Multiple transactivation domains of EZH2 bind to the TAZ2 domain of p300 and stimulate the acetyltransferase function of p300 Open Access

Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2), a major transcriptional regulator that restricts lineage-specific gene activation and maintains cell identity [1,2]. The PRC2 complex methylates lysine 27 of histone H3, producing the epigenetic mark H3K27me3, a hallmark of gene repression [1,3-5]. EZH2 and the scaffolding subunits SUZ12 and EED form a minimal assembly that retains methyltransferase activity and, together with another subunit RBBP7/4, comprise the PRC2 core [6]. In addition, several non-core accessory proteins, such as AEBP2, JARID2 and PCL1-3, can associate with this core, leading to the formation of distinct subcomplexes [7-12]. Recruitment of PRC2 to the particular genomic loci and its release require multiple co-operative and competitive contacts with DNA and histones, often involving several subunits of the complex [13,14].

The methyltransferase activity of the su(var)3–9, enhancer of zeste, trithorax (SET) domain of EZH2 can be allosterically regulated intramolecularly through α-helical motifs, or modulated intermolecularly by other PRC2 subunits, and pre-existing H3K27me3 [7-10]. Generally, EZH2 is viewed as a transcriptional silencer; however, recent studies have linked it to PRC2-independent gene activation. EZH2 stimulates gene transcription in cancer cells and has been found to be overexpressed in a wide array of cancers [15-18]. Although much less is known regarding this non-canonical PRC2-independent function of EZH2, biochemical studies identified a series of partially disordered helices of EZH2 that can serve as transactivation domains (TADs) to recruit transcriptional activators, such as c-Myc and p300 [19-21].

p300 and its paralogue CREB-binding protein (CBP) are histone H3K18- and H3K27-specific acetyltransferases and transcriptional coactivators [22-26]. p300 contains a diverse set of protein- and DNA-binding modules that interact with activators and basal transcription factors forming multi-subunit complexes required for cell proliferation, differentiation, apoptosis and many other normal cellular processes [27-30]. p300 enzymatic activity is regulated through the autoinhibitory loop of the catalytic histone acetyltransferase (HAT) domain, which, in a hypoacetylated form, suppresses this activity but releases the inhibition upon hyper-autoacetylation, and through other domains present in p300. These include the acetyllysine binding bromodomain [31-33], the RING-PHD fingers region [34], the ZZ domain that stimulates acetylation of H3K27 and H3K18 in cis [35], and the TAZ2 domain that associates with transcription factors [36-38].

In the present study, we show that multiple TADs of EZH2 (EZH2_TADs) are recognized by the TAZ2 domain of p300 (p300_TAZ2), and this recognition promotes the enzymatic activity of p300. The increased apparent concentration of the binding sites due to the close proximity of EZH2_TADs and multivalent engagement suggest a mechanism for rapid accumulation of p300 at the EZH2 binding sites. This binding mechanism could explain the observed substantial co-localization of p300 and EZH2 at EZH2’s non-canonical genomic targets.

p300_TAZ2 has been shown to recognize the hydrophobic ΦΦxxΦΦ motif, also known as the TAD, where Φ represents a hydrophobic residue and x represents any residue [39]. EZH2 contains three such motifs, encompassing amino acids 145–150, 170–175 and 223–227 of EZH2 (Figure 1a and b). In the structure of the PRC2 complex [10], the region containing all three motifs is in close proximity to the EED subunit of the complex, although the motifs themselves do not directly interact with EED (Figure 1c). Each motif is found in a separate short α-helix, with the second and third motif-containing α-helices packing against each other and the first motif-containing α-helix positioned away from the other two. To determine whether the EZH2 motifs represent TADs for p300_TAZ2, we produced p300_TAZ2 and tested its binding to three EZH2 peptides, each containing a single motif, by microscale thermophoresis (MST). The MST measurements showed that p300_TAZ2 binds well to either EZH2 peptide, hereafter referred to as EZH2_TAD1, EZH2_TAD2 and EZH2_TAD3 (Figure 1d and e). The interactions of p300_TAZ2 with the EZH2_TAD peptides yielded K_ds (dissociation constants) of 0.52–0.53 μM for EZH2_TAD1 and EZH2_TAD3, respectively, and 0.31 μM for EZH2_TAD2. These values were in the range of binding affinities observed for the association of p300_TAZ2 with TADs from other proteins, indicating that the p300_TAZ2–EZH2_TAD interactions are physiologically relevant [36,39,40].

