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• W3136200267 abstract "•p300/CBP and deacetylase activities regulate dynamic (de)activation of enhancers•p300/CBP-catalyzed acetylation promotes PIC assembly and RNAPII recruitment•BRD4 acts as a p300/CBP downstream effector to promote RNAPII pause release•Coupling of RNAPII recruitment and pause release enables rapid enhancer activation The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a “recruit-and-release” mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription. The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a “recruit-and-release” mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription. Among different regulatory mechanisms, distal-acting enhancers are extensively implicated in the activation of developmental and perturbation-induced genes in metazoans (de Laat and Duboule, 2013de Laat W. Duboule D. Topology of mammalian developmental enhancers and their regulatory landscapes.Nature. 2013; 502: 499-506Crossref PubMed Scopus (324) Google Scholar; Spitz and Furlong, 2012Spitz F. Furlong E.E. Transcription factors: from enhancer binding to developmental control.Nat. Rev. Genet. 2012; 13: 613-626Crossref PubMed Scopus (1100) Google Scholar). How enhancers are dynamically activated and deactivated and how they promote gene transcription is not fully understood. The metazoan-specific acetyltransferase paralogs p300 and CBP (hereafter p300/CBP) are frequently implicated in enhancer-dependent transcription regulation. Early studies reported a structural role of p300/CBP in mediating the interaction with general transcription factors (GTFs) and RNA polymerase II (RNAPII, or Pol2) (Kim et al., 1998Kim T.K. Kim T.H. Maniatis T. Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro.Proc. Natl. Acad. Sci. USA. 1998; 95: 12191-12196Crossref PubMed Scopus (93) Google Scholar; Wang et al., 2013Wang F. Marshall C.B. Ikura M. Transcriptional/epigenetic regulator CBP/p300 in tumorigenesis: structural and functional versatility in target recognition.Cell. Mol. Life Sci. 2013; 70: 3989-4008Crossref PubMed Scopus (194) Google Scholar). In recent years, p300/CBP activity has been directly implicated in enhancer activation, and p300/CBP-catalyzed H3K27ac is widely used as a hallmark for distinguishing transcriptionally active enhancers from inactive and paused enhancers (Calo and Wysocka, 2013Calo E. Wysocka J. Modification of enhancer chromatin: what, how, and why?.Mol. Cell. 2013; 49: 825-837Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar; Creyghton et al., 2010Creyghton M.P. Cheng A.W. Welstead G.G. Kooistra T. Carey B.W. Steine E.J. Hanna J. Lodato M.A. Frampton G.M. Sharp P.A. et al.Histone H3K27ac separates active from poised enhancers and predicts developmental state.Proc. Natl. Acad. Sci. USA. 2010; 107: 21931-21936Crossref PubMed Scopus (2361) Google Scholar; Heintzman et al., 2007Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. et al.Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (2336) Google Scholar; Rada-Iglesias et al., 2011Rada-Iglesias A. Bajpai R. Swigut T. Brugmann S.A. Flynn R.A. Wysocka J. A unique chromatin signature uncovers early developmental enhancers in humans.Nature. 2011; 470: 279-283Crossref PubMed Scopus (1467) Google Scholar). Recent proteome-wide analyses show that p300/CBP activity additionally targets hundreds of acetylation sites on transcription regulators, including many enhancer-associated proteins, and p300/CBP-catalyzed acetylation is rapidly turned over (Weinert et al., 2018Weinert B.T. Narita T. Satpathy S. Srinivasan B. Hansen B.K. Schölz C. Hamilton W.B. Zucconi B.E. Wang W.W. Liu W.R. et al.Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.Cell. 2018; 174: 231-244.e12Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Mechanistically, it is understood that p300/CBP-catalyzed acetylation promotes the recruitment of BRD4, a member of the bromo and extra-terminal (BET) family, which then promotes the recruitment and/or activation of P-TEFb and the release of promoter-proximal-paused RNAPII. Notably, promoter-proximal RNAPII pausing is a distinctive feature of metazoan transcription whose evolution appears to coincide with the metazoan-specific emergence of p300/CBP, and the release of paused RNAPII is thought to be a rate-limiting step in enhancer activation (Adelman and Lis, 2012Adelman K. Lis J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans.Nat. Rev. Genet. 