ATP-dependent chromatin remodeling is an essential process required for the dynamic organization of chromatin structure. Here we describe the genome-wide location and activity of three remodeler proteins with diverse physiological functions in the mouse genome: Brg1, Chd4 and Snf2h. The localization patterns of all three proteins substantially overlap with one another and with regions of accessible chromatin. Furthermore, using inducible mutant variants, we demonstrate that the catalytic activity of these proteins contributes to the remodeling of chromatin genome wide and that each of these remodelers can independently regulate chromatin reorganization at distinct sites. Many regions require the activity of more than one remodeler to regulate accessibility. These findings provide a dynamic view of chromatin organization and highlight the differential contributions of remodelers to chromatin maintenance in higher eukaryotes.
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The authors thank D. Picketts (University of Ottawa) and J. Svaren (University of Wisconsin) for the kind gift of remodeler cDNA constructs (hSNF2H and mChd4, respectively), A. Indrawan for technical assistance, the US National Cancer Institute Advanced Technology Program Sequencing Facility for sequencing services and Epitomics, Inc. for generation of the monoclonal rabbit antibody to BRG1. This research was supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute Center for Cancer Research and by a UNCF/Merck Postdoctoral Science Research Fellowship to S.A.M.
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Integrated supplementary information
(a) Left-side, Western blot detection of Brg1 in nuclear extracts with monoclonal antibody. Right-side, top graph, ChIP-QPCR analysis of initial Brg1 antiserum at regions bound by Brg1 (Orm2 and Fabp4) and not bound by Brg1 (tRNA). Right-side, bottom graph, ChIP-QPCR analysis of final monoclonal Brg1 antibody at the same regions described above. (b) Specificity of Brg1, Chd4 and Snf2h antibodies by Western blot analysis of whole cell extracts. MW, molecular weight markers. Asterisk, unidentified band that does not appear following immunoprecipitation with the Chd4 antibody. (c) Additional examples of ChIP-seq genome browser views of Brg1, Chd4, and Snf2h occupancy. Images represent tag densities relative to genome coordinates. Input tracks of sonicated, genomic DNA are aligned below each ChIP-seq track. (d) Box plots of tag density enrichment (log2 scale) of remodeler binding sites located at the indicated genomic regions. Sites are classified as promoter (< 2.5 kb upstream or downstream from the nearest TSS), exon (> 2.5 kb downstream from the nearest TSS , overlapping an exon), intron (> 2.5 kb downstream from the nearest TSS , in an intron not overlapping an exon), distal upstream (> 2.5 kb upstream from the nearest TSS), or downstream (> 2.5 kb downstream from the nearest TSS).
(a) Additional example of ChIP-seq genome browser view of Brg1, Chd4, and Snf2h occupancy at the same genomic coordinates on chromosome 4. Tag density is indicated on the y-axis. (b) ChIP-QPCR analysis of Brg1, Chd4, and Snf2h at the indicated genomic regions. Bars represent the average of two replicates normalized to input DNA. No Ab, no antibody control; tRNA, negative control. Error bars represent the SEM of two independent replicates. (c-e) Genome browser view of regions analyzed in panel [(b)]. Sites amplified are highlighted in grey. Input track is included in the alignment.
(a) To detect potential interactions of soluble complexes, Western blot analysis of co-immunoprecipitation (Co-IP) experiments was performed using nuclear extracts and Brg1, Snf2h, and Chd4 specific antibodies. As a control, known protein interactions were analyzed by Western (Baf155 (Brg1-associated), Wstf (Snf2h-associated), and Hdac1 (Chd4-associated). Input, 15 μg of IP nuclear extract; IP, immunoprecipitation; and control IP, lysate from 'no antibody' control sample. (b) Aggregate plot of the average tag density of Brg1 binding sites (hotspot) at regions occupied by: Brg1, Chd4, and Snf2h (black), Brg1 and Chd4 only (brown), Brg1 and Snf2h only (light blue), or Brg1 only (pink). (c) Same as in [(b)], except for Chd4 binding sites. (d) Same as in [(b and c)] for Snf2h binding sites. (e) Potential remodeler interactions at the template were examined by sequential ChIP (re-ChIP) analysis. Primary ChIP with anti-Brg1, anti-Snf2h, and anti-Chd4 was performed at the Orm2 and Ccl2 sites described in Supplementary Fig. 2b. (f) Positive control; secondary re-ChIP was performed with the anti-Brg1 IP from panel (e). (g) Secondary re-ChIP was performed with anti-Snf2h and anti-Chd4 on the anti-Brg1 IP from panel (e). Glul is a negative control site where none of the remodeler proteins were detected.
