Volume 61, Issue 1, Pages 27-38 (January 2016) EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation  Suman K. Pradhan, Trent Su, Linda Yen, Karine Jacquet, Chengyang Huang, Jacques Côté, Siavash K. Kurdistani, Michael F. Carey  Molecular Cell  Volume 61, Issue 1, Pages 27-38 (January 2016) DOI: 10.1016/j.molcel.2015.10.039 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Variant Chromatin Stimulates Transcription In Vitro in an EP400-Dependent Manner (A) In vitro transcription. End-biotinylated G5E4T was assembled into chromatin using canonical, single-variant, or double-variant octamers as indicated and immobilized on paramagnetic beads. Chromatin was acetylated by Gcn5 and Esa1 (HAT) as indicated and incubated with nucleotides (NTPs) in HeLa nuclear extract with or without GAL4-VP16 (Activator). A phosphorimage of the primer-extension gel is shown and graphed using mean and SD spectral units of 3 replicates. ∗∗p < 0.01 by Student’s t test. n.s., not significant. (B) IT capture of PICs. Immobilized canonical or double-variant chromatin was as in (A) minus NTPs. Purified PICs were immunoblotted with antibodies to indicated proteins. (C) Quantitation of select proteins from immunoblot in (B) of acetylated canonical or double-variant chromatin. (D) Levels of H3 acetylation measured for canonical and double-variant chromatin using pan-H3Ac antibody: B (bound) and S (supernatant/wash) fractions. (E) EP400 immunoblots of Mock (M) and EP400-depleted (EP400Δ) extracts over a 9-fold titration range. (F) IT analysis of select PIC components in Mock and EP400Δ extracts. (G) Silver-stained gel of EP400 preparation. (H) In vitro transcription of acetylated chromatin in Mock or EP400Δ extracts. Graphed as in (A) for 3 replicates but with or without recombinant EP400 as indicated. ∗∗p value < 0.01 by Student’s t test. Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 EP400 Is Necessary for Transcription in a Model Cell-Based Reporter System (A) Schematic of U2OS Tet-On VP16 reporter system. (B) Quantitation of F-Luc RT-PCR products at indicated time points (hours after Dox induction [h.p.i.]) in untreated, Mock, and EP400 siRNA-KD cells 72 hr after transfection of siRNA. (C) ChIP-quantitative PCR (qPCR) of promoter at the indicated time points with antibodies against VP16, Pol II, MED1 (Mediator), EP400, and H3.3. Bar graph indicates enrichment at time points relative to input DNA. (D) EP400 immunoblot of Mock versus siRNA KD normalized via GAPDH levels. (E) Immunoblot analysis of H2AZ, H3.3, H3, and H4 for crosslinked chromatin fractions or whole-cell extracts. The numbers indicate the intensity of signal in KD normalized to 1 for Mock (M); representative blots are shown. (F) ChIP of Pol II, Mediator, and H3.3 in Mock versus EP400 KD cells time post Dox. ∗∗p < 0.01 by Student’s t test. Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Genome-Wide Analysis of EP400 and Variant Chromatin at Promoters (A) Heatmaps of EP400, Pol II, Mediator (MED26), H3.3, and H2AZ at U2OS promoters (3 kb flanking TSSs). P values for enrichment are plotted and ranked by EP400. The mRNA heatmaps plot FPKM of Mock alongside fold change (FC) of log2 KD/Mock FPKM (green is downregulated, red is upregulated, and black is no change). P value of differences by Wilcoxon rank-sum test = 9 × 10−19. (B) Representative genome browser view. (C) Metagene analyses of (A) clustered by gene expression. The colored lines indicate enrichment for each protein of top (red; C1), middle (green; C2), and bottom (purple; C3) 10% of expressed genes; black line (C4) is average for all genes. (D) Fold change in enrichment upon EP400 KD for ChIP experiments in (A) plotted as log2 (KD/Mock) on EP400-bound gene promoters by significant peaks (TSS ± 3 kb). Green indicates reduced binding of indicated protein. Total H3 (H3) ChIP data are added to this plot but not shown in (A). (E) Boxplots of −log2 Poisson p values of H3.3, total H3, H2AZ, Pol II, and MED26 ChIP signals in M and KD conditions. Error by Wilcoxon rank-sum test is shown. Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Analysis of EP400 KD on Variant Chromatin Gene Expression (A) Venn diagram of differentially expressed genes in EP400 KD versus Mock. RNAs from the top 9,964 genes (by >1 FPKM value) analyzed for changes ≥1.5 fold; 4,024 were downregulated (green) and 2,086 (red) were upregulated in EP400 KD conditions. (B) Boxplot representing downregulated (green) and upregulated (red) genes by FPKM in M and KD conditions using a log2 scale. (C) Heatmaps showing the downregulated (green) and upregulated (red) genes in M and KD by FPKM and ranked by fold change for clarity. (D) Gene Ontology analysis of affected genes by category. P values were corrected for multiple hypothesis testing using Benjamini correction. Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Analysis of EP400 KD on Enhancer Occupancy and Gene Expression (A) Heatmaps of H3K4me1, H3K18ac, EP400, H3.3, H2AZ, and Pol II at active distal intergenic U2OS enhancers (±5 kb from the center of H3K4me1 peaks) in KD and/or Mock cells. Active enhancers were scored by the H3K4me1 and H3K18ac marks, excluding +3 kb upstream of TSS or downstream of TTS, and sorted by H3K4me1 enrichment in Mock and EP400 siRNA KD cells. (B) Browser track of putative enhancers. Those showing markers consistent with active enhancers are indicated by arrows below the tracks (i.e., identifiable peaks of Pol II, Mediator, EP400, H3.3, H2AZ, H3K18ac, and H3K4me1). (C) Fold change in enrichment of H3.3, H2AZ, and Pol II as in Figure 3D for all EP400-bound enhancers by significant peaks, ranked by H3K4me1 abundance. Wilcoxon rank-sum p values for change in H3.3 and H2AZ are both <2 × 10−308 (saturated), and p value for Pol II is 2 × 10−148. (D) Boxplots of −log2 Poisson p values of H3.3, H2AZ, and Pol II in M and KD conditions for top 10% enhancers with p values. (E) Nearest neighbor effects by GREAT analysis. Average FPKM was plotted for nearest enhancer-proximal genes for top, middle, and bottom 10% of putative enhancers as sorted by H3K4me1 enrichment. Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Histone Exchange by EP400 In Vitro (A and B) Three hundred nanograms immobilized canonical chromatin was incubated as indicated with Apyrase-treated EP400 and 300 ng H3.3-H4 tetramers or H2AZ-H2B dimers ± 1 mM ATP. Chromatin was captured, washed, and subjected to immunoblotting for H2AZ (A) or H3 (B). FLAG-H3.3 is distinguished from H3.1 by mobility and detected using a pan H3 antibody; H2B on beads as a control. (C) Alternatively, 150 ng of immobilized naked DNA or 300 ng canonical chromatin was incubated with EP400 and 300 ng of histone variant octamers as above and FLAG-immunoblotted for H3.3. Graphs in (A–C) represent ≥3 independent experiments. Input and conditions for (D) and (E) were as above. (D) H2AZ exchange into bead-bound H2A-containing chromatin (I); reverse reaction of H2A deposition into H2AZ chromatin (II). (E) H3.3 exchange into H3.1 chromatin (I); reverse reaction of H3.1 deposition into H3.3 chromatin (II); exchange of His-H3.3 into Flag-H3.3 chromatin (III). Molecular Cell 2016 61, 27-38DOI: (10.1016/j.molcel.2015.10.039) Copyright © 2016 Elsevier Inc. Terms and Conditions