To examine the molecular mechanism by which p300_TAZ2 recognizes three EZH2_TADs, we carried out NMR experiments. ¹H,¹⁵N heteronuclear single quantum coherence (HSQC) spectra of ¹⁵N-labeled p300_TAZ2 were collected, while unlabeled EZH2_TAD peptides were titrated into NMR samples (Figure 2a). Addition of each EZH2_TAD resulted in chemical shift perturbations (CSPs) in the spectra of p300_TAZ2, confirming the formation of the complexes between p300_TAZ2 and either EZH2_TAD1, EZH2_TAD2 or EZH2_TAD3 (Figure 2a). To identify p300_TAZ2 residues involved in contact with EZH2_TAD, we reassigned backbone amide resonances of p300_TAZ2 [37] and plotted CSPs observed in ¹H,¹⁵N HSQC spectra of p300_TAZ2 upon the addition of EZH2_TAD1, EZH2_TAD2 and EZH2_TAD3 per residue (Figure 2b–d). Overall, each EZH2_TAD peptide induced CSPs roughly in four regions of p300_TAZ2, encompassing residues 1730–1740, 1760–1770, 1780–1790 and 1800–1810. Although patterns of CSPs in three NMR experiments were similar, they were not identical. In comparison with EZH2_TAD1 and EZH2_TAD3, EZH2_TAD2 induced CSPs larger in magnitude, and saturation was reached faster, supporting MST data that among the three EZH2_TADs, EZH2_TAD2 binds most tightly. The patterns of CSPs observed upon binding of EZH2_TAD2 and EZH2_TAD3 were very similar, whereas CSPs observed upon binding of EZH2_TAD1 were unique in comparison.

To gain insight into the formation of the complexes, we mapped the most perturbed residues shown in Figure 2b–d onto the structure of p300_TAZ2 and analyzed all the structures of the p300_TAZ2-TAD complexes reported to date. We found that CSPs caused by EZH2_TAD peptides are well aligned with the binding pockets for E1A or STAT1 peptides in their respective complexes with p300_TAZ2 (Figure 3a–c). The substantial overlap of the binding sites for EZH2_TAD2 and EZH2_TAD3 suggested that these TADs do not concurrently interact with one p300_TAZ2 domain; however, the more extensive binding site for EZH2_TAD1 prompted us to investigate whether EZH2_TAD1 and EZH2_TAD2 can bind concomitantly (Figure 3d).

We tested the binding of p300_TAZ2 to a longer EZH2 peptide, containing both EZH2_TAD1 and EZH2_TAD2 (EZH2_TAD1-TAD2) by NMR. Titration of the dual EZH2_TAD1-TAD2 led to large CSPs in ¹H,¹⁵N HSQC spectra of p300_TAZ2 (Figure 4a). Binding affinity of p300_TAZ2 to EZH2_TAD1-TAD2, measured by MST (K_d of 0.15 µM) and tryptophan fluorescence (K_d of 0.18 µM), revealed a ~two-fold increase in binding affinity compared with the binding affinity of p300_TAZ2 to EZH2_TAD2 and a ~three-fold increase compared with the binding affinity to EZH2_TAD1 (Figure 4b and c). Such an increase is likely due to an increase in apparent concentration of the binding sites in the linked EZH2_TADs.

