itself required for robust H3 acetylation. either the AIL deletion or the 3K/Q mutation impaired CBX4 In summary, PC inhibits CBP HAT activity by binding to the AIL binding (Fig. S2B, lanes 4 and 5). Similarly, substitution of the and competing with histone H3 for binding to the CBP HAT

Tie et al. PNAS | Published online January 22, 2016 | E747 Downloaded by guest on September 27, 2021 domain, presumably directly or indirectly preventing the interac- tion of H3 with the active site of the enzyme.

Altering PC Levels in Vivo Affects the H3K27ac Level. To explore the effect of PC on CBP HAT activity in vivo, we altered PC levels by RNAi knockdown in S2 cells. We assayed protein levels and histone modifications by Western blots. For comparison, we also performed double knockdown of the PcG proteins ESC and ESC-like (ESCL) and single knockdown of PCL. ESC and ESCL are alternative core subunits of PRC2 that are essential for PRC2 methyltransferase activity (41), whereas PCL is present in a larger PRC2 complex (42, 43) and required for the high levels of H3K27me3 at some Polycomb target genes (43). As we pre- viously showed, simultaneous knockdown of ESC and ESCL abolished H3K27me3 (8, 41) and resulted in a very substantial increase of the alternative H3K27ac modification (8) (Fig. 6A, e and f, compare lane 2 with lane 1). Knockdown of PCL caused little change in bulk H3K27me3 and H3K27ac levels (Fig. 6A, e and f, compare lane 4 with lane 1). In contrast to knockdown of PRC2 core subunits (ESC + ESCL), knockdown of PC caused no detectable effect on the bulk H3K27me3 level (Fig. 6A, e, lane 3) but resulted in approximately a twofold increase in the total H3K27ac level (Fig. 6A, f,compare lane 3 with lane 1). The effect of PC knockdown is thus also distinct from the effect of PCL knockdown. The levels of CBP and H3K14ac (which is acetylated by GCN5 in Drosophila)werealso barely changed after PC knockdown (Fig. 6A, panels 1 and g), suggesting that the effect is specific to CBP HAT activity. Conversely, moderate constitutive overexpression of PC from a hs-Pc transgene by raising homozygotes continuously at 29 °C (a subheat shock temperature that promotes substantial leaky ex- pression of the Hsp70 promoter) resulted in a decrease in the bulk H3K27ac level in adult male flies but little detectable change in the bulk H3K27me3 level (Fig. 6B, lane 2). We also examined the effect of PC overexpression (Fig. S4A) on the H3K27ac level by immu- nostaining hs-Pc and wt embryos raised at 32 °C. Whereas the CBP level appeared unchanged (Fig. S4B), the H3K27ac level was sub- stantially reduced (Fig. S4C). This result is unlikely to be due to increased deacetylation, because the level of the H3K27ac deacetylase RPD3 in wt control embryos and hs-Pc embryosisin- distinguishable (Fig. S4 G and H). These results of PC knockdown and overexpression are consistent with the in vitro results shown in Fig. 5 and suggest that PC inhibits CBP HAT activity in vivo. The lack of a detectable change in total H3K27me3 when PC is knocked down or overexpressed does not rule out the possibility that some individual genes may have altered H3K27me3 levels (discussed below). However, it does suggest that the concomitant substantial changes in the H3K27ac level may be occurring predominantly at transcriptionally active PC-regulated genes, where an increase in H3K27ac would not have to occur at the expense of H3K27me3, as isthecaseatsilent(H3K27me3-marked) PC-regulated genes, due Fig. 4. CBP autoacetylation impairs PC binding. (A) GST-CBP pull-down us- to the mutually exclusive nature of these two modifications. ing GST-CBP catalytic core wt and mutants in which three conserved Lys To determine if the changes in the bulk H3K27ac level after residues in the AIL are mutated to Ala (3K/A) or Gln (3K/Q) (sequence is PC knockdown reflect changes at individual genes, particularly shown in C). (B) GST-CBP wt and 3K/A mutant, with (+) or without (−) pre- incubation with acetyl-CoA (Ac-CoA) to allow in vitro autoacetylation, were active genes, we also examined the effect of PC RNAi knock- used to pull down PC75–228. Relative PC75–228 levels (lane 4/lane3, lane 6/ down on the H3K27ac and H3K27me3 levels at promoter re- lane 5), quantified by a GE Typhoon TRIO Imager, are shown. (Middle) gions of seven individual genes representing four classes of genes Autoacetylated GST-CBP core proteins (wt and mutants) were detected by an co-occupied by PC and CBP in ChIP-chip (36) and ChIP- antiacetylated Lys (Kac) Ab. GST and GST-CBP core (wt and mutations) were sequencing (seq) datasets (21, 44, 45) (Fig. S5 and Table S1) and stained with Coomassie blue (A, Bottom and B, Bottom). (C) Partial se- distinguished by histone marks, RNA levels, RNA polymerase II quences of human p300 and Drosophila CBP AIL are shown. Four autoace- (Pol II) occupancy, and short RNAs [sRNAs; an indicator of Pol tylation sites in p300 are marked by asterisks above the sequence. The 3K/A II pausing (45)]. Among seven PC-regulated genes examined and 3K/Q mutations are shown below sequences. (D and E) PC is associated [abd-A, cad, pnr, Psc, Su(z)2, Thor, and tsh], Thor, tsh, and pnr with unacetylated CBP in vivo. (D) Schematic for IPs of CBP: (i) IP of CBP by anti-PC from embryo NE treated with EtBr and (ii) IP of CBP by anti-CBP Abs from the unbound by anti-PC IP. (E) Western blots were performed for CBP, acetylated CBP (ac-CBP), and PSC. IP of CBP by anti-CBP Abs (lanes 2 and 3) lated) AIL and HAT domain (Left) but is unassociated with hyperacetylated was diluted to the similar amount of CBP IP by anti-PC Ab (lane 1). (F) Model AIL and HAT (Right) after CBP autoacetylation, which may induce confor- of PC interaction with CBP HAT. PC binds to unacetylated (or hypoacety- mational changes within the HAT domain (33).

