Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: Relationship between pS118-ER and DNA binding. (A) Western blot analysis of ER and pS118-ER in MCF-7 cells treated with 10 nM E2 for various amounts of time. β-Actin is shown as a loading control. (B) Western blot analysis of MDA-MB-231 cells containing doxycycline-inducible wt ER or S118A ER. MDA-MB-231 cells were treated with 5.0 µg/ml (wt ER) or 0.5 µg/ml (S118A ER) dox for 24 h, followed by 30 min of treatment with vehicle (Veh; 0.1% EtOH) or 10 nM E2. β-Actin serves as a loading control. (C) ER ChIP-qPCR of MDA-MB-231 wt ER or S118A ER-expressing cells. Data are displayed as percentages of input. n = 3. Means ± standard deviations (SD) are shown. *, P < 0.05. (D) HEK293 cells transfected with either vector, wt ER, or the DNA binding ER mutant C202/205H were treated for 30 min with either vehicle or 10 nM E2 and analyzed via immunoblotting.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: Binding Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: pS118-ER ChIP-seq with multiple antibodies. (A) Venn diagrams displaying the overlap in ER or pS118-ER ChIP-seq sites between vehicle (0.1% EtOH)- and E2-treated MCF-7 cells. Three separate pS118-ER antibodies were used for ChIP-seq analysis and are denoted as pS118-ER #1, pS118-ER #2, and pS118-ER #3. (B) ChIP-qPCR validation of pS118-ER occupancy sites. The number after a gene name denotes the approximate distance, in base pairs if not otherwise specified, between the region tested and the transcription start site of the gene, with a negative number being upstream and a positive number being downstream. Data are displayed as a percentage of input. n = 3. Means ± SD are shown. *, P < 0.05 versus vehicle. (C) ChIP-seq track display from the UCSC Genome Browser showing ER and pS118-ER occupancy sites at the GREB1 promoter. For each track, vehicle-treated (yellow) and E2-treated samples (blue) are overlaid. Overlapping tracks are displayed in green. The y axis displays tag density normalized to 107 tags.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: ChIP-sequencing

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: (A) Combination of pS118-ER sites into one data set. E2 peaks from pS118-ER #2 and pS118-ER #3 ChIP-seq were combined into one peak set and overlapped with ER sites. Ab, antibody. (B) Location annotation of pS118-ER and ER occupancy sites. Sites were annotated to either a promoter region, intron, intergenic region, or other. A promoter region was defined as bp −1000 to bp +100 from a transcription start site. The outer ring displays the distribution of ER sites, and the inner ring displays the distribution of pS118-ER sites. (C) ER and pS118-ER sites were annotated to their nearest transcription start site and binned into various distances. (D) Average H3K27ac signal at ER and pS118-ER sites. H3K27ac data for MCF-7 cells treated with E2 were acquired from a previously published data set (58) (GEO accession no. GSE45822). (E) Volcano plot of RNA-seq data from MCF-7 cells treated with 1 nM E2 for 24 h acquired from a previous published data set (59) (GSE89888). Fold change represents E2 treated versus vehicle treated. Each dot represents a gene, and red dots are genes with a pS118-ER occupancy site within 100 kb of its transcription start site. The horizontal dashed line denotes a cutoff of significance at P = 0.001. (F) Proportion of genes upregulated or downregulated by E2 with a pS118-ER site within 100 kb of their respective transcription start sites.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: ChIP-sequencing, RNA Sequencing Assay

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: Motif analysis of ER and pS118-ER sites. (A) De novo motif analysis of ER sites and pS118-ER sites using HOMER. TF, associated transcription factor for each motif. (B) Differential motif analysis of pS118-ER sites. Enriched motifs were searched for using the CentriMo tool in the MEME suite (60). All ER sites (44,050) were used as control (background) sequences, and pS118-ER sites were used as primary input sequences. (C) Proportion of sites containing each specific motif from known motif analysis using HOMER. P values from chi-squared test are displayed when significant. (D) Distribution of specific motifs around ER and pS118-ER sites.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: SNAP array analysis reveals direct and indirect ER binding events. (A) Schematic for identifying direct and indirect binding events. If a site shows binding in both ChIP-seq and the SNAP array, it is categorized as a direct binding event. If a peak identified in ChIP-seq is not detected on the SNAP array, it is categorized as an indirect binding event. (B) Representative examples of a direct and indirect binding event. (Top) ChIP-seq signal from ER and pS118-ER at the TFF1 promoter and a site within the IGF1R gene. The color key and y axis are the same as those in Fig. 2C. (Bottom) Relative ER binding intensity at these regions on the SNAP array. Each region contains multiple DNA probes tiled across the region. (C) Breakdown of regions assayed on the SNAP array. Pie charts display the proportion of regions assayed on the SNAP array and present at either the pS118-ER or ER-only ChIP-seq sites. Analysis revealed that directly bound sites were more likely to be occupied by pS118-ER than ER. (D) Distribution of the number of ER binding events present in the SNAP array genomic regions assayed. The number of ER binding events was calculated for each genomic region and is displayed as a histogram. A relative ER binding intensity of 5 was used as a cutoff for a binding event. (E) Proportion of SNAP array regions present in ER or pS118-ER ChIP-seq sites. SNAP array regions were binned by the number of ER binding events, and these regions were overlapped with ER and pS118-ER sites.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: Binding Assay, ChIP-sequencing

Journal: Molecular and Cellular Biology

Article Title: The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

doi: 10.1128/MCB.00417-18

Figure Lengend Snippet: Analysis of GRHL2 occupancy at pS118-ER sites. (A) Overlap of GRHL2 sites with pS118-ER sites and ER sites identified by ChIP-seq. (B) Average GRHL2 signal at ER and pS118-ER sites. GRHL2 ChIP-seq data were acquired from a previously published data set (61). (C and D) ChIP-qPCR analysis of ER, pS118-ER, and GRHL2 after 30 min of treatment with E2 at sites cooccupied by pS118-ER and GRHL2 (n = 4) (C) or occupied by GRHL2 only (D). IgG was used as a control. n = 3. Means ± standard errors (SE) are shown. *, P < 0.05.

Article Snippet: Antibodies used for ChIP-seq were ER (HC-20, sc-543; Santa Cruz), pS118-ER #1 (16J4; Cell Signaling), pS118-ER #2 (ab32396; Abcam), and pS118-ER #3 (sc-12915; Santa Cruz).

Techniques: ChIP-sequencing