BRG1 governs glucocorticoid receptor interactions with chromatin and pioneer factors across the genome
- National Institute of Environmental Health Sciences, National Institutes of Health, United States
Figures
Figure 1 with 2 supplements
BRG1 chromatin interaction defines distinct classes of GR binding site.
(A) Heatmap demonstrating that GR is detected at 29934 binding sites following 1 hr Dex treatment. (B) Heatmaps illustrating detection of BRG1 at a subset of GR binding sites prior to hormone …
Figure 1—figure supplement 1
Hormone treatment reorganizes BRG1 chromatin localization.
(A) Heatmap depicting BRG1 ChIP-seq coverage at BRG1 peaks that are called in EtOH only, Dex only, or both conditions. (B) Heatmap depicting BRG1 ChIP-seq coverage at BRG1 peaks as in (A), but with …
Figure 1—figure supplement 2
Biological replication of Next Generation Sequencing data sets.
Heatmaps depicting consistency between biological replicate ChIP-seq and ATAC-seq experiments over GR peak classes
Figure 2 with 3 supplements
Class II GR peaks are associated with open and transcriptionally active chromatin.
(A) ATAC-seq accessibility/short reads are enriched over Class II GR peaks independently of hormone treatment. (B) H3K27ac ChIP-seq coverage is specifically enriched over Class II GR peaks …
Figure 2—figure supplement 1
BRG1 peaks without GR are accessible and have active chromatin marks.
BRG1 peaks without GR that are hormone-independent (labeled ‘Both’) have enrichment patterns for ATAC accessibility, H3K27ac, and H3K4me1 that are similar to Class II peaks. EtOH-specific and …
Figure 2—figure supplement 2
GR is weakly enriched around TSSs.
Heatmaps showing GR ChIP-seq coverage at (A) active gene TSSs (same plot as Figure 2e with adjusted y and z scales), (B) TSSs of RNA-seq identified DEGs following 1 hr Dex treatment, (C) TSSs of …
Figure 2—figure supplement 3
BRG1 is enriched around TSSs.
Heatmaps showing BRG1 +/- Dex at ChIP-seq coverage at (A) TSSs of RNA-seq identified DEGs following 1 hr Dex treatment, (B) TSSs of microarray-identified DEGs following 1 hr Dex treatment, (C) TSSs …
Figure 3 with 1 supplement
BRG1 is required for the Dex-induced transcriptional response.
(A) Venn diagram showing overlap between DEGs in 8 hr Dex treatment vs EtOH in WT and BRG1-KD cells. (B) Heatmap depicting log2 fold change of DEGs in 8 hr Dex treatment vs EtOH in WT and BRG1-KD …
Figure 3—figure supplement 1
BRG1 and GR knockdown in A1A3 cells.
(A) Western blots depicting protein levels of BRG1, cytoplasmic GR, nuclear GR, GATA3, FOXA1, and KU70 in A1A3 cells following BRG1 knockdown by doxycycline treatment. (B) Western blots depicting …
Figure 4 with 1 supplement
GR peak classes have distinct underlying DNA sequences and transcription factor motifs.
(A) Class II GR peaks have fewer perfect GRE motifs than Class I or Class III peaks. (B) Heatmap of -log10 p-values for enrichment of hormone receptor motifs under GR peak classes. (C) Heatmap of …
-
Figure 4—source data 1
Table of MCF7 ENCODE ChIP-seq data sets.
Table of ENCODE ChIP-seq data sets from MCF7 cells used to calculated average transcription factor ChIP-seq meta-plot in Figure 4E.
- https://doi.org/10.7554/eLife.35073.014
Figure 4—figure supplement 1
Transcription factor motifs enriched under distinct GR peak classes.
Box plots of -log10 p-values of families of transcription factor motifs under Class I, II, and III GR peaks. Note that Fox, Gata, SP/KLF, and Pou family motifs are most strongly enriched under Class …
Figure 5
BRG1 is required for Dex-induced recruitment of pioneer factors.
(A–B) FOXA1 and GATA3 ChIP-seq coverage is enriched across all GR peak classes, but shows strongest enrichment over Class II peaks. (C–D) Meta-profiles of FOXA1 and GATA3 ChIP-seq signal over GR …
Figure 6 with 1 supplement
BRG1 binding to pioneer factor binding sites is predictive of GR binding upon Dex treatment.