E748 | www.pnas.org/cgi/doi/10.1073/pnas.1515465113 Tie et al. Downloaded by guest on September 27, 2021 down. It did not alter the robust H3K27me3 signal at the pro- PNAS PLUS moter region of the repressed abd-A gene, which remained devoid of H3K27ac, as would be expected if the H3K27me3 mark already occupies all of the H3K27 substrates there. In contrast, the H3K27ac levels were significantly increased at the promoter regions of the already active Thor and tsh genes, which contain little or no H3K27me3. Interestingly, the active pnr gene, with its already very strong and broad H3K27ac signal encom- passing the entire gene, did not exhibit any increase of H3K27ac in its promoter region after PC knockdown. The inactive cad gene, with a strong H3K27me3 signal and paused Pol II, exhibits a very substantial increase in H3K27ac after PC knockdown and a moderate drop in its H3K27me3 signal. The adjacent and divergently transcribed Psc and Su(z)2 genes, which exhibit substantial broad H3K27ac and H3K27me3 signals and low ex- pression, show very substantial increases in H3K27ac and loss of H3K27me3. H3K27ac levels at two control regions, one down- stream of Thor gene (with a high H3K27me3 level) (control 1) and another upstream of Su(z)2 (control 2), were unchanged after PC knockdown (Fig. 6C), suggesting that changes of H3K27ac levels observed above are specific to promoter regions of these PC- regulated genes. These ChIP-qPCR results reveal a variety of be- haviors of different classes of CBP-occupied PcG-regulated pro- moter regions in response to RNAi knockdown of PC. The elevated H3K27ac levels observed for the already active Thor and tsh genes are consistent with the observed in vitro inhibition of CBP by PC and the hypothesis that PC inhibition of CBP modulates Fig. 5. PC inhibits histone H3 acetylation by CBP in vitro. (A) Competition H3K27ac levels at active genes. The unchanged high H3K27ac between PC and histone H3 for binding to CBP. PC (total of 1 μg) and histone H3 (1–3 μg) were pulled down by GST-CBP1603–2678 and were analyzed by WBs. (B) HAT activity of recombinant GST-CBP1603–2678 on free histone H3 in the absence or presence of PC. WBs were performed with anti-H3K27ac, anti-H3K18ac, and anti-H3 Abs. (C) PC inhibits CBP HAT activity regulated by the CBP AIL. The wt GST-CBP core (lanes 2 and 4) and its AIL-deleted (ΔAIL,

lanes 3 and 5) were used in HAT assays in the absence (lanes 2 and 3) or GENETICS presence (lanes 4 and 5) of PC. (Left) Autoacetylated GST-CBP core (ac-CBP) and acetylated histone H3 (ac-H3) were detected by WBs with anti-Kac Ab. (Right) PC and recombinant H3 were detected by anti-His Ab. GST-CBP core was detected by anti-GST Ab.