(a) Shown are the most significantly enriched motifs associated with Brg1 unique sites (not bound by Chd4 or Snf2h) identified by MEME analysis (P < 10-4) using the top 2,000 sites . The AP-1 motif was found to be the most highly enriched (MEME E value = 1.4e-1841). (b) Venn diagrams representing the overlap of binding sites for Chd4 or Snf2h with AP-1 sites. (c-d) Similar de novo motif analysis as above was performed on Chd4 and Snf2h unique sites. The CTCF motif was found to be the most highly enriched motif for Chd4 unique sites (MEME E value = 3.6e-815) and Snf2h unique sites (MEME E value = 1.1e-1026). (e) Venn diagrams of sites shared between remodelers and CTCF. Left-side, Venn diagram of the overlap between Brg1 and CTCF sites. Right-side, three-way Venn diagram representing the overlap between remodeler sites that specifically co-localize with CTCF sites. (f) Lost and gained DHS sites; Left-side, distribution at annotated genic regions of DHS sites lost following the expression of dnBrg1. Bottom; Shown are the most significantly enriched motifs identified by MEME analysis (P < 10-4) using the top 1,000 DHS sites (based on tag density) lost following dnBrg1 expression. The motif identified as AP-1 was found to be the most highly enriched motif (MEME E value = 1.8e-184). (g) Lost and gained DHS sites; Right-side, distribution at annotated genomic regions of DHS sites gained following the expression of dnChd4. Bottom, the most significantly enriched motifs identified using the top 1,000 DHS sites (based on tag density) gained following dnChd4 expression. The motif identified as Sp1 was found to be the most highly enriched motif (MEME E value = 6.5e-167).
(a) Additional example of genome browser view displaying Brg1, Chd4, and Snf2h ChIP-seq occupancy and DNase I hypersensitivity patterns at a region on chromosome 6. (b-d) Box plots of tag density enrichment (log2 scale) of the indicated remodeler binding sites overlapping DNase I hypersensitive sites (DHS) and remodeler binding sites not associated with DHS sites. (e) Box plot of tag density enrichment (log2 scale) of DHS sites overlapping remodeler binding sites (Remodeler) and DHS sites not associated with remodeler binding. The number of each site type is indicated below the corresponding box plot. (f) Aggregate plot of the average tag density of DHS sites (hotspot) at regions occupied by: all remodelers (any/all combinations of remodeler binding, black), no remodelers (red), Brg1 only (light blue), Chd4 only (brown), or Snf2h only (pink).
(a) Top, alignment of hBRG1, mouse Chd4, and hSNF2H ATPase domain region containing a conserved lysine (K) residue essential for catalytic activity. Bottom, schematic representation of dominant-negative remodeler constructs. Each remodeler contains either a single or triple FLAG tag at its N- or C-terminus for the monitoring of expression. Highlighted is the mutation of the conserved lysine within the ATPase domain to either arginine (R) or cysteine (C). (b) Schematic of tetracycline-regulated expression system. Each dominant-negative remodeler construct was stably transfected into cells containing the tetracycline transactivator (tTA) system. The tTA construct is positioned downstream of a tetracycline (Tet) response element (TRE) bound to a minimal CMV promoter and is itself regulated by the tTA protein (Vp16-TetR) providing autoinducible control. (c) Western blot analysis of dominant-negative (dn) protein expression derived from whole cell extracts of cultured cells in the presence or absence of tetracycline (Tet). Each remodeler was FLAG-tagged at either the N- or C-terminus and detected using a FLAG-specific antibody. As a positive control, the expression of tetracycline transactivator (tTA) was analyzed (only expressed in the absence of Tet). Actin was analyzed to determine equal loading. (d) mRNA expression of dominant-negative remodelers in the presence (+Tet, white bars) or absence of tetracycline (-Tet, black bars). Expression is shown relative to the +Tet condition (no expression) with normalization to Actin expression. Error bars represent the SEM of three independent replicates. (e-g) Effect of dn-remodeler expression on levels of alternate remodeling proteins. Western blot analysis of selected remodeler expression levels with activation of alternate dn factors. Nuclear extracts from cells either expressing (- tet), or not expressing (+tet, 48 hr.), FLAG-tagged versions of dnBrg1, dnSnf2h, and dnChd4 were tested for effects on the other remodeling proteins. Input, 15 μg of IP nuclear extract; western blots were developed with antibodies specific to FLAG, Brg1, Chd4, Snf2h, the tet regulator, or GAPDH as loading control. Black arrows indicate position of size markers; blue arrows indicated position of factor tested. (e) Cells expressing dnCHD4; Brg1 and Snf2h levels are unaffected. (f) Cells expressing dnBrg1; Chd4 and Snf2h levels are unaffected. Strong induction of the tet regulator is also shown at the top of the panel. (g) Cells expressing dn Snf2h; Brg1 and Chd4 levels are unaffected.