2012; 13: 720-731Crossref PubMed Scopus (735) Google Scholar; Chen et al., 2018Chen F.X. Smith E.R. Shilatifard A. Born to run: control of transcription elongation by RNA polymerase II.Nat. Rev. Mol. Cell Biol. 2018; 19: 464-478Crossref PubMed Scopus (162) Google Scholar; Core and Adelman, 2019Core L. Adelman K. Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation.Genes Dev. 2019; 33: 960-982Crossref PubMed Scopus (142) Google Scholar). Transcription initiation requires the assembly of pre-initiation complex (PIC), which consists of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. The first steps in PIC assembly involve the recognition of promoter DNA by TFIID and subsequent binding of TFIIA and TFIIB (Sainsbury et al., 2015Sainsbury S. Bernecky C. Cramer P. Structural basis of transcription initiation by RNA polymerase II.Nat. Rev. Mol. Cell Biol. 2015; 16: 129-143Crossref PubMed Scopus (231) Google Scholar). TFIIB recruits TFIIF-RNAPII and successive binding of TFIIE and TFIIH complete the core PIC. Mediator is recruited through enhancer-associated transcription factors (TFs), but it contacts with multiple PIC components, including TFIID and RNAPII, and plays an important role in the assembly and/or stabilization of PIC (Malik and Roeder, 2010Malik S. Roeder R.G. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.Nat. Rev. Genet. 2010; 11: 761-772Crossref PubMed Scopus (516) Google Scholar; Soutourina, 2018Soutourina J. Transcription regulation by the Mediator complex.Nat. Rev. Mol. Cell Biol. 2018; 19: 262-274Crossref PubMed Scopus (228) Google Scholar). While BRD4-dependent RNAPII pause release has emerged as a central regulator of enhancer activation, much less is known about the dynamic regulation of transcription initiation. For example, very little is known about whether and how signal-induced perturbations regulate PIC assembly and RNAPII recruitment and whether this plays a role in dynamic transcription regulation. For this reason, transcription initiation is rarely portrayed as a regulatory step from a dynamic control point of view, and RNAPII pause release is depicted as the main regulatory step in signal-induced dynamic enhancer activation. Recent studies proposed a role of phase-separated condensates in the activation of super-enhancers (SEs) but not typical enhancers (TEs) (Boija et al., 2018Boija A. Klein I.A. Sabari B.R. Dall’Agnese A. Coffey E.L. Zamudio A.V. Li C.H. Shrinivas K. Manteiga J.C. Hannett N.M. et al.Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains.Cell. 2018; 175: 1842-1855.e16Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar; Cho et al., 2018Cho W.K. Spille J.H. Hecht M. Lee C. Li C. Grube V. Cisse I.I. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.Science. 2018; 361: 412-415Crossref PubMed Scopus (537) Google Scholar; Sabari et al., 2018Sabari B.R. Dall’Agnese A. Boija A. Klein I.A. Coffey E.L. Shrinivas K. Abraham B.J. Hannett N.M. Zamudio A.V. Manteiga J.C. et al.Coactivator condensation at super-enhancers links phase separation and gene control.Science. 2018; 361: eaar3958Crossref PubMed Google Scholar). However, it remains unclear whether and how RNAPII recruitment at enhancers is dynamically regulated and whether reversible acetylation has a role in this process. Here, we systematically investigated the function and mechanisms of p300/CBP-catalyzed acetylation in dynamic enhancer activation. Our work reveals that p300/CBP-catalyzed acetylation is a key driver of rapid enhancer activation, and unmasks a critical role of lysine deacetylases (KDACs) in kinetic enhancer deactivation. Notably, p300/CBP inhibition abrogates enhancer activation without altering the binding of TFs and p300. We uncover a hitherto unappreciated function of p300/CBP-catalyzed acetylation in RNAPII recruitment and transcription initiation and uncouple this function from the previously known role of p300/CBP in BRD4 recruitment and pause release. Mechanistically, we identify a crucial role of p300/CBP activity in the dynamic recruitment of TFIID, PIC assembly, and transcription initiation. We propose that p300/CBP uses a “recruit-and-release” mechanism to simultaneously promote RNAPII recruitment and pause release; it thereby functions as a master regulator of enhancer-dependent transcription. To investigate the kinetics and mechanisms of p300/CBP-regulated enhancer activation, we used A-485, a highly selective inhibitor of p300/CBP catalytic activity (Lasko et al., 2017Lasko L.M. Jakob C.G. Edalji R.P. Qiu W. Montgomery D. Digiammarino E.L. Hansen T.M. Risi R.M. Frey R. Manaves V. et al.Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.Nature. 2017; 550: 128-132Crossref PubMed Scopus (305) Google Scholar; Weinert et al., 2018Weinert B.T. Narita T. Satpathy S. Srinivasan B. Hansen B.K. Schölz C. Hamilton W.B. Zucconi B.E. Wang W.W. Liu W.R. et al.Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.Cell. 2018; 174: 231-244.e12Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The inhibition of p300/CBP activity was confirmed in mouse embryonic stem cells (ESCs) by quantifying the acetylome and proteome changes using mass spectrometry. A-485 treatment reduced the acetylation of several hundred sites, including many sites on histones and transcription regulators (Table S1). Acetylome changes in ESCs were highly correlated (r = 0.76) with A-485-induced changes in mouse embryonic fibroblasts (MEFs) (Weinert et al., 2018Weinert B.T. Narita T. Satpathy S. Srinivasan B. Hansen B.K. Schölz C. Hamilton W.B. Zucconi B.E. Wang W.W. Liu W.R. et al.Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.Cell. 2018; 174: 231-244.e12Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) (Figures S1A and S1B), confirming a robust A-485-induced acetylation inhibition. To investigate the kinetics of p300/CBP-dependent transcription, we performed time-resolved transcriptome analyses in ESCs by in vivo nascent RNA labeling by 5-ethynyl uridine (5-EU) and next-generation sequencing (hereafter EU-seq) (Table S2). Transcription changes were quantified at 30, 60, and 120 min after A-485 treatment. Previously, we used a 3-μM concentration of A-485 for cellular assays (Lasko et al., 2017Lasko L.M. Jakob C.G. Edalji R.P. Qiu W. Montgomery D. Digiammarino E.L. Hansen T.M. Risi R.M. Frey R. Manaves V. et al.Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.Nature. 2017; 550: 128-132Crossref PubMed Scopus (305) Google Scholar; Weinert et al., 2018Weinert B.T. Narita T. Satpathy S. Srinivasan B. Hansen B.K. Schölz C. Hamilton W.B. Zucconi B.E. Wang W.W. Liu W.R. et al.Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome.Cell. 2018; 174: 231-244.e12Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, during the course of this work, we noted that p300/CBP is incompletely inhibited at this concentration and used a higher (10 μM) concentration of A-485. Although A-485 does not inhibit other acetyltransferases at 10 μM, other off-target effects are possible at this concentration (Lasko et al., 2017Lasko L.M. Jakob C.G. Edalji R.P. Qiu W. Montgomery D. Digiammarino E.L. Hansen T.M. Risi R.M. Frey R. Manaves V. et al.Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.Nature. 2017; 550: 128-132Crossref PubMed Scopus (305) Google Scholar). At each time point, cells were pulsed with 5-EU for 20 min before harvesting. A-485 treatment almost exclusively caused transcription downregulation, demonstrating an exquisite role of p300/CBP activity in transcription activation (Figure S1C). Based on their kinetic profiles, we categorized genes into three classes: A-485-downregulated (Down), slightly downregulated (Slt. Down), and not changed (N.C.; unregulated) (Figure 1A). This classification of A-485 regulated genes was used throughout for different comparisons. Notably, transcription was strongly inhibited within 30 min of A-485 treatment (Figure 1A). To obtain an even better temporal resolution, we added A-485 and 5-EU simultaneously and analyzed transcription after 20 min. Remarkably, even in this condition, transcription of p300/CBP-dependent genes was robustly downregulated (Figures 1B and 1C). To directly assess the role of p300/CBP activity in enhancer activation, we analyzed the expression of enhancer RNAs (eRNAs), which closely mirrors enhancer activity (Kim et al., 2010Kim T.K. Hemberg M. Gray J.M. Costa A.M. Bear D.M. Wu J. Harmin D.A. Laptewicz M. Barbara-Haley K. Kuersten S. et al.Widespread transcription at neuronal activity-regulated enhancers.Nature. 2010; 465: 182-187Crossref PubMed Scopus (1628) Google Scholar). eRNAs are expressed at low levels and are difficult to capture in bulk nascent transcriptome analyses; therefore, we analyzed eRNA expression in SE regions, which are more highly transcribed. eRNA expression was rapidly attenuated (Figures 1D and 1E), demonstrating that p300/CBP inhibition results in very rapid enhancer deactivation. To test a cell-type specificity of A-485-downregulated genes, we compared the specificity of A-485-downregulated and nonregulated genes with genes expressed in 151 different mouse tissues and developmental stages (Hon et al., 2017Hon C.C. Ramilowski J.A. Harshbarger J. Bertin N. Rackham O.J. Gough J. Denisenko E. Schmeier S. Poulsen T.M. Severin J. et al.An atlas of human long non-coding RNAs with accurate 5′ ends.Nature. 