belong to the “active” class (marked by H3K27ac and expressed), but only pnr is embedded within a broad H3K27ac region that coverstheentiregene(Fig. S5A). abd-A, a homeotic/HOX gene in the Bithorax complex, belongs to the “repressed” class, which is inactive, lacking Pol II and sRNA, and embedded within a broad H3K27me3 region (Fig. S5C). cad is in the “poised” class, marked by H3K27me3 and not expressed, but containing Pol II and sRNA (paused Pol II) at promoter regions (Fig. S5D). Psc and Su(z)2 were included because they are distinctive. They related Fig. 6. Altering PC levels in vivo affects histone acetylation by CBP. alternative core subunits of PRC1 and must be expressed in all (A) Immunoblots of S2 cells after knockdown of GFP (control), ESC + ESCL, cells, but are themselves PcG-regulated. They have been placed in PC, and PCL as indicated at the top of lanes 1–4. Two different exposures of the so-called “balanced” (38) class because they appear to contain one WB of H3K27ac are shown in f.(B) Immunoblots of extracts from Ore- both H3K27me3 and H3K27ac over their promoter and gene gon-R (control) and hs-Pc adult male flies raised at 29 °C. PC level in hs-Pc body regions (Fig. S5B). This pattern has been hypothesized to flies was increased 75–80% in comparison to wt control at 29 °C. The H3K27ac reflect dynamic regulation or oscillating on/off expression result- level is shown in two separate WBs. Note that the H3K27ac level was de- ing from their negative autoregulation (46). Some genes in the creased but the CBP level was not reduced in hs-Pc flies. (C) ChIP-qPCR analysis. IPs were performed with anti-H3K27ac (Top) and anti-H3K27me3 (Bottom) poised and balanced classes [including cad, Psc,andSu(z)2]andin Abs. The qPCR assays were performed with primers at promoter regions of the active class (including tsh) have been previously reported to be seven genes [abd-A, cad, pnr, Psc, Su(z)2, Thor,andtsh]. Primer sets for a re- up-regulated upon knockdown of PC and PH in Kc cells (47). We gion 3′ of Thor gene (control 1) and for a region 5′ of Su(z)2 gene (control 2) previously showed that the Thor gene, which is expressed in S2 were used for controls. Rabbit preimmune serum was also used for control IP cells, was expressed at a higher level after knockdown of PC (48). from S2 cells (knockdown with GFP dsRNA) to estimate background. The av- We used PC knockdown and control S2 cells to perform chro- erage ChIP-qPCR signal in these controls was 0.1% at promoters of the seven matin IP with anti-H3K27ac and anti-H3K27me3 Abs coupled genes, and signals below 0.2% were considered to be background. In com- paring the relative ChIP efficiency from control and PC knockdown (KD) S2 with real-time quantitative PCR (ChIP-qPCR) to examine changes cells, the significant increases of the H3K27ac level at Psc, Su(z)2, cad, Thor,and in the H3K27ac/H3K27me3 levels at these seven genes. tsh and the significant decreases of the H3K27me3 level at Psc, Su(z)2,andcad As shown in Fig. 6C, five of the seven genes examined are marked by asterisks. Note that H3K27me3 levels at promoters of two active exhibited significantly elevated H3K27ac levels after PC knock- genes Thor and tsh are at background in PC KD and S2 cells.

Tie et al. PNAS | Published online January 22, 2016 | E749 Downloaded by guest on September 27, 2021 Table 1. PC overexpression suppresses the Pc phenotypes of TRX overexpressors Genotype (male progeny) T2 to T1 leg transformation T3 to T1 leg transformation

Act-Gal4/+;trxEP3541/+ 32/78 (41%) 10/78 (12%) + + hs-Pc/Y;Act-Gal4/ ;trxEP3541/ 19/70 (27%) 5/70 (7%)

Crosses were done at a constant 29 °C. Adult males were scored for the presence of extra sex combs on T2 and T3 legs. hs-Pc, heat-inducible Pc transgene; trxEP3541, GAL4-inducible trx allele.