The finding that each EZH2_TAD can be bound by a separate p300_TAZ2 was substantiated in mass photometry (MP) assays (Figure 5). The MP measurements showed that in the apo-state, GST-p300_TAZ2 exists in a dimeric form (MP peak of ~80 kDa) due to the ability of GST to dimerize (Figure 5a). The addition of the dual EZH2_TAD1-TAD2 resulted in the formation of the complex of molecular mass corresponding to four molecules of GST-p300_TAZ2 and one molecule of EZH2_TAD1-TAD2 (MP peak of ~155 kDa). These data indicated that one dimer of GST-p300_TAZ2 interacts with EZH2_TAD1, whereas another dimer of GST-p300_TAZ2 interacts with EZH2_TAD2. Furthermore, the addition of the triple EZH2_{TAD1-TAD2-TAD3} led to the formation of the complex of molecular mass corresponding to six molecules of GST-p300_TAZ2 and one molecule of EZH2_{TAD1-TAD2-TAD3} (MP peak of ~224 kDa) (Figure 5b). Collectively, MP data revealed that each EZH2_TAD can be bound by a separate p300_TAZ2 (Figure 5e). This was further corroborated by MP measurements using GST-EZH2_TAD1 which formed a dimer in the apo-state again due to the ability of GST to dimerize (MP peak of ~70 kDa). The addition of p300_TAZ2 to GST-EZH2_TAD1 led to a ~14 kDa shift of the MP peak, suggesting the presence of two complexes, dimeric GST-EZH2_TAD1 bound by one molecule of p300_TAZ2 and by two molecules of p300_TAZ2.

We have previously shown that p300_TAZ2 mediates acetyltransferase function of p300 [41]. To explore whether binding of EZH2_TADs affects the catalytic activity of the p300 HAT domain, we purified FLAG-HA-tagged p300 catalytic core (aa 1035–1830 of p300) from 293 T cells and tested it in HAT assays using the recombinant nucleosome as a substrate (Figure 6). As shown in Figure 6a and b, the FLAG-HA-tagged p300 catalytic core alone inefficiently acetylates histone H3K18 and H3K27. The addition of GST-tagged EZH2_{TAD1-TAD2-TAD3} (aa 119–267, EZH2 WT) or homologous GST-tagged EZH1_{TAD1-TAD2-TAD3} (aa 120–280, EZH1 WT) to the reactions led to a dose-dependent increase in H3K18 and H3K27 acetylation. The EZH1-dependent stimulation of the p300 enzymatic activity was decreased when F176 and F237 in TAD2 and TAD3 of EZH1 (EZH1 2A in Figure 6a) were mutated to alanine and was further decreased when additionally F146 in TAD1 and Y257 of EZH1 (EZH1 4A in Figure 6b) were substituted to alanine. We note that F145A and F171A mutations in EZH2 (equivalent to F146A and F176A in EZH1) were previously shown to disrupt binding to p300 in pull-down assays [20]. Collectively, these data indicated that binding of EZH1/2_TADs to p300_TAZ2 stimulates the acetyltransferase function of p300.

To determine whether the genomic binding sites of p300 and EZH2 correlate in the cellular context, we performed cleavage under targets and tagmentation (CUT&Tag) experiments for p300 in 22Rv1 cells that are commonly used as a prostate cancer cell model. For comparative genomic profiling analyses, we used the publicly available datasets, such as assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq), and cleavage under targets and release using nuclease (CUT&RUN) in the same 22Rv1 cells. Using the EZH2 and H3K27me3 peaks as input, an unsupervised clustering analysis was performed to define categories of genomic sites showing differential binding patterns. First, we observed a co-binding pattern for EZH2 and H3K27me3, a repressive PTM associated with the catalytic activity of the canonical EZH2:PRC2 complex, at a set of genomic sites (8307 peaks), termed the EZH2 ensemble sites (Figure 7a and b). As expected, these canonical EZH2:PRC2 target sites were lacking p300 and other markers of transcriptionally active chromatin, including H3K27ac, H3K4me2, H3K4me3, the transcriptional coactivator BRD4, and RNA polymerase II (Pol II) (Figure 7a–c). Surprisingly, we also identified a large set of the EZH2 peaks (termed EZH2-solo) lacking H3K27me3 (11,657 peaks) (Figure 7d–f). In contrast to the canonical EZH2:PRC2 binding sites, EZH2-solo binding sites were characterized by substantial p300 co-occupancy and high levels of H3K27ac (an active histone mark deposited by p300), H3K4me2, H3K4me3, BRD4, and RNA Pol II. Co-localization of EZH2, p300, and BRD4 and high level of H3K27ac but the lack of H3K27me3 were obvious at CDK2 and MYBL2, two well-recognized oncogenes (Figure 7g). Together, these results indicated that EZH2 and p300 co-localize at a large set of genes in 22Rv1 prostate cancer cells.

