Volume 44, Issue 3, Pages (November 2011)

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Volume 44, Issue 3, Pages 410-423 (November 2011) SAGA and ATAC Histone Acetyl Transferase Complexes Regulate Distinct Sets of Genes and ATAC Defines a Class of p300-Independent Enhancers  Arnaud R. Krebs, Krishanpal Karmodiya, Marianne Lindahl-Allen, Kevin Struhl, Làszlò Tora  Molecular Cell  Volume 44, Issue 3, Pages 410-423 (November 2011) DOI: 10.1016/j.molcel.2011.08.037 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 The Function of ATAC and SAGA Is Mutually Exclusive on Most of Their Binding Loci (A) Dot plot representing ATAC versus SAGA enrichment over their high-confidence binding sites. High-confidence binding site lists of both complexes were pooled, correcting for sites being present in both lists (within 500 bp) to establish a combined list of binding sites. Then enrichments over input tracks were collected and plotted for SAGA and ATAC. (B) Randomly chosen loci, belonging to the isolated subsets (High SAGA, Equal SAGA and ATAC, High ATAC), were validated by ChIP-qPCR. Sequence information for each locus (labeled 1 to 4) in each subset can be found in Table S3. Enrichments over background are plotted for SAGA (gray bar) and ATAC (black bar). The horizontal red dotted line indicates the 2-fold enrichments over the control genomic region. Error bars represent the standard deviation for three technical replicates. (C) Average gene profiles of ATAC (upper panel) and SAGA (lower panel). Enrichments over input were plotted around an average gene structure for ATAC (black) or SAGA (black), as well as for RNA Pol II (red) and H3K36me3 (blue). (D) Average binding profile of ATAC (upper panel) and SAGA (lower panel) around the TSS. Same color code as in (C) was used. See also Figure S1, Table S2, and Table S3. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 ATAC Associates with Both Promoter- and Enhancer-Type Elements (A) Heat map of the signal densities observed on regions surrounding the 372 high-confidence ZZZ3 (ATAC) binding sites (±5 kb) for different genomic features (as indicated). The density map was subjected to clustering in order to create groups of loci sharing the same genomic profile. Loci were classified as putative enhancers (red box and labeled 1) when enriched for H3K4me1 and harboring reduced H3K4me3 signal; promoters (black box and labeled 2) when enriched for H3K4me3 and Pol II, and unassigned (blue box and labeled 3) when none of the tested features were enriched significantly (see also Figure S3). (B) Frequency plot representing the distance of the ATAC binding sites to the transcription start site (TSS) of the closest gene in the genome in each category previously isolated. Binding sites in both the enhancer (1, red) and unassigned (3, blue) categories do not show any preferential location close to TSSs, while ATAC-binding sites are preferentially found next to TSSs in the promoter category (2, black). (C) UCSC genome browser tracks of representative examples of loci from each category previously isolated (1, enhancer; 2, promoter; 3, unassigned). Colors of the filled boxes highlight the features used to define the category of the loci (red, enhancer; black, promoter; blue, unassigned). See also Figures S2 and S3 and Table S2. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 SAGA Associates Preferentially with Promoters (A) Heat map of the signal densities observed on regions surrounding the 380 high-confidence SPT20 (SAGA) binding sites (±5 kb) for different genomic features (as indicated). The density map was subjected to clustering in order to create groups of loci sharing the same genomic profile. Loci were classified similarly as in Figure 2 (see also Figure S3). (B) Frequency plot representing the distance of the SAGA binding sites to the transcription start site (TSS) of the closest gene in the genome in each category previously isolated. Binding sites in the enhancer (1, red) category does not show any preferential location next to TSS, while SAGA-binding sites in the promoter category (2, black) are preferentially found next to TSSs. (C) UCSC genome browser tracks of representative examples of loci from each category previously isolated (1, enhancer; 2, promoter). See also Figures S2 and S3 and Table S2. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 ATAC and SAGA Are Binding a Restricted Set of Active Genes (A) Gene expression correlates genome-wide with the RNA Pol II enrichment levels at promoters. Whisker plots representing the enrichment of RNA Pol II at the promoters of genes sharing similar levels of expression. Genes were categorized in increasing expression level (based on RPKM), enrichments over the control were collected at corresponding promoters and Whisker plots were plotted for each category. (B and C) ATAC (B) and SAGA (C) bind a limited set of active genes. Similar methodology as described above for Pol II was applied to ZZZ3 or SPT20 data set, revealing an absence of global correlation between the recruitment of ATAC, or SAGA, and expression. (D and E) The