level at the active pnr promoter region may reflect a lack of PC greater in cluster 1 and attenuated in cluster 2, possibly due to inhibition of CBP there and already saturating acetylation of all inhibition by PC. As expected, there is no detectable H3K27ac available H3K27 in its promoter region. The substantial increase associated with the weaker CBP peaks in clusters 7 and 4, where in H3K27ac at both Psc and Su(z)2 is consistent with PC in- PC and broad H3K27me3 peaks are present. H3K27ac is present hibition of CBP at their promoters, but this interpretation is in clusters 5 and 6, where medium-height CBP peaks are asso- complicated by the concomitant drop in H3K27me3, which ciated with low PC peaks, suggesting that CBP is enzymatically could reflect an indirect effect, or, as suggested previously, two active at these sites (Fig. S5 E and F). Interestingly, paused Pol II different populations of S2 cells where Psc and Su(z)2 are ei- (indicated by peaks of sRNAs) is strongly correlated with CBP ther active or repressed. An important goal of future studies and PC in clusters 1, 2, 5, and 6 and weakly correlated with will be to investigate genome-wide changes of H3K27ac and CBP and PC in cluster 7 (which include PREs, enhancers. and H3K27me3 at CBP and PC co-occupied genes by ChIP-seq promoters), suggesting that inhibition of CBP HAT activity by PC after PC knockdown. is associated with Pol II pausing in both active and repressed states. We further examined the physical association of PC and Overexpression of PC Suppresses CBP-Dependent Perturbation of paused Pol II by co-IP. CBP/p300 is present in a Pol II complex Polycomb Silencing Caused by TRX Overexpression. We also exam- (49) that preferentially contains a non- or hypophosphorylated ined the phenotypic effect of the reduced H3K27ac caused by PC overexpression in another genetic assay. We previously showed that CBP interacts directly with TRX (23) and that TRX over- expression in vivo increases the level of H3K27ac in bulk histones and antagonizes Polycomb silencing (8). TRX overexpression also results in phenotypes characteristic of Pc mutants, including the appearance of sex combs on second and third thoracic (T2 and T3) legs due to derepression of the Scr gene, which is normally expressed only in T1. Moderate RNAi knockdown of CBP sup- presses both the elevated H3K27ac level and the impaired si- lencing phenotypes caused by TRX overexpression, indicating that the phenotypic manifestations of this antagonistic effect of TRX overexpression on Polycomb silencing requires CBP (23). Given that PC overexpression also decreases the H3K27ac level, we reasoned that it may suppress these CBP-dependent pheno- types caused by TRX overexpression, thus providing additional evidence for PC inhibition of CBP activity in vivo. We found that the same moderate overexpression of PC from the hs-Pc transgene (29 °C) partially suppresses the impaired Polycomb silencing induced by constitutive TRX overexpression from a + GAL4-inducible trx allele (Materials and Methods). As shown in Table 1, the frequencies of sex comb transformations from T2 to Fig. 7. PC and CBP are associated with paused Pol II in vivo. (A)Heatmap T1 (41%) or from T3 to T1 (12%) in TRX overexpressers were of all PC peaks correlated with peaks of CBP, H3K27ac, H3K27me3, and decreased to 27% and 7%, respectively, by simultaneous over- sRNA in S2 cells, forming seven clusters. The 5 kb on each side of peak expression of PC. This phenotypic suppression further suggests midpoints is shown. Note that PC and CBP peaks are correlated with sRNA that PC overexpression also inhibits CBP in vivo. (paused Pol II) in clusters 1, 2, 5, 6, and 7. (B) Immunoblots of Pol II (Ser5-P, Ser2-P, and non-P forms) from IP by rabbit anti-PC (lane 3) and anti-CBP PC Is Physically Associated with Paused Pol II in Vivo. Genome-wide (lane 4) Abs from embryo NE. Lane 1 (input) is 5–10% of NE. Rabbit pre- chromatin occupancy patterns of PC and CBP in cultured S2 immune serum was used in lane 2 for a negative control. Note that the non- cells have revealed that PC and CBP cobind many sites, primarily P and Ser5-P forms of Pol II were detected in IPs by anti-CBP and anti-PC Abs. (C) Model of PC function in inhibition of CBP HAT activity. (Left)Inthe PREs, enhancers, and promoters of many active and repressed repressive state, chromatin is condensed. CBP, PC, and PRC complexes are genes (23, 36), and that PC is associated with many active pro- present at poised and PRE (marked by H3K27me3 and low-level moters that exhibit Pol II pausing (21, 22). To examine the ge- ). PC is associated with unacetylated CBP. CBP HAT activity to nome-wide correlation between PC, CBP, CBP HAT activity histones (and nonhistone proteins) is likely inhibited by PC. Furthermore, (H3K27ac), and paused Pol II, we generated a heat map using PC- and CBP-associated RPD3 may maintain H3K27 in a deacetylated state. ChIP-chip data for CBP, PC, H3K27ac, and H3K27me3 (36), (Right) In the active state, chromatin is relaxed. H3K27 acetylation occurs, and sRNAs produced by promoter-proximal (i.e., paused) Pol II but its level is inversely proportional to PC level (cluster 2 in A). Autoace- (45). All PC peaks on all chromosomes were clustered with the tylated CBP, Pol II, and TrxG proteins, and a relatively lower level of PC are other four factors into seven groups (Fig. 7A). CBP is present present at active enhancers (marked by H3K27ac and high-level H3K4me1) that are transcribed into enhancer RNA (eRNA). Mediator (Med), binding to with PC in all clusters except cluster 3. Although CBP peaks are transcription factors, CBP and Pol II, is involved in looping of active en- strong in both clusters 1 and 2, H3K27ac levels are different: In hancer and promoter. Paused Pol II in the Ser5-P (S5P) form and sRNA are cluster 1, H3K27ac is strong and broad, whereas PC is present at present at the proximal transcription start site (TSS). Hyperautoacetylated a moderate level; in cluster 2, where PC peaks are stronger, CBP and H3K27ac near the TSS facilitate TrxG-mediated transcription elon- H3K27ac peaks are weaker, suggesting that CBP HAT activity is gation (RNA product) by Ser2-P (S2P) Pol II.