An analysis of mRNA expression levels of EZH2 and p300 in prostate adenocarcinoma (PRAD), kidney renal clear cell carcinoma (KIRC), uterine corpus endometrial carcinoma (UCEC), and lung squamous cell carcinoma (LUSC) from The Cancer Genome Atlas (TCGA) showed that EZH2 was consistently overexpressed in tumor tissues compared with normal tissues (Figure 8). The differences were statistically significant (P<0.05) in agreement with previous reports demonstrating the canonical oncogenic activity of EZH2 [42]. PRAD patients with high EZH2 expression had a poor prognosis, with significantly shorter progression-free survival compared with patients with low EZH2 expression (Supplementary Figure S2). In contrast, no significant differences in the expression level of p300 were detected between normal tissues and these four cancer types (Figure 8). Furthermore, correlation analysis across tumor samples revealed no significant association between p300 and EZH2 expression, indicating that their transcriptional regulation occurs independently (Supplementary Figure S3).

Both loss-of-function and gain-of-function alterations of EZH2 have been identified in various cancer types. Many of these alterations reflect the cell type-specific tumor-suppressive or oncogenic activities of PRC2, which usually rely on H3K27me3-mediated gene repression [1,43]. In contrast, emerging evidence suggests that EZH2 functions beyond the PRC2 context and plays a role in activating gene expression. In the present study, we show that in 22Rv1 prostate cancer cells, EZH2 occupies a large set of gene loci lacking H3K27me3. These non-canonical genomic sites of EZH2 are instead co-bound by p300, RNA Pol II, and BRD4 and are rich in histone marks associated with transcriptional activation. Our findings suggest a mechanism for such a high degree of genetic correlation through the direct binding of p300_TAZ2 to EZH2_TADs and the subsequent stimulation of acetyltransferase activity of p300. Both the close proximity of EZH2_TADs that increases the apparent concentration of the ligand and the ability of three p300_TAZ2 to simultaneously interact with all three EZH2_TADs could play a role in bridging EZH2 and p300. As p300_TAZ2 has been shown to bind DNA, in future work, it will be interesting to assess the contribution of this binding to the EZH2-p300 complex formation. Elucidation of the molecular basis for the EZH2-p300-chromatin assembly is also essential for our understanding of the etiology of cancers associated with non-canonical PRC2-independent function of EZH2.

Human p300_TAZ2 [aa 1722–1812, pGEX-4T-1 vector with an additional tobacco etch virus (TEV) cleavage site] with a tryptophan substituted at A1723 and p300_TAZ2-6×His constructs were purified essentially as in Becht et al. [37]. Briefly, both ¹⁵N-labeled and unlabeled constructs were expressed in Escherichia coli Rosetta-2 (DE3) pLysS cells grown in either M9 minimal media or TB, both supplemented with 50–150 μM ZnCl₂. After induction with isopropyl b-D-1-thiogalactopyranoside (IPTG), harvesting, and lysis in buffer [50 mM Tris pH 7.0, 1 M NaCl, 5 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonylfluoride (PMSF) and DNase] by sonication, the proteins were purified on glutathione agarose beads (Pierce) and eluted with buffer containing reduced L-glutathione (Fisher). The GST tag was either cleaved with TEV protease overnight at 22°C or kept intact during subsequent purification steps. Both GST- and His-tagged proteins, as well as untagged p300_TAZ2, were further purified by cation exchange (HiTrap SP HP, Cytiva) or heparin affinity chromatography (HiTrap Heparin HP, Cytiva).