(A) Overlap between BRG1 EtOH and FOXA1 EtOH peaks. (B) Overlap between BRG1 EtOH and GATA3 EtOH peaks. (C) Three-way Venn diagram showing overlap of all three factors (D) Heatmaps depicting FOXA1, …
Figure 6—figure supplement 1
Proportion of GREs under pioneer factor peaks.
Percentage of GATA3 and FOXA1 peaks that have perfect GRE motifs within 500 bp of the center of the peak. The percentage of peaks with perfect GRE motifs is similar between peaks ‘with BRG1’ and …
Figure 7
Overview of three classes of GR binding site.
(A) Class I GR binding sites (GBSs) reside within relatively closed regions of chromatin and have little dex-dependent chromatin remodeling or recruitment of BRG1 and pioneer factors. Despite this, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H.sapiens) | A1-2 | PMID: 7838148 | RRID:CVCL_0I95 | T47D derivative with incorporated rat GR |
| Cell line (H.sapiens) | A1-A3 | PMID: 22451486 | A1-2 derivative with incorporated BRG1 shRNA | |
| Antibody | anti-BRG1 | PMID: 26055322 | lab-made, ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml | |
| Antibody | anti-GR | Santa Cruz | M-20, sc-1004, RRID:AB_2155786 | ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml |
| Antibody | anti-FOXA1 | Abcam | ab23738, RRID:AB_2104842 | ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml |
| Antibody | anti-GATA3 | Cell Signaling | D13C9, RRID:AB_10834528 | ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml |
| Antibody | anti-H3K27ac | Abcam | ab4729, RRID:AB_2118291 | ChIP = 1 ug/100 ug chromatin |
| Antibody | anti-H3K27me3 | Active Motif | 39155, RRID:AB_2561020 | ChIP = 1 ug/100 ug chromatin |
| Antibody | anti-H3K4me1 | Abcam | ab8895, RRID:AB_306847 | ChIP = 1 ug/100 ug chromatin |
| Antibody | anti-KU70 | Santa Cruz | H-308, sc-9033, RRID:AB_650476 | Western Blot = 0.1 ug/ml |
| Sequence-based reagent | GR siRNA | Dharmacon | ON-TARGETplus J-089504–07 | UUACAAAGAUUGCAGGUAU |
| Sequence-based reagent | Non-targeting Control siRNA | Dharmacon | ON-TARGETplus Not-targeting Pool D-001810-10-20 | |
| Commercial assay or kit | Nextera XT library generation kit | Illumina | 15032350 | |
| Commercial assay or kit | SuperScript III First-Strand kit | Invitrogen | 18080–051 | |
| Commercial assay or kit | iScript cDNA Sythesis kit | Bio-Rad | 170–8891 | |
| Commercial assay or kit | ssoAdvanced Universal SYBR Green Supermix | Bio-Rad | 172–5274 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | 74104 | |
| Commercial assay or kit | RNA 6000 RNA Pico Kit | Agilent Technologies | 5067–1513 | |
| Commercial assay or kit | QiaQuick PCR purification kit | Qiagen | 28104 | |
| Commercial assay or kit | HALT protease inhibitors | ThermoFisher | 78430 | |
| Chemical compound, drug | Dexamethasone | Sigma | D4902 | 100 nM |
| Chemical compound, drug | Doxycycline | Sigma | D9891 | 10 ug/ml |
| Software, algorithm | Cutadapt | DOI: http://dx.doi.org/10.14806/ej.17.1.200 | RRID:SCR_011841 | |
| Software, algorithm | Sickle | https://github.com/najoshi/sickle | RRID:SCR_006800 | |
| Software, algorithm | Bowtie2 | PMID: 22388286 | RRID:SCR_005476 | |
| Software, algorithm | Samtools | PMID: 19505943 | RRID:SCR_002105 | |
| Software, algorithm | MACS2 | PMID: 18798982 | RRID:SCR_013291 | |
| Software, algorithm | Homer | PMID: 20513432 | RRID:SCR_010881 | |
| Software, algorithm | Bedtools | PMID: 20110278 | RRID:SCR_006646 | |
| Software, algorithm | Deeptools | PMID: 27079975 | ||
| Software, algorithm | AME | DOI: https://doi.org/10.1186/1471-2105-11-165 | RRID:SCR_001783 | http://meme-suite.org/tools/ame |
| Software, algorithm | STAR | PMID: 23104886 | RRID:SCR_015899 | |
| Software, algorithm | Salmon | PMID: 28263959 | ||
| Software, algorithm | limma-voom | PMID: 24485249 | RRID:SCR_010943 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.35073.019