E750 | www.pnas.org/cgi/doi/10.1073/pnas.1515465113 Tie et al. Downloaded by guest on September 27, 2021 form of Pol II (50–52). As shown in Fig. 7B for IP from embryo future goal will be to determine the genome-wide locations of PNAS PLUS NE, the unphosphorylated (non-P) form and Ser5-phosphory- acetylated (active) and unacetylated (inactive) CBP as well as the lated (Ser5-P) form, but not the Ser2-phosphorylated (Ser2-P) elevated H3K27ac level caused by loss of PC and whether they form, of Pol II were found in association with Drosophila CBP are consistent with genome-wide PC inhibition of CBP activity at (Fig. 7B, lane 4), similar to what was found for human p300 (50). active as well as repressed genes. Moreover, a small portion of Pol II with non-P and Ser5-P forms What are the roles of PC inhibition of CBP activity? CBP is (but not the Ser2-P form) is also associated with PC (Fig. 7B, recruited to specific sites on chromatin, notably enhancers and lane 3), suggesting that PC may be transiently associated with promoters, by a multitude of different transcription factors. CBP these forms of Pol II at promoters and active enhancers (53). catalytic activity plays many roles in transcription regulation, Ser5-P Pol II also coimmunoprecipitated with PC from Sg4 cell including acetylation of histones at active enhancers and pro- extracts (Fig. S6). Treatment of Sg4 cells with the RNA elon- moters, and acetylation of many transcription factors, in- gation inhibitor 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside cluding the Pol II C-terminal domain, which is essential for appeared to impair the association of Ser5-P Pol II with PC (Fig. stable association of Pol II with some promoters (57). PC in- S6, compare lane 6 with lane 3). This result is consistent with the hibition of CBP HAT activity could have biological conse- previous finding that PC/PRC1 is associated with some general quences in any of the contexts in which PC and CBP become transcription factors (54, 55). This result suggests that PC in- physically associated. Enhancers, PREs, and promoters figure hibition of CBP HAT activity may play a role in Pol II pausing. prominently as candidates, given the genome-wide pattern of PC This view is supported by recent evidence that RNAi knockdown and CBP cobinding and the effects of PC knockdown and over- of PC results in the appearance of greater amounts of Pol II on expression on global H3K27ac levels. The requirement of CBP the bodies of actively transcribed genes (22). for long-distance enhancer/promoter interactions (58) also sug- gests that PC inhibition of CBP might regulate gene expression by Discussion affecting local chromatin structure. The results presented above reveal a previously unidentified evo- CBP/p300 occupancy is a key feature of the chromatin signa- lutionarily conserved function of the PC subunit of PRC1: in- ture of enhancers, along with the H3K4me1 modification and hibition of CBP acetyltransferase activity. PC binds directly to the clusters of bound transcription factors. CBP/p300 is responsible CBP HAT domain, making a major contact with its previously for the H3K27ac mark that distinguishes active enhancers from identified AIL. The multiple Lys of the AIL are the predominant poised enhancers, some of which are marked by PRC2 with targets of CBP autoacetylation, which acts like a switch to enhance H3K27me3, a hallmark of Polycomb silencing. Although the greatly the weak HAT activity of unacetylated CBP and enable presence of H3K27me3 at cis-regulatory regions of silent Poly- robust acetylation of histones and other CBP substrates. The AIL comb target genes is sufficient to block H3K27ac and to facilitate is required for strong binding of PC to CBP and for PC inhibition recruitment of PC/PRC1 for chromatin compaction and other of histone acetylation. To our knowledge, PC is the first protein activities, binding of PC to H3K27me3 and its inhibition of CBP shown to bind to the AIL. PC inhibition of H3 acetylation might at these enhancers could act as an important backup mechanism simply be due to the observed competition between PC and H3 for to prevent unscheduled acetylation of H3K27 in the event of