2017; 543: 199-204Crossref PubMed Scopus (558) Google Scholar; Yu et al., 2015bYu N.Y. Hallström B.M. Fagerberg L. Ponten F. Kawaji H. Carninci P. Forrest A.R. Hayashizaki Y. Uhlén M. Daub C.O. Fantom ConsortiumComplementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium.Nucleic Acids Res. 2015; 43: 6787-6798Crossref PubMed Scopus (61) Google Scholar). A-485-downregulated genes were more specifically expressed in ESCs, while unregulated genes were commonly expressed across all tissues (Figure 1F). Strongly downregulated genes were the most cell-type specific, and slightly downregulated genes showed intermediate cell-type specificity. Notably, the transcription of key ESC-specific TFs, including Nanog, Oct4 (Pou5f1), Sox2, Nr0b1, Klf2, Esrrb, Rex1, and Tet1, was virtually switched off within 30 min (Figure S1D). To test the reversibility of A-485-induced transcription inhibition, we analyzed the expression of GFP-tagged NANOG. NANOG-GFP expression was reduced within hours and became almost undetectable after 17 h of A-485 treatment. Notably, the expression of NANOG-GFP was restored within 24 h after A-485 withdrawal (Figure S2A), indicating that enhancers are deactivated transiently and remain poised for reactivation, at least within the time frame of our experiments. These results support a key function of p300/CBP in CTS transcription and demonstrate that continuous p300/CBP catalytic activity is critical for maintaining metazoan cell identity. We hypothesized that if transcription inhibition by A-485 results directly from reduced acetylation, then the inhibition of KDACs should have the opposite effect and reciprocally increase the expression of the same genes. To test this idea, we analyzed transcription changes in ESCs treated with the KDAC inhibitor TSA (trichostatin A). TSA treatment (60 min) caused modest transcription changes, resulting in both transcription up- and downregulation, and TSA-regulated changes showed no correlation (r = −0.07) with A-485-induced changes (Figure S2B). TSA-regulated genes showed little overlap with A-485-downregulated genes (Figure S2C), and A-485-downregulated genes remained unaffected by TSA (Figure S2D). Although TSA treatment alone did not increase the expression of enhancer-regulated genes, remarkably, co-treatment with TSA prevented A-485-induced transcription downregulation (Figure 2A, right panel). The transcription profiles of TSA and A-485 co-treated cells were similar to those of cells treated with TSA alone (Figure 2A, left panel). In contrast, the transcription profile of TSA-regulated genes was not altered by A-485 co-treatment (Figure 2B, left panel). TSA-regulated genes remained unaffected by A-485 treatment (Figure 2B, right panel). These results show that A-485-induced transcription downregulation depends entirely on the concurrent KDAC activity, whereas TSA-induced transcription changes are largely independent of p300/CBP activity. To further test whether KDAC inhibition could restore gene expression post-A-485 treatment, transcription was analyzed in cells treated sequentially with A-485 and TSA. Treatment with A-485 alone (30 min) caused robust transcription inhibition; strikingly, the subsequent addition of TSA rapidly restored A-485-induced transcription arrest (Figure 2C). Inspection of exemplary gene tracks of Esrrb and Pald1 show that within 30 min of A-485 addition transcription is inhibited at >50 kb across the gene body, and within 30 min of subsequent TSA addition, transcription is restored to the same length (Figure 2D). Considering the median elongation speed (∼2.0 kb/min) of mammalian RNAPII (Gregersen et al., 2020Gregersen L.H. Mitter R. Svejstrup J.Q. Using TTchem-seq for profiling nascent transcription and measuring transcript elongation.Nat. Protoc. 