(a-c) Genome browser views of ChIP-seq remodeler binding at DNase I hypersensitive (DHS, DNaseI-seq) sites affected by dominant-negative remodeler expression. In each image tag densities are displayed relative to the indicated genomic coordinates. The left image is an example of an affected site bound only by the wild-type version of the mutant remodeler. The right image displays binding by all three remodelers at the affected DHS site. Changes in hypersensitivity in the absence (+Tet) or presence (-Tet) of the indicated dominant-negative remodeler are overlayed and highlighted by arrows. (d-f) Overlay of remodeler binding at DHS sites in the presence or absence of the indicated dominant-negative remodeler protein. Displayed are scatter plots of DHS site tag density in the presence of each dominant-negative protein (y-axis) compared to DHS site tag density in the absence of this protein. Inset, original scatter plots of DNase I hypersensitivity from Fig. 5.
(a) The scatter plot compares the maximum tag density of -Tet versus +Tet DHS hotspots in the control 3134 dataset. (b) The venn diagram shows the overlap between -Tet DHS hotspots and +Tet DHS hotspots in the control 3134 dataset. The area of each subset is proportional to the number of sites.
Venn diagrams present the occurrence frequencies for the 27 trend classes expected for dnBrg1, dnSnf2h and dnChd4 interactions at DHS sites. For each of these classes, the positional distribution is shown here for categories with more than 50 sites genome-wide. Notation; Brg1-UP indicates sites increased by more than 2-fold in -Tet (with dnBrg1 expression); Brg1-DN indicates means sites decreased by more than 2-fold in -Tet; NC indicates no change. The same notation is used for the dnSnf2h and dnChd4 classes. Sites are classified as promoter (< 2.5 kb upstream or downstream from the nearest TSS), exon (> 2.5 kb downstream from the nearest TSS , overlapping an exon), intron (> 2.5 kb downstream from the nearest TSS , in an intron not overlapping an exon), distal upstream (> 2.5 kb upstream from the nearest TSS), or downstream (> 2.5 kb downstream from the nearest TSS).
RNA levels were examined by microarray analysis in cells either expressing (- tet), or not expressing (+tet) dnBrg1, dnSnf2h, and dnChd4. (a) Example 1; Gipr locus is strongly up-regulated by dnCHD4. In a parallel cell line with tet regulated w.t. Chd4, strong induction of the w.t. protein has no effect of Gipr exression. (b) DHS track for Gipr locus; pronounced DHS element immediately downstream of TSS (arrow) is activated by dnCHD4. (c) Example 2; Kcne1 locus is up-regulated by dnCHD4. Again, w.t. Chd4 has no effect. (d) DHS track for Kcne1 locus; DHS element downstream of TSS (arrow) is activated by dnCHD4. (e) In parallel cell lines, -tet activation produces 30-35 fold increases in dnChd4 and w.t. Chd4 RNA levels. (f) Global effects on gene expression. The number of genes either up- or down-regulated (fc > 1.2; p<0.05) is shown.
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Morris, S., Baek, S., Sung, MH. et al. Overlapping chromatin-remodeling systems collaborate genome wide at dynamic chromatin transitions. Nat Struct Mol Biol 21, 73–81 (2014). https://doi.org/10.1038/nsmb.2718
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