binding to the CBP HAT domain. However, because PC binds to its adventitious demethylation. Furthermore, inhibiting acetyla- GENETICS the AIL, it might also reduce CBP catalytic activity by inhibiting tion of other CBP histone and nonhistone substrates at these autoacetylation of the AIL. Consistent with this possibility, PC is enhancers, including transcriptional activators, may also be im- preferentially associated with unacetylated CBP in vivo, whereas portant for maintaining their poised or repressed state and autoacetylation of CBP in vitro impairs its binding to PC. preventing unscheduled activation. PC inhibition of CBP HAT PC binding to the CBP HAT domain does not require the PC activity may also play a role in switching from active to silent chromodomain, but it requires an adjacent short motif with a states, where it may be required in conjunction with the removal KRG core that is conserved in mammalian PC orthologs, which of CBP-dependent active modifications by deacetylases to maintain also bind to CBP HAT domain and inhibit its histone acetylation the deacetylated state and allow time for acquisition of the activity. The conservation of CBP HAT binding, the KRG-binding H3K27me3 mark and maturation of a silent chromatin state. PC motif, and in vitro inhibition of CBP HAT activity by mammalian inhibition of CBP at enhancers of PC-regulated genes may also PC orthologs further suggests that PC plays a conserved role in the regulate the onset of enhancer transcription, which has been regulation of CBP/p300 activity in vivo. Interestingly, despite its reported to be impaired by p300 knockdown (59). Recent genome- intrinsic autoacetylation activity, the bulk of the p300 in mam- wide temporal profiling of enhancer transcription revealed that a malian cell extracts, as well as recombinant p300 purified from rapid burst of it, accompanied by enrichment of the H3K27ac mark insect cells, is unacetylated or hypoacetylated (56), indicating that on enhancers, occurs just before transcription from the promoters its acetylation state is tightly regulated. SIRT2 has been identified of their associated genes and is the most common initial tran- as the principal p300/CBP deacetylase in mammalian cells (31). scription event genome-wide (60). The p300 that is unacetylated on the AIL has been shown to be PC is also found associated with the promoters of both re- the form that is preferentially recruited by transcription factors to pressed and active Polycomb-regulated genes. At the former, chromatin, where it subsequently becomes activated by autoace- H3K27me3 binding by the PC chromodomain may facilitate lo- tylation in situ (56). The results reported here, including the ge- cal inhibitory interactions of PC/PRC1 with CBP. Active Poly- nome-wide colocalization of PC and CBP at many active as well as comb-regulated genes, which lack the H3K27me3 mark, are repressed genes, PC inhibition of H3 binding and acetylation by enriched for paused Pol II. The presence of PC/PRC1 at these CBP in vitro, and the preferential association of PC with unac- genes suggests that PC/PRC1 is recruited to them by a mecha- etylated CBP in vivo, suggest that PC also plays a role in inhibiting nism that is independent of H3K27me3 and that PC inhibition of or modulating CBP activity in situ on chromatin. The elevated CBP HAT activity may also modulate the transcription of these H3K27ac level caused by PC RNAi knockdown, both in bulk active genes at one or more of the multiple steps leading to histones and on five of seven PC-regulated genes analyzed, and mature mRNA production. The changes we observed in global the reduced H3K27ac level caused by PC overexpression provide H3K27ac levels in response to PC overexpression and knock- support for this conclusion, as does the ability of PC overexpression down, which occur without detectable changes in H3K27me3, to suppress the CBP-dependent effects of TRX overexpression, suggest that these changes may be occurring predominantly at including both the elevated H3K27ac level and the homeotic active genes, where an increase of H3K27ac need not occur at phenotypes due to impaired Polycomb silencing. An important the expense of H3K27me3. This possibility is further supported

Tie et al. PNAS | Published online January 22, 2016 | E751 Downloaded by guest on September 27, 2021 by the observation of increased H3K27ac levels at promoter a 1.5-mL tube and frozen in liquid nitrogen and then ground into power. regions of the active PC-regulated genes Thor and tsh, measured Proteins were extracted with 0.2 mL of nuclear extraction buffer (plus pro- by ChIP-qPCR after PC knockdown in S2 cells (Fig. 6C). Recent teinase and inhibitors and 10 mM Na butyrate) from ground evidence suggests that cohesin, which interacts physically with flies, and histones were extracted from nuclear pellets with 0.4 mL of 0.2 M H SO as previously described (8). PRC1 (61), is required for the recruitment of PRC1 to pro- 2 4 moters of some active genes, many