2020; 15: 604-627Crossref PubMed Scopus (17) Google Scholar), these results imply that A-485 causes almost immediate transcription arrest and subsequent addition of TSA instantaneously restores transcription. The results also show that, even when p300/CBP activity is severely attenuated, KDAC inhibition rapidly restores transcription, possibly through the increased p300/CBP autoacetylation (Thompson et al., 2004Thompson P.R. Wang D. Wang L. Fulco M. Pediconi N. Zhang D. An W. Ge Q. Roeder R.G. Wong J. et al.Regulation of the p300 HAT domain via a novel activation loop.Nat. Struct. Mol. Biol. 2004; 11: 308-315Crossref PubMed Scopus (319) Google Scholar) and target acetylation. These results establish that robust KDAC activity is critical for dynamic enhancer deactivation. Next, we investigated the possible mechanisms by which p300/CBP activity promotes transcription activation. TFs recruit p300/CBP to enhancers, and p300/CBP activity is suggested to reinforce TF binding (Li et al., 2019Li J. Dong A. Saydaminova K. Chang H. Wang G. Ochiai H. Yamamoto T. Pertsinidis A. Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells.Cell. 2019; 178: 491-506.e28Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Also, p300/CBP bromodomain (BRD) can bind to acetylated proteins, suggesting that p300/CBP activity may promote its chromatin retention through a feedforward mechanism (Zeng et al., 2008Zeng L. Zhang Q. Gerona-Navarro G. Moshkina N. Zhou M.M. Structural basis of site-specific histone recognition by the bromodomains of human coactivators PCAF and CBP/p300.Structure. 2008; 16: 643-652Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). To test whether A-485 impairs TF or p300/CBP binding, OCT4, NANOG, and p300 binding was analyzed using chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq). OCT4 and NANOG binding remained unaltered, whereas p300 binding was increased upon A-485 treatment (Figure 3A). p300 binding was specifically increased at SE, TE, and transcription start sites (TSSs) of A-485-downregulated genes. Increased p300 binding at TSS was inversely correlated (r = −0.32 to −0.41) with reduced expression of downstream genes (Figure 3B). These results show that p300/CBP inhibition does not impair TF binding, p300/CBP activity appears to contribute to its dynamic dissociation from chromatin, and chromatin binding of TFs and p300/CBP per se is not sufficient to activate enhancers. p300/CBP-catalyzed H3K27ac directly opposes the polycomb repressive complex 2 (PRC2)-catalyzed repressive H3K27me3 mark (Tie et al., 2009Tie F. Banerjee R. Stratton C.A. Prasad-Sinha J. Stepanik V. Zlobin A. Diaz M.O. Scacheri P.C. Harte P.J. CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing.Development. 2009; 136: 3131-3141Crossref PubMed Scopus (360) Google Scholar), and lysine-specific histone demethylase 1 (LSD1)-dependent H3K4me1/2 demethylation causes enhancer decommissioning (Whyte et al., 2012Whyte W.A. Bilodeau S. Orlando D.A. Hoke H.A. Frampton G.M. Foster C.T. Cowley S.M. Young R.A. Enhancer decommissioning by LSD1 during embryonic stem cell differentiation.Nature. 2012; 482: 221-225Crossref PubMed Scopus (407) Google Scholar). We hypothesized that p300/CBP inhibition may lead to an increase in H3K27me3 or rapid H3K4me1 demethylation, which could result in transcription inhibition. Anticipating a rapid response, H3K27ac and H3K4me1 were analyzed after 15 min and H3K27me3 after 60 min of A-485 treatment. H3K27ac and H3K27me3 peaks were mutually exclusive (Figure S3A), and H3K27ac was reduced by A-485, but H3K27me3 and H3K4me1 remained unchanged (Figures 3C, S3A, and S3B). H3K27ac was specifically reduced at SE, TE, and A-485-downregulated gene TSSs (Figure 3C). Although H3K27ac was reduced only at a subset (23%) of promoters, decreased promoter H3K27ac was correlated (p = 0.50–0.53) with transcription downregulation of proximal genes (Figure 3D). These results show that A-485-induced transcription inhibition correlates with decreased H3K27ac, and rapid enhancer deactivation occurs independently of a change in H3K27me3 and H3K4me1. To investigate whether A-485-induced transcription inhibition results from a change in chromatin accessibility, we analyzed DNA accessibility using assay for transposase-accessible chromatin using sequencing (ATAC-seq). Analysis of high-confidence differentially accessible regions (DARs) revealed that chromatin accessibility was changed only at a small fraction (0.08% at 30 min and 0.45% at 60 min) of regions (peaks) (Figure 3E). Nonetheless, a majority (93%) of peaks downregulated at 30 min overlapped with peaks downregulated at 60 min (Figure S3C). ATAC-seq signal was reduced at SE, TE, and A-485-downregulated gene TSSs, but not at unregulated gene TSS (Figure 3F), suggesting that p300/CBP-catalyzed acetylation and/or ongoing transcription likely contributes to chromatin accessibility. However, the observed change in chromatin accessibility, especially at early