Journal: Nucleic Acids Research

Article Title: Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation

doi: 10.1093/nar/gkw1163

Figure Lengend Snippet: Functional analysis of the correlation between H3K9ac levels and GR binding: correlation ≠ causation. ( A ) Schematic diagram depicting the design of the study to test if the correlation between H3K9ac levels and GR binding reflects a causative link. ( B ) Comparison of % input precipitated by ChIP with an H3K9ac-specific antibody versus IgG control at GR-bound genomic loci as indicated was assayed by qPCR (average enrichment + error from 7 pooled biological replicates) in either wild type MEFs (blue) or in GCN5/PCAF double knock-out (dko) MEFs (salmon). ( C ) Normalized tag density from ChIP-seq for GR after hormone treatment for wild type MEFs (blue) and dko MEFs (salmon) for genomic region near RAC1 gene: GR-bound in both wt MEFs and dko MEFS; SUMF1 : dko-specific GR binding and TXK : wt-specific GR binding. ( D ) Venn diagram showing the number and overlap of GR-bound loci in wt and dko MEFs. ( E ) Comparison of the % of GR ChIP-seq peaks mapping to promoter regions for wt-specific, common and dko-specific peaks.

Article Snippet: ChIP assays were done essentially as described using either the N499 GR-antibody ( , 2 μl/ChIP) an H3K9ac antibody (C5B11, Cell Signaling, 6 μl/ChIP) or as control IgG (cat#: C15410206, Diagenode, 6 μl/ChIP).

Techniques: Functional Assay, Binding Assay, Comparison, Control, Knock-Out, ChIP-sequencing

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: Macrophage GRIP1 is a GR coactivator of anti-inflammatory genes. GRIP1 was depleted in a THP1 cells (using a human-specific small-hairpin (sh)GRIP1 vs. scrambled (shSCR) control) or b mouse BMMΦ (from GRIP1 KO vs. WT control mice), and the induction of indicated genes by dexamethasone (Dex; 100 nM, 2 h) was analyzed by RT-qPCR, normalized to the expression of β-actin and expressed relative to untreated (=1). GRIP1 depletion efficiency is shown by immunoblotting with HSP90 as a loading control on the left (quantified in Supplementary Fig. ; see Supplementary Fig. for full-size bots). ChIP-seq for GR and GRIP1 was performed in THP1 cells ( c ) or mBMMΦ ( d ) treated ±Dex for 1 h (or 45 min for GR in BMMΦ), as indicated, and peaks were called as described in Methods. (Top) The overlap between peaks for GRIP1±Dex or GRIP1 and GR (Venn diagrams) was determined using subsetByOverlap function from GenomicRanges package (Bioconductor) with the minimum overlap of 1 bp (see “Methods”). (Middle) Ab initio sequence motif overrepresentation in GR–GRIP1 overlapping peaks was determined using MEME-ChIP. Genomic location of GRIP1 binding sites relative to known genomic features was determined by ChIPpeakAnno (Bioconductor). The union of peaks for two GRIP1 replicas in each cell type, the consensus for two THP1 GR replicas and a single mBMMΦ GR experiment are shown (Supplementary Figs. – ). (Bottom) Read density profiles of genes analyzed in a , b show peaks for GR and GRIP1. e CD14 + hMΦ were untreated (U) or treated with Dex for 2 h, and the expression of representative genes was measured as in a . f GR and GRIP1 occupancy at relevant sites in hMΦ was assessed by ChIP-qPCR (normalized to the 28s rDNA housekeeping gene, untreated=1). Samples in a , b and e , f were compared by unpaired two-tailed Student’s t -test. Shown are means+standard deviation (SD) error bars ( n ≥ 3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Control, Quantitative RT-PCR, Expressing, Western Blot, ChIP-sequencing, Sequencing, Binding Assay, ChIP-qPCR, Two Tailed Test, Standard Deviation

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: GRIP1 is a substrate for dexamethasone (Dex)-induced phosphorylation by CDK9 in macrophages. a hMΦ, THP1 cells or BMMΦ were treated with Dex for the times indicated and the levels of GRIP1 (total or phosphorylated (p) at S469, S487, S493, and S499), GR, CDK9 and HSP90 as a loading control were assessed by immunoblotting (quantified in Supplementary Fig. ). b THP1 cells or mBMMΦ were treated with Dex±flavopiridol (FVP) at indicated concentrations for 2 h and GRIP1 phosphorylation was visualized by immunoblotting (quantified in Supplementary Fig. ). c Immortalized BMMΦ were depleted of CDK9 (single guide (sg)CDK9 vs. scrambled sgSCR control) as described in Methods and treated ±Dex for 1 h. The levels of GRIP1, phospho-(p)GRIP1, GR, CDK9 and HSP90 were assayed by western blot and quantified by densitometry; CDK9 levels or ratios of pGRIP1/GRIP1 in sgCDK9 cells are expressed relative to those in sgSCR cells (=1). Samples were compared by unpaired, two-tailed Student’s t -test; means + standard error of the mean (SEM) are shown ( n ≥ 3, * P < 0.05, ** P < 0.01). d E . coli -produced affinity-purified GST–GRIP1 322-631 WT and S469A/S487A/S493A/S499A (4A) were subjected to in vitro kinase assays with baculovirus-expressed cyclin T1-CDK9. GRIP1 phosphorylation was assessed by autoradiography (left) and immunoblotting (right) with Coomassie blue staining (left) or immunoblotting for GST-tag (right) showing equal loading. e BMMΦ or THP1 cells were treated ±Dex for 1 h, double cross-linked and GR immunoprecipitations (IP) performed (see “Methods”). Inputs and IPs were immunoblotted for GRIP1, GR and CDK9 (with HSP90 as an input loading control). Full-size western blots are shown in Supplementary Fig.