of which exhibit Pol II IP. A 1:1 mixture of Protein A and G beads (30 μL; GE Healthcare) was pre- pausing (22). Loss of PC or other PRC1 components has been incubated with binding buffer containing 1 mg/mL BSA (64), incubated with shown to lead to an increase in Pol II on the bodies of these ∼20 μL of Abs or preimmune serum in the presence of 0.3 mL of binding genes, suggesting that PRC1 may influence Pol II pausing and/or buffer for 1–2 h at 4 °C, and then washed four times with buffer containing elongation (22). The association of PC with the initiating form of 0.5 M NaCl. Beads were resuspended in 0.2 mL of binding buffer. NE (0.1 mL) Pol II (Ser5-P) that we observed may point to such a role. or samples (0.2 mL) from Superose 6 column fractions were incubated with In summary (Fig. 7C), we have shown that the PC protein, the prepared Protein A/G beads for 2 h at 4 °C on a rotator. Beads were presumably in the context of PRC1, binds directly to the CBP washed four times with buffer containing 0.3 M NaCl and once with buffer containing 0.1 M NaCl. Bound proteins on beads were eluted by addition of HAT and AIL and inhibits histone acetylation activity, and – μ × μ competes with histone H3 for binding to the CBP catalytic do- 30 40 Lof2 SDS sample buffer. The samples were boiled, and 10- L ali- quots were used for Western blots. main. Moreover, PC is associated with unacetylated CBP in vivo and negatively regulates the total H3K27ac level and the GST Pull-Down Assay. GST and GST fusion proteins immobilized on gluta- H3K27ac level at individual genes in vivo. Taken together, these thione beads (15–30 μL) were incubated with protein extracts or purified results point to a new mechanism by which PC/PRC1 exerts a protein (∼1 μg), core histones (total of 2–4 μg of calf thymus histones) in 0.2– repressive effect on transcription. 0.3 mL of binding buffer (containing 1 mg/mL BSA) for 90 min at 4 °C. The beads were then washed four times with washing buffer [50 mM Hepes, 0.25 Materials and Methods or 0.3 M NaCl, 0.2% Nonidet P-40, 5% (vol/vol) glycerol] and once with Abs. Rabbit anti-PC Abs were raised against recombinant full-length PC (with washing buffer containing 0.1 M NaCl. Bound proteins were eluted with 20– a6× His-tag at the C terminus). Affinity purification of anti-PC Abs on 30 μLof2× SDS sample buffer for Western blots. PC-coupled Sepharose beads was performed as previously described (42). Guinea pig and rabbit anti-CBP Abs and other Abs were described previously ChIP-qPCR Analysis. S2 (Drosophila Genomics Resource Center) cells were (8). Goat anti-H3 (ab12079), rabbit anti-H3K27ac (ab4729), and anti-H3K18ac treated with dsRNA for 7 d and then cross-linked with 1% formaldehyde for (ab1191) Abs were from Abcam. Rabbit anti-H3K27me3 (07-449) and anti- 10 min at room temperature at a concentration of 2 × 107 cells per milliliter. H3K14ac (06-911) Abs were from Millipore. Anti-FLAG mAb was from Sigma. Each chromatin sample was prepared from 2.5 × 106 cells after sonication Rabbit anti-acetyl Lys (9441) Ab was from Cell Signaling. The mAbs anti-Pol II (size of DNA fragments: 0.3–1 kb) and was incubated with 0.1 mL of Protein (8WG16), phosphoserine 5 (Ser5-P) Pol II (H14), and phosphoserine 2 (Ser2-P) Gfor1–2 h at 4 °C for precleaning. IP using 3–5 μg of anti-H3K27ac Ab Pol II (H5) were from Covance. Goat anti-GST Ab was from GE Healthcare. (Abcam) or anti-H3K27me3 Ab (Millipore) and qPCR (100- to 120-bp size fragments) were performed as previously described (8) in duplicate. In- Constructs. An EST clone (RE66837) with the full length of PC coding sequence formation about qPCR primers for abd-A, cad, pnr, Psc, Su(z)2, Thor, and tsh was obtained from the Berkeley Drosophila Genome Center. Fragments and two controls is provided in Table S2. encoding PC amino acid residues 1–390 (full-length), 1–86, 75–390, 75–228, and 222–390 were generated by PCR and inserted into a modified pET-11 Heat Map Generation from ChIP-chip and ChIP-seq Data. ChIP-chip (36) vector (cut by Nde I and Nsi I). CBP constructs were previously described (8, (genetics.case.edu/Harte_ChIP-chip/) and sRNA-seq (GSM463298) (45) data 36). Other CBP constructs were made by inserting PCR fragments into the from S2 cells were loaded on the Integrated Genome Browser for Dro- pGEX-2T vector using the In-Fusion HD Cloning Kit (Clontech). PCΔ134–167, sophila chromosomes 2L, 2R, 3L, 3R, 4, and X. Peaks were called by value PC75–228(KRG/AAA), CBP 3K/A and 3K/Q mutations, and ΔAIL were gener- (1.5 for PC) or percentile (98% for CBP) and saved in a BED (Browser Ex- ated with In-Fusion