time points, is relatively modest compared to the strong transcription inhibition, implying that rapid transcription inhibition by A-485 is unlikely due to a direct consequence of altered chromatin accessibility. Given the key role of acetylation in BRD4 recruitment, we hypothesized that p300/CBP inhibition may abrogate BRD4 binding and thereby impair transcription. To test this premise, BRD4 binding was analyzed after 15 and 60 min of A-485 treatment. As a reference, we used JQ1, which selectively inhibits acetylation-dependent binding of BET-BRDs (Filippakopoulos et al., 2010Filippakopoulos P. Qi J. Picaud S. Shen Y. Smith W.B. Fedorov O. Morse E.M. Keates T. Hickman T.T. Felletar I. et al.Selective inhibition of BET bromodomains.Nature. 2010; 468: 1067-1073Crossref PubMed Scopus (2708) Google Scholar). BRD4 binding was similarly reduced by A-485 and JQ1 at SE, TE, and at A-485-downregulated gene TSSs (Figure 4A). However, at A-485-unregulated genes, BRD4 binding was reduced only by JQ1 (Figure 4A). A greater fraction of BRD4 peaks were downregulated by JQ1 (60%) than by A-485 (37%) (Figure S3D). Nonetheless, a vast majority (88%) of A-485-downregulated peaks overlapped with JQ1-downregulated peaks (Figure S3E). A similar number of BRD4 peaks were reduced after 15 and 60 min of A-485 treatment, and 83% of downregulated peaks at 60 min were already significantly regulated at the 15 min time point (Figure S3E). These results show that p300/CBP activity selectively promotes BRD4 binding at enhancers and enhancer-regulated gene promoters. To test whether transcription downregulation by p300/CBP inhibition can be fully explained by impaired BET-BRD recruitment, we analyzed nascent transcription after acute JQ1 treatment and compared the resulting changes with those induced by A-485. JQ1- and A-485-induced transcription changes were correlated (r = 0.40–0.52) (Figure 4B). However, the transcription of A-485-downregulated genes was less severely reduced by JQ1 than by A-485 (Figures 4C and S3F). Transcription inhibition by JQ1 remained weak, even if cells were treated with a 20-fold higher concentration (10 μM) of the inhibitor (Figures S3G and S3H). Next, we asked whether JQ1-induced transcription inhibition was sensitive to KDACs. Unlike the restoration of A-485-induced transcription inhibition (Figure 2C), JQ1-induced transcription inhibition was not restored by the subsequent addition of TSA (Figures 4D and 4E). This is likely because JQ1 competitively inhibits BRD4 binding, and therefore, it abrogates BRD4 binding regardless of the dynamics of the underlying acetylation. In contrast, the effect of A-485 is indirect; it inhibits p300/CBP activity, and the transcription shutdown depends on the removal of acetylation by KDACs. Strikingly, despite this fundamental mechanistic difference, A-485 induces immediate transcription arrest, suggesting a highly dynamic acetylation turnover at enhancers. Thus, the dynamics of A-485-induced, but not JQ1-induced, transcription inhibition reflects the kinetics at which enhancers are regulated in their native context. To understand the basis of potent transcription regulation by p300/CBP activity, we analyzed the recruitment of unphosphorylated RNAPII (Pol2) as well as phosphorylated forms of RNAPII. RNAPII Ser5P (Pol2 S5P) marks TSS-bound/-paused RNAPII, whereas RNAPII Ser2P (Pol2 S2P) is associated with elongating RNAPII. Because a majority of genes remain unaffected by A-485 treatment (Figure S1C), we assumed that RNAPII recruitment at these genes should be insensitive to A-485, and normalized the data using RNAPII ChIP signal within gene body regions of N.C. genes. RNAPII binding was analyzed at Mediator subunit 1 (MED1)-occupied regions, which include enhancers and gene promoters. A-485 treatment strongly reduced the RNAPII S2P (Pol2 S2P) signal, but, surprisingly, unphosphorylated RNAPII and RNAPII S5P (Pol2 S5P) signals were also equally severely attenuated (Figure 5A). Unphosphorylated RNAPII and phosphorylated RNAPII signal were strongly reduced at SE, TE, and A-485-downregulated gene TSSs. Reduced RNAPII binding was robustly correlated (r = 0.81–0.83) with attenuated RNAPII S2P and RNAPII S5P ChIP" @default.
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• W3136200267 date "2021-05-01" @default.
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• W3136200267 title "Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release" @default.
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