BRG1 governs glucocorticoid receptor interactions with chromatin and pioneer factors across the genome

  1. Jackson A Hoffman
  2. Kevin W Trotter
  3. James M Ward
  4. Trevor K Archer  Is a corresponding author
  1. National Institute of Environmental Health Sciences, National Institutes of Health, United States
7 figures, 1 table and 1 additional file

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 …

https://doi.org/10.7554/eLife.35073.003
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 …

https://doi.org/10.7554/eLife.35073.004
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

https://doi.org/10.7554/eLife.35073.005
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 …

https://doi.org/10.7554/eLife.35073.006
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 …

https://doi.org/10.7554/eLife.35073.007
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 …

https://doi.org/10.7554/eLife.35073.008
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 …

https://doi.org/10.7554/eLife.35073.009
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 …

https://doi.org/10.7554/eLife.35073.010
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 …

https://doi.org/10.7554/eLife.35073.011
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 …

https://doi.org/10.7554/eLife.35073.012
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 …

https://doi.org/10.7554/eLife.35073.013
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 …

https://doi.org/10.7554/eLife.35073.015
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, …

https://doi.org/10.7554/eLife.35073.016
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 …

https://doi.org/10.7554/eLife.35073.017
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, …

https://doi.org/10.7554/eLife.35073.018

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (H.sapiens)A1-2PMID: 7838148RRID:CVCL_0I95T47D derivative with incorporated rat GR
Cell line (H.sapiens)A1-A3PMID: 22451486A1-2 derivative with incorporated BRG1 shRNA
Antibodyanti-BRG1PMID: 26055322lab-made, ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml
Antibodyanti-GRSanta CruzM-20, sc-1004, RRID:AB_2155786ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml
Antibodyanti-FOXA1Abcamab23738, RRID:AB_2104842ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml
Antibodyanti-GATA3Cell SignalingD13C9, RRID:AB_10834528ChIP = 1 ug/100 ug chromatin, Western Blot = 0.1 ug/ml
Antibodyanti-H3K27acAbcamab4729, RRID:AB_2118291ChIP = 1 ug/100 ug chromatin
Antibodyanti-H3K27me3Active Motif39155, RRID:AB_2561020ChIP = 1 ug/100 ug chromatin
Antibodyanti-H3K4me1Abcamab8895, RRID:AB_306847ChIP = 1 ug/100 ug chromatin
Antibodyanti-KU70Santa CruzH-308, sc-9033, RRID:AB_650476Western Blot = 0.1 ug/ml
Sequence-based reagentGR siRNADharmaconON-TARGETplus J-089504–07UUACAAAGAUUGCAGGUAU
Sequence-based reagentNon-targeting Control siRNADharmaconON-TARGETplus Not-targeting Pool D-001810-10-20
Commercial assay or kitNextera XT library generation kitIllumina15032350
Commercial assay or kitSuperScript III First-Strand kitInvitrogen18080–051
Commercial assay or kitiScript cDNA Sythesis kitBio-Rad170–8891
Commercial assay or kitssoAdvanced Universal SYBR Green SupermixBio-Rad172–5274
Commercial assay or kitRNeasy Mini KitQiagen74104
Commercial assay or kitRNA 6000 RNA Pico KitAgilent Technologies5067–1513
Commercial assay or kitQiaQuick PCR purification kitQiagen28104
Commercial assay or kitHALT protease inhibitorsThermoFisher78430
Chemical compound, drugDexamethasoneSigmaD4902100 nM
Chemical compound, drugDoxycyclineSigmaD989110 ug/ml
Software, algorithmCutadaptDOI: http://dx.doi.org/10.14806/ej.17.1.200RRID:SCR_011841
Software, algorithmSicklehttps://github.com/najoshi/sickleRRID:SCR_006800
Software, algorithmBowtie2PMID: 22388286RRID:SCR_005476
Software, algorithmSamtoolsPMID: 19505943RRID:SCR_002105
Software, algorithmMACS2PMID: 18798982RRID:SCR_013291
Software, algorithmHomerPMID: 20513432RRID:SCR_010881
Software, algorithmBedtoolsPMID: 20110278RRID:SCR_006646
Software, algorithmDeeptoolsPMID: 27079975
Software, algorithmAMEDOI: https://doi.org/10.1186/1471-2105-11-165RRID:SCR_001783http://meme-suite.org/tools/ame
Software, algorithmSTARPMID: 23104886RRID:SCR_015899
Software, algorithmSalmonPMID: 28263959
Software, algorithmlimma-voomPMID: 24485249RRID:SCR_010943

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