Human EZH2_{TAD1-TAD2-TAD3} (aa 142–231) construct with a C-terminal 6×His tag was cloned into a pGEX-6P-1 vector (GenScript). The EZH2_TAD1 (aa 142–160) construct was generated by deletion using Q5 Hot Start High-Fidelity kit (NEB) and confirmed by DNA sequencing. Both constructs were expressed in E. coli Rosetta-2 (DE3) pLysS cells grown in LB media and induced at an OD₆₀₀ of 0.6–0.8 with 1 mM IPTG for 16 h at 16°C. Cells were harvested by centrifugation, suspended in buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1% CHAPS, 2 mM DTT, 1 mM PMSF, Roche cOmplete Protease Inhibitor Cocktail, and DNase), and lysed by sonication. The doubly-GST- and -His-tagged EZH2_TAD constructs were purified on glutathione agarose beads (Pierce), eluted with buffer containing 50 mM reduced L-glutathione, then exchanged into relevant experimental buffers using 10 kDa MWCO centrifugal concentrators (Millipore). For MP, EZH2_TAD constructs were either cleaved with PreScission protease or left with GST tag intact, followed by purification on Ni-NTA beads (HisPur, Thermo Sci.), washed with buffer (50 mM Tris pH 7.5, 500 mM NaCl, 30 mM imidazole, 1% CHAPS, 2 mM DTT), and eluted with buffer containing 300 mM imidazole. When necessary, EZH2_TAD constructs were further purified using a Superdex 200 Increase 10/300 GL column (Cytiva) with monitoring at 205 and 280 nm.

EZH2_TAD1 (aa 142–154 of EZH2), EZH2_TAD2 (aa 167–185 of EZH2), EZH2_TAD3 (aa 220–231 of EZH2), and EZH2_TAD1-TAD2 (aa 142–185 of EZH2) peptides were synthesized by SynPeptide Co. Ltd.

NMR experiments were performed at 298 K on a Varian INOVA 600 MHz spectrometer equipped with a cryoprobe. The ¹H,¹⁵N HSQC spectra of 50–100 μM uniformly ¹⁵N-labeled proteins were collected in the presence of an increasing amount of peptide (SynPeptide) in buffer containing 50 mM MES pH 6.5, 150 mM NaCl, 5 mM DTT. NMR data were processed and analyzed with NMRPipe and NMRDraw as previously described [44]. Normalized chemical shift changes were calculated using the following equation:

where Δδ is the change in chemical shift in parts per million (ppm). CSP graphs were analyzed with CcpNmr Analysis v3.2 and generated in GraphPad PRISM.

Fluorescence spectra were collected at 25°C on a Fluoromax spectrofluorometer (HORIBA). Sample contained 1 μM protein in a buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, and 2.5 mM DTT. p300_TAZ2 in the absence and presence of increasing concentrations of the EZH2_TAD1-TAD2 peptide was excited at 295 nm, and emission spectra were recorded between 320 and 360 nm with a 0.5-nm step size and a 0.5-s integration time. The apparent [45] K_d estimated for EZH2_TAD1-TAD2 peptide was determined by a nonlinear least-squares analysis using a 1:1 stoichiometry and the following equation:

where [L] is concentration of the peptide, [P] is concentration of the protein, ΔI is the observed change of signal intensity, and ΔI_max is the difference in signal intensity of the free and bound states of the protein. The K_d value was averaged over three independent experiments with error calculated as the standard deviation between the runs.

MST experiments were carried out on a Monolith NT.115 instrument (NanoTemper). All experiments were performed using SEC-purified p300_TAZ2-6×His protein in 25 mM Tris pH 7.5 buffer, 150 mM NaCl, and 2.5 mM DTT. p300_TAZ2-6×His was labeled using a His-Tag Labeling Kit RED-tris-NTA (2nd Generation, NanoTemper) and kept constant at 10 nM. Dissociation constants were determined using a direct binding assay in which peptide was varied in concentration by serial dilution of discrete samples. The measurements were performed at 50% LED and medium MST power with 3-s pre-laser time, 20-s laser on-time, and 1-s off-time. The K_d values, including apparent K_d estimated for EZH2_TAD1-TAD2 peptide, were calculated using MO Affinity Analysis software (NanoTemper) using a 1:1 stoichiometry and averaged over three or four separate experiments with error reported as SD. Plots were generated in GraphPad PRISM.