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Phospho-proteomics, Control, Western Blot, Two Tailed Test, Produced, Affinity Purification, In Vitro, Autoradiography, Staining

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: GRIP1 phosphorylation potentiates GR-mediated transcriptional activation. a THP1 cells or BMMΦ were treated ±dexamentasone (Dex)±50 nM flavopiridol (FVP) for 2 h and “fold induction” of indicated genes analyzed by RT-qPCR, normalized to β-actin and expressed relative to untreated or FVP alone (=1); mean+SEM are shown; n ≥ 3. b The induction by Dex (30 min) of GR target genes in single guide (sg)CDK9 and scrambled sgSCR control IBMMΦ was analyzed by RT-qPCR as in a ; mean + SD is shown, n = 4. c THP1 cells transduced with SCR shRNA (-) or those depleted of endogenous hGRIP1 (+) from Fig. , were stably transduced with WT or 4A mGRIP1 (see “Methods”), as indicated, and GRIP1 expression tested by immunoblotting with HSP90 is a loading control (left). WT- and S469A/S487A/S493A/S499A (4A)-expressing THP1 cells were treated ±Dex for 2 h and GRIP1 (total and phospho-isoforms), GR, CDK9 and HSP90 were visualized by immunoblotting (right). Quantification was performed as in Fig. and expressed relative to WT (=1); mean + SEM are shown; n ≥ 3. Full-size western blots are shown in Supplementary Fig. g. d WT and 4A THP1 cells were treated ±Dex for 2 h and the expression of GR target genes was analyzed by RT-qPCR as in a ; mean + SEM are shown, n ≥ 3. In each panel * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; unpaired, two-tailed Student’s t -test

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Phospho-proteomics, Activation Assay, Quantitative RT-PCR, Control, Transduction, shRNA, Stable Transfection, Expressing, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: GRIP1 phospho-isoforms occupy GR target genes in a site-specific manner. THP1 cells ( a ) or BMMΦ ( b ) were incubated with dexamentasone (Dex) for 1 h, and the occupancy of phospho- (p)S469-, pS487-, pS493-, and pS499-GRIP1 as well as CDK9 was assessed at GRIP1-occupied sites from Fig. by ChIP-qPCR as in Fig. . Mean + SEM are shown ( n ≥ 3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; unpaired, two-tailed Student’s t -test). c ChIP-seq for pS469-GRIP1 was performed in THP1 MΦ and Dex-induced peaks were called using MACS2 with pS469 peaks in untreated THP1 cells set as a background. (Left, top) Overlaps between pS469 and GR peaks in Dex-treated cells were determined as in Fig. (Venn diagram), and the fraction of total GRIP1-bound sites that overlapped with pS469 peaks is shown (pie chart; see “Methods”). The union of peaks detected in two pS469-GRIP1 ChIP-seq experiments was used for the analysis (Supplementary Fig. ). (Left, bottom) Ab initio sequence motif discovery and overrepresentation was assessed as in Fig. . Red rectangles indicate overlapping NR3C half-sites. (Right) Gene-peak associations were analyzed using GREAT as described in “Methods”. d THP1 MΦ ChIP-seq read density profiles for genes showing GR and GRIP1 peaks with ( NFKBIA ) or without ( IL1R2 ) S469 phosphorylation or e a pair of GR:GRIP1 binding sites nearby the A20 locus both with (red arrow) and without (blue arrow) pS469

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Incubation, ChIP-qPCR, Two Tailed Test, ChIP-sequencing, Sequencing, Phospho-proteomics, Binding Assay

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: GRIP1 phosphorylation affects its localization at a subset of genomic sites. THP1 cells ( a ) and BMMΦ ( b ) were treated ±dexamentasone (Dex) ±50 nM flavopiridol (FVP) for 1 h, and the occupancy of GR, pS469-GRIP1 and GRIP1 was analyzed by ChIP-qPCR as in Fig. . c THP1-mutant-MΦ expressing WT or S469A/S487A/S493A/S499A (4A) mGRIP1 were treated ±Dex for 1 h and the expression of indicated genes was analyzed by RT-qPCR as in Fig. and expressed relative to untreated cells for each genotype (=1). Means + SEM are shown ( n ≥ 3, * P < 0.05, ** P < 0.01; one-way ANOVA with Bonferroni’s multiple comparison test)