HD Cloning Plus (Clontech). Construct pGEX-5X-CBX4 tensible Data) file. The sgr (simcity 4 graphics rules) files from ChiP-chip (human) was a gift from Jinke Cheng (Shanghai Jiao Tong University, Shang- were converted to WIG (wiggle) files based on a standard peak finding hai, China). pGEX-5X-CBX4ΔN20 was made from pGEX-5X-CBX4 by deleting a protocol involving BWA (Burrows-Wheeler Aligner), samtools, and MACS BamHI-BamHI fragment. Constructs pGEX-2T-CBX4ΔN78, pGEX-2T-CBX4(KRG/ (Model-based Analysis of ChIP-Seq). The z score for peaks from all data (PC, AAA), pET-CBX4-H6, and pET-CBX4ΔN66-H6 were generated by inserting a CBP, H3K27Ac, H3K27me3, and sRNAs) was calculated based on the BED PCR fragment into pGEX-2T and pET vectors, respectively. Mouse CBX6, CBX7, file and corresponding WIG files (Perl code kindly provided by Alina and CBX8 constructs were generated similarly. Saiakhova, Case Western Reserve University, Cleveland). Based on the z scores, peaks were clustered by genes (fix cluster number = 7) using Eu- In Vitro Acetylation Assay. The acetylation assay was performed in 30-μL clidean distance in Cluster 3.0 (65). The png (portable network graphics) volumes of total reaction buffer containing 5 mM sodium butyrate and tree file was then opened using Java TreeView to generate the heat map. 1 mM PMSF for 30–90 min at 30 °C (8). Recombinant GST-CBP1603–2678 or GST-CBP1689–2330 (∼50 ng) and free histone H3 (1–2 μg) (with a 6× His-tag Genetic Crosses. To test whether overexpression of PC suppresses the Pc at the C terminus) or recombinant human nucleosomes containing histone mutant-like phenotypes caused by TRX overexpression, males of the geno- + H3.1 (1.5 μg) (active motif) as a substrate were used in these assays. The type Act-Gal4/Act-Gal4;trxEP3541/trxEP3541, which carry a GAL4-inducible trx reaction was quenched by adding 30 μLof2× SDS sample buffer. For the allele (trxEP3541) and the constitutive and nearly ubiquitous Act4-GAL4 driver, inhibition assay, PC, CBX4, or BSA (0.1–1 μg) was mixed with GST-CBP for 40– were crossed to females carrying an X chromosome insertion of a hs-Pc 60 min at 4 °C before acetylation assay. transgene (66) composed of a Pc cDNA under the control of the Hsp70 promoter. Adult male progeny of the genotype hs-Pc/Y;Act-Gal4/+;trxEP3541/+ Cell Culture. Drosophila S2 and Sg4 cells (from the Drosophila Genomics Resource (experimental) were scored for homeotic transformations of T2 and T3 legs Center) were cultured in Schneider’s medium supplemented with 10% (vol/vol) FBS to T1 legs based on the appearance of sex comb teeth on T2 and T3. As a control at 25 °C. S2 cells were cotransfected for transient expression of FLAG-PC and FLAG- Act-Gal4/Act-Gal4;trxEP3541/trxEP3541 females were crossed (GE Healthcare:) to the CBPΔN as previously described (8, 62). Cells extracts were used for IP. Oregon-R wt strain, and the male progeny of the genotype Act-Gal4/+;trxEP354/+ were similarly scored. The hs-Pc transgene itself causes no homeotic transfor- Protein Extracts and Fractionation. Embryo NE (0.2 mL) was fractionated on a mations or other obvious morphological phenotypes. All of the crosses were Superose 6 column (10/300 mm; GE Healthcare) as previously described (63) doneat29°C.ThetrxEP3541 and hs-Pc lines were obtained from the Bloomington and collected at a rate of 0.5 mL per tube, with the void volume ending with Drosophila Stock Center (NIH P40OD018537). fraction 6. Immunostaining of polytene chromosomes and embryos and RNAi knock- down in S2 cells with 15 μg/mL dsRNA for 7 d were performed as previously Protein and Histone Extracts from Adult Flies Overexpressing PC. Embryos were described (8). The dsRNAs of Pc and Pcl are correspondent to 676 bp of the Pc collected from wt (Oregon-R) and homozygous hs-Pc transgenic flies and coding region (base pairs 223–898) and 705 bp of the Pcl coding region (base raised continuously at 29 °C for 2 wk. One hundred male flies were placed in pairs 1,267–1,971), respectively.

E752 | www.pnas.org/cgi/doi/10.1073/pnas.1515465113 Tie et al. Downloaded by guest on September 27, 2021 ACKNOWLEDGMENTS. We thank Dr. Sarah Smolik for chicken anti-CBP Ab; for polytene chromosome staining; Alina Saiakhova for Perl code; and Patrick PNAS PLUS Dr. Jeffrey Simon for anti-SCM Ab; Dr. Jinke Cheng for human CBX4 construct; Wu, Emily Bentley, and Brendan Mullen for assistance in plasmid construction. Dr. Danny Reinberg for cDNA of mouse CBX genes; Dr. Jayashree Prasad-Sinha This work was supported by NIH Grant R01GM39255 (to P.J.H.).

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