MP experiments were conducted using a Refeyn TwoMP mass photometer (Refeyn Ltd, Oxford, U.K) to monitor the formation of oligomeric states involving p300_TAZ2 and TADs of EZH2. To overcome the minimum size limit, a GST-fusion construct was used for one of the components in each sample. In summary, samples contained either 20 nM GST-p300_TAZ2 in the absence and presence of EZH2_TAD1-TAD2 (5 μM) or EZH2_{TAD1-TAD2-TAD3} (1 μM) or 20 nM GST-EZH2_TAD1 with or without p300_TAZ2 (0.1 μM). Ten microliter of each sample was loaded into the sample wells of the silicon cassettes assembled onto MassGlass UC coverslips (Refeyn, Ltd.). Measurements were performed at room temperature in buffer (50  mM Tris pH 7.5, 150  mM NaCl, 5  mM DTT). β-amylase was used as calibration standard (56  kDa, 112  kDa, and 224  kDa). After the focus was set and locked, movies were captured for 60  s (2800 frames) using AcquireMP software (Refeyn Ltd). Data were processed using DiscoverMP (Refeyn Ltd).

For HAT assays, FLAG-HA-tagged p300 core (aa 1035–1830 of p300) in a pCDH vector was expressed in 293 T cells for two days. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1% Triton100, 10% glycerol, 1 mM PMSF, and protease inhibitors) and sonicated. Cell lysates were incubated with anti-Flag M2 beads (Sigma) at 4° for 4 h. The beads were then washed three times with buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% NP40, 10% glycerol, and 1 mM PMSF), and the protein was eluted with elution buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT, 10% glycerol, and 0.4 mg/ml Flag peptide).

GST-tagged EZH2_TADs (aa 119–267), EZH1_TADs (aa 120–280), F176A/F237A EZH1_TADs (EZH1 2A), and F146A/F176A/F237A/Y257A EZH1_TADs (EZH1 4A) constructs in the pGEX-6p-1 vector were transformed into RosettaTM2 (DE3) pLysS competent cells (Novagen). Protein production was induced by incubating with 0.4 mM IPTG at 16°C for 20 h in LB. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1 mM PMSF, and 1 × cOmpleteTM EDTA-free Protease Inhibitor Cocktail) and lysed by sonication. Lysates were centrifuged at 17,000 g for 15 min, and supernatants were incubated with Glutathione Sepharose® 4B beads (Sigma) at 4°C for 2 h. Beads were washed with lysis buffer twice, and proteins were eluted with elution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, and 15 mg/ml GSH).

Purified FLAG-HA-tagged p300 catalytic core fragment (1 μg) was incubated with increasing amounts of GST-tagged EZH2_TADs and GST-tagged EZH1_TADs (2.5, 5, or 10 μg) and the nucleosome (1 μg) in HAT reaction buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT, 100 mM acetyl-CoA, and 1× cOmplete EDTA-free Protease Inhibitor Cocktail) at 30°C for 15 min. Reactions were stopped by boiling at 95°C for 5 min in 2×SDS loading buffer, and samples were analyzed by SDS-PAGE and Western blot. The following antibodies were used: HA (CST, 3724, 1:2000), GST (Santa Cruz, sc-459, 1:2000), H3 (Abcam, ab1791, 1:80,000), H3K18ac (ActiveMotif, 39755, 1:1000), and H3K27ac (Abcam, ab4729, 1:1000).

The 22Rv1 cells, a classic prostate cancer cell model, were obtained from American Tissue Culture Collection (ATCC, CRL-2505) and cultured according to the vendor-provided protocol. CUT&Tag was performed using the commercial EpiCypher CUTANA kit and following the manufacturer’s detailed protocols. Briefly, 100,000 human tumor cells were used in each assay followed by sample preparation, multiplex library preparation, and deep sequencing (Illumina NextSeq 1000). The primary antibodies of p300 (Cell Signaling cat# 54,062S) were used at a 1:100 concentration. The public genomic datasets of 22Rv1 cells included those of NCBI Gene Expression Omnibus (GEO) database under the accession number GSE205107 (EZH2 and H3K27me3 CUT&RUN data of 22Rv1 cells), GSE99378 (ATAC-seq data of 22Rv1 cells), GSM2827408 and GSE85558 (ChIP-seq for histone mark in 22Rv1 cells), and GSE94013 (ChIP-seq for BRD4 in 22Rv1 cells).