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Phospho-proteomics, ChIP-qPCR, Mutagenesis, Expressing, Quantitative RT-PCR, Comparison

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: GRIP1 phosphorylation does not potentiate its GR corepressor function. a THP1 cells (small hairpin scrambled (shSCR) or shGRIP1) or BMMΦ (WT or GRIP1 KO) were treated ± 10 ng ml −1 LPS and ± Dex for 1 h and the expression of indicated genes was analyzed by RT-qPCR as described in Fig. . “Fold repression” was defined as the quotient of the transcript level at lipopolysaccharide (LPS) over LPS+dexamethasone (Dex) condition and is shown as Tukey box-and-whisker plots ( n = 4). b THP1 cells expressing WT or S469A/S487A/S493A/S499A (4A) mGRIP1 were treated as in (a) and fold repression of indicated genes is shown. ns, non-significant. c THP1 MΦ or BMMΦ were treated ± Dex ± LPS for 2 h, as indicated, and the levels of GRIP1, GRIP1 phospho-isoforms, GR, CDK9 and HSP90 were evaluated by immunoblotting (quantified in Supplementary Fig. ; full-size blots are shown in Supplementary Fig. ). THP1 MΦ ( d ) or BMMΦ ( e ) treated with ±Dex ±LPS for 1 h were analyzed for GR, GRIP1, CDK9 and phospho-GRIP1 occupancy by ChIP-qPCR as in Fig. (see Supplementary Fig. for more genes). Shown are mean + SD, n ≥ 3, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Phospho-proteomics, Expressing, Quantitative RT-PCR, Whisker Assay, Western Blot, ChIP-qPCR, Comparison

Journal: Nature Communications

Article Title: Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages

doi: 10.1038/s41467-017-01569-2

Figure Lengend Snippet: Differential utilization of GRIP1 phosphorylation at GR-regulated genes. Diagramed is a macrophage (MΦ) with GR bound at a palindromic GC response element (GRE) activating transcription of anti-inflammatory genes or “tethered” to AP1 or NF-κB repressing pro-inflammatory genes. GRIP1 is recruited to both types of complexes but undergoes phosphorylation by CDK9 at the palindromic GRE only

Article Snippet: Inputs were taken from cleared lysates and the rest were incubated for 4 h at 4 °C with the following antibodies to precipitate protein:DNA complexes: GR (Santa Cruz, M20x, sc-1004x or N499), GRIP1 (Abcam, ab10491 or Bethyl A300-346A), phospho-specific GRIP1 antibodies, or CDK9 (Santa Cruz, H169x, sc-8338x).

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

doi: 10.1101/2020.10.15.340877

Figure Lengend Snippet: (a) Scatterplot depicting enrichment of GR- and AR-RIME experiments over IgG control. Interactors recruited: ≥ 2 Label-free Quantification (LFQ) enriched over IgG (dotted line) and significant (-log(padj)>1,3; green). n=4. U2OS-GR and U2OS-AR cell lines were treated with either 1μM Dex or 5 nM R1881 for 4h, respectively. (b) Gene Set Enrichment Analysis (GSEA) enrichment ranks for transcription coactivator activity, nuclear receptor binding, chromatin remodeling (M19139), and RNA polymerase transcription factor binding genesets based on GR- and AR-RIME datasets. n=4. (c) Volcano plot depicting differentially enriched interactors for AR and GR. n=4. (d) LFQ enrichment of mediator complex members in GR- and AR-RIME experiments. n=4. (e) Med1 occupancy at loci as indicated was analyzed by ChIP followed by qPCR for cells treated with vehicle control (ethanol for U2OS-GR, dmso for U2OS-AR) or 1 μM Dex, 1.5h (U2OS-GR) or 5 nM R1881, 1.5h (U2OS-AR). Average percentage of input precipitated ± SEM from three independent experiments is shown. (f) same as (e) except that ChIP was for EP300.

Article Snippet: ChIP assays were performed using the following antibodies: GR, N499 (2 μl/ChIP); AR, polyclonal antibody PG-21 (Anti-AR; Sigma Aldrich; 06-680, 2 μl/ChIP); H3K27ac, C15410196 (Diagenode 1μg /ChIP); Med1, A300-793A (Bethyl Laboratories, 5 μl/ChIP); EP300, C15200211 (Diagenode, 5 μl/ChIP); H3K4me1, C15410194 (Diagenode 1μg/ChIP); H3K9me3, C15410193 (Diagenode 1μg/ChIP); H3K27me3, C1541095 (Diagenode 1μg/ChIP).

Techniques: Control, Quantitative Proteomics, Activity Assay, Binding Assay