distribution of the expression levels of the genes, where ATAC and SAGA were detected at promoters, is displayed by Whisker plot. It shows that ATAC and SAGA identified binding sites are associated with promoters of active genes. Moreover, ATAC or SAGA enrichment over promoters does not correlate genome-wide with the corresponding gene expression. (F) Depletion of ATAC subunits (ZZZ3, ATAC2, GCN5/PCAF) in HeLa cells decreases the expression of three randomly selected ATAC-bound genes by 30%–50% as compared to the scrambled siRNA control. (G) Depletion of SAGA subunits (SPT20, GCN5/PCAF) in HeLa cells decreases the expression of three randomly selected SAGA-bound genes by 50%–60% as compared to the scrambled siRNA control. Average results of three biologically independent experiments are shown with standard deviation as error bars. p values marked above the bars with asterisks correspond to an unpaired t test for triplicates (p < 0.01). See also Figure S4, Table S2, and Table S3. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 ATAC and SAGA Binding Exert Higher Tissue Specificity on Enhancers Compared to Promoters (A) Left: Comparative heatmap of the ATAC (ZZZ3) ChIP-seq signal density in GM12878 and HeLa cells around the high confidence ZZZ3 binding sites. Similar loci organization as in Figure 2A was conserved in order to illustrate differences in cell-type binding specificity between enhancer (red), promoter (black), and undefined (blue) type of loci. Right: Similar analysis has been carried out for SAGA-bound loci. (B) Table presenting Pearson correlation coefficients calculated by comparing ChIP-seq signal in GM12878 and HeLa cells for each of the described categories (as indicated). (C) Piled bar diagram representing the type of genes, bound by ATAC or SAGA compared to the distribution of these genes over the reference gene set (RefSeq transcripts). It reveals that neither SAGA nor ATAC show a preferential association with any of the tested categories. The proportion of genes regulated by ATAC and SAGA from the total set in each category is depicted in the corresponding subbars (in percentage). See also Figure S5, Table S1, and Table S2. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 A Screen of Transcription Factor Motifs Identifies Candidate Transcription Factors for SAGA and ATAC Recruitment (A) Table summarizing the JASPAR motifs identified as enriched in the sequence of each category of ATAC or SAGA binding sites compared to the corresponding control set (see the complete analysis in the Supplemental Information). The motifs are ranked by enrichment over the control set in each category. The expression level category of the significant matches (enrichment > 2) is represented based on RNA-seq data in GM cells (RPKM categories: – [–1 < ]; + [–1:1]; ++ [1:2]; +++ [2 < ]). (B) Dot plot highlighting the significant enrichments of GABPα ChIP-seq signal (fold enrichment > 2 in red) over the ATAC and SAGA bound sites. (C) Average profile of enrichment of GABPα (red) and SAGA (compared to input) over a 5 kb window centered on the SAGA sites, where co-occupancy was observed. See also Figure S6 and Table S2. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 ATAC Is Defining a Class of Enhancers that Are Independent of p300 (A and B) Analysis of the distribution of p300 enrichment over enhancers and promoters of ATAC and SAGA-bound loci. For each category, the enrichment for p300 were calculated and compared to ATAC (A) or SAGA (B) enrichment, respectively. For each category a fixed step distribution histogram was plotted representing back-to-back the enrichments (log2) of ATAC or SAGA (left, red) and p300 (right, blue). The p value was calculated using a Wilcoxon test. (C) UCSC Genome browser tracks of representative examples of ATAC specific putative enhancers (left), or enhancers bound by both ATAC and p300 (right). The color of the filled boxes highlights the features used to define the category of the loci (red, enhancer). (D) Validation of ZZZ3- and/or p300-bound enhancers by ChIP-qPCR quantification. Sequence information for each enhancer in each subset can be found in Table S3. Enrichment of ATAC and p300 over loci, where no binding is expected (control), is calculated for ZZZ3 (black bar) and p300 (white bar). The red line indicates the 2-fold enrichments over the background. (E) Enhancer reporter (luciferase) assay was carried out to measure the activity of ATAC (ZZZ3)-bound enhancers (E1–E4) and randomly selected genomic regions (N1–N2) compared to empty vector in GM (white bars) and HeLa cells (black bars). Known p300 bound HeLa cell-specific enhancers (H4 and H9) (Heintzman et al., 2009) were used as positive controls. ATAC bound enhancers identified in GM cells are not active in HeLa cells. The red line indicates the highest activity obtained with negative controls. Error bars represent the standard deviation for three biological replicates. See also Figure S7, Table S2, Table S3, and Table S4. Molecular Cell 2011 44, 410-423DOI: (10.1016/j.molcel.2011.08.037) Copyright © 2011 Elsevier Inc. Terms and Conditions