Genomic data were analyzed as before [20]. Briefly, fastq files (trim_galore) were mapped to the reference genome (hg38) using bowtie2 (v.2.4.4). The non-primary alignment and PCR duplicates were removed from aligned data using Samtools (v.1.10) (-q 30 F 1804 f 2 for unique mapping reads), the Picard ‘MarkDuplicates’ function (v.2.18.2), and bedtools (v.2.30.0) (‘intersect’ function used to exclude genome blacklist regions), respectively. Peak calling was performed using MACS2 (v.2.2.6) (macs2 callpeak -f BAMPE -g hs/mm –keep-dup 1 --cutoff-analysis -q 0.05). For spike-in samples, fastq files were mapped to the reference genome using STAR (v.2.7.11a). Then, non-primary alignment and PCR duplicate alignments were removed by Samtools (v.1.10) (-F 256) and Picard ‘MarkDuplicates’ function (v.2.18.2). Deeptools (v3.3.0) was used to generate bigwig files. Genomic binding profiles were generated by using the deepTools ‘bam-Compare’ functions. An unsupervised clustering function of deepTools was used to analyze the EZH2 and H3K27me3 peaks and define categories of genomic sites showing different binding patterns, such as the EZH2 ensemble sites and the EZH2 solo sites.

Gene expression data for p300 and EZH2 were retrieved from cBioPortal for Cancer Genomics (https://www.cbioportal.org/) and UCSC Xena (https://xena.ucsc.edu/). Datasets from TCGA for PRAD, KIRC, UCEC, and LUSC were used in analysis. RNA-Seq expression data, normalized using RSEM and batch-corrected, were analyzed across tumor samples for each cancer type. Correlation analysis between p300 and EZH2 mRNA expression was performed using Pearson or Spearman correlation coefficients, with statistical significance set at P<0.05. For the PRAD dataset, samples were stratified into quartiles based on EZH2 expression levels, and patient progression-free survival was compared between the highest (top 25%) and lowest (bottom 25%) quartiles.

All relevant data supporting the key findings of this study are available within the article and the Supplementary Information file.

The authors declare no competing interests.

This work was supported in part by grants from the NIH: CA252707 and AG067664 to T.G.K., CA268519 to G.G.W., CA262903 to L.C., CA268440 to X.S., GM128705 to C.A.M., and GM136308 and NS137323 to X.L, from NSF: MCB 2203311 to C.A.M. and from the Canadian Institutes of Health Research to E.B.A. The cores affiliated to Duke Cancer Institute were partly supported by the NCI Cancer Center Support Grant award P30CA014236.

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D.C.B., S.B., C.X., M.K., and H.X. performed experiments and together with L.C., C.A.M., X.L, X.S., E.B.A., G.G.W., and T.G.K. analyzed the data and prepared figures. T.G.K. wrote the manuscript with input from all authors.

ATAC-seq

assay for transposase-accessible chromatin using sequencing

CUT&RUN

cleavage under targets and release using nuclease

CUT&Tag

cleavage under targets and tagmentation

ChIP-seq

chromatin immunoprecipitation followed by sequencing

DTT

dithiothreitol

EZH2

enhancer of zeste homologue 2

HAT

histone acetyltransferase

IPTG

isopropyl b-D-1-thiogalactopyranoside

KIRC

kidney renal clear cell carcinoma

LUSC

lung squamous cell carcinoma

MP

mass photometry

PMSF

phenylmethanesulfonylfluoride

PRAD

prostate adenocarcinoma

PRC2

polycomb repressive complex 2

TADs

transactivation domains

TCGA

The Cancer Genome Atlas

TEV

tobacco etch virus

UCEC

uterine corpus endometrial carcinoma