Figure 1.
Genome-wide profile of Gro recruitment in Kc167 cells.
A) Venn diagram showing the relationship between 2 ChIP-seq biological replicates generated using the anti-Gro antibody. B) Venn diagram illustrating the relationship between ChIP-seq peaks derived from untreated Kc167 cells, and Kc167 cells depleted of Gro by RNAi. C) Venn diagram showing the relationship between 2 ChIP-seq biological replicates generated using the anti-GFP antibody in Kc167 cells transfected with Gro-GFP. D) Venn diagram illustrating the overlap between peaks of endogenous Gro and Gro-GFP recruitment.
Figure 2.
Characterization of high confidence Gro binding sites in Kc167 cells.
A) Histogram showing the frequency of peak widths (100 bp bins) of high confidence Gro binding sites in Kc167 cells. B) Histogram showing the number of peaks observed within 5 kb of another Gro peak. C) Plot showing the position of Gro recruitment in Kc167 cells with respect to annotated transcripts. Note: ‘includeFeature’ means the Gro peak covers the entire transcript and ‘inside’ means the peak is within the transcript boundary. D) Pattern of Gro recruitment in the E(spl)-C in wild-type and Kc167 cells depleted of Gro by RNAi. Peaks of Su(H) binding from ChIP-chip analysis (FDR ≤1 from [28]) are marked as blue bars under the gene names. E) Plot showing up regulation of E(spl)mβ-HLH and E(spl)m3-HLH expression in Kc167 cells treated with gro RNAi detected by quantitative PCR. vtd and Su(H) were included as controls. Jinghua Li and Sarah Bray contributed the data for this panel. F) Centrimo analysis of Gro motif binding in Kc167 cells (Bailey and Machanick, 2012). G) Gene Ontology Analysis of genes associated with Gro peaks. Terms were selected by taking the most significant term (p value<10−4) in a cluster and the most significant unclustered terms generated from an analysis with DAVID [67].
Figure 3.
Characterization of Gro recruitment in S2 cells.
A) Venn diagram showing the relationship between biological replicates with the most aligned reads from ChIP using anti-Gro and ChIP using anti-GFP (to Gro-GFP) in S2 cells. B) Venn diagram illustrating the overlap of high confidence Gro ChIP peaks in Kc167 and S2 cell lines. C) Plot showing the position of Gro recruitment with respect to annotated transcripts in S2 cells. D) Histogram showing the frequency of peak widths (100 bp bins) of Gro binding sites in S2 cells. E) Centrimo analysis of Gro motif binding in S2 cells (Centrimo; [30]). F) Gene Ontology Analysis of genes associated with Gro peaks in S2 cells. Terms were selected by taking the most significant term (p value<10−4) in a cluster and the most significant unclustered terms generated from an analysis with DAVID [67].
Figure 4.
Blocking oligomerization of Gro does not affect peak width in Kc167 or S2 cells.
A) Heat maps illustrating the relationship of Gro-GFP and GroL38D,L87D-GFP peaks in Kc167 and S2 cells. Plots extend 500 bp either side of the center of each peak (0) and are ordered by the width of the peak. B) Plot of the average ChIP peak widths obtained for endogenous Gro, Gro-GFP and GroL38D,L87D-GFP in Kc167 cells and for human TLE3 in the MCF7 cell line (TLE3 ChIP-seq data from [32]; GEO accession no. GSM1019137). Error bars represent 95% confidence intervals on the estimates of the means. C) Binding of Gro-GFP and GroL38D,L87D-GFP around the Rh5 and E(spl)mβ-HLH loci in Kc167 cells.
Figure 5.
Relationship between Gro recruitment and acetylation status of histones H3 and H4.
A) Venn diagram showing the overlap between Gro (superset sites) and HDAC1/Rpd3 binding sites in Kc167 cells. Rpd3 peaks are derived from ChIP-chip data available through modENCODE ([24] http://www.modencode.org). B) Relationship between Gro recruitment and acetylation status of histones H3 and H4 around the E(spl)mβ-HLH gene in wild-type and Gro depleted Kc167 cells. C) Average profile of acetylated histone H3 binding with respect to the location of Gro peaks in wild-type Kc167 cells. (location of Gro sites is indicated by red - TSS, black - upstream of a gene, blue - inside a gene, green - downstream of a gene; the profiles are arranged by the strand of the nearest transcript). D) Profile of acetylated histone H3 binding with respect to the location of Gro peaks in Kc167 cells treated with gro RNAi. E) Profile of acetylated histone H4 binding with respect to the location of Gro peaks in wild-type Kc167 cells. F) Profile of acetylated histone H4 binding with respect to the location of Gro peaks in Kc167 cells treated with gro RNAi. In C)–F) data from a single ChIP-seq replicate is shown. G) Profile of H3K27ac binding with respect to the location of Gro peaks in Kc167. ChIP-seq data for H3K27ac was from [35]. H) Profile of H3K4me3 binding with respect to the location of Gro peaks in Kc167 cells. ChIP-seq data for H3K4me3 was from [35].
Figure 6.
Analysis of the relationship between Gro, chromatin class and RNAP II recruitment in Kc167 cells.
A) Enrichment of Gro peaks in the different classes of chromatin defined by [23]. The class of chromatin is indicated by the colour of the bar and in text underneath. The plot is based on the percentage of Gro binding sites (100 bp near the summit of each peak) within each chromatin class. The plot also includes the per cent of the genome based on the number of base pairs that can be mapped to each chromatin class. B) Average profile of DNase hypersensitive sites with respect to the location of Gro peaks in Kc167 cells. The data for DNase hypersensitive sites was obtained from modENCODE [24]. Location of Gro sites is indicated by red - TSS, black - upstream of a gene, blue - inside a gene, green - downstream of a gene; the profiles are arranged by the strand of the nearest transcript). C) Histogram showing where Gro is binding relative to annotated transcript TSSs. The distance is from the summit of the Gro peak to the nearest TSS, adjusted for strand used for transcription. D) Average profile of RNAP II (Ser2-P form) binding at Gro peaks at different locations. E) Density plot showing expression levels of genes with respect to the site of Gro recruitment. red - TSS, black - upstream of a gene, blue - inside a gene, green - downstream of a gene. The expression level of all annotated genes is shown in grey. F) Plot of the average amount of Gro binding at different locations with respect to genes (location of Gro sites is indicated by red - TSS, black - upstream of a gene, blue - inside a gene, green - downstream of a gene).
Figure 7.
Gro is enriched at genes that exhibit RNAP II promoter proximal pausing.
A) Plot showing binding of Gro with respect to polymerase pausing. The pause ratio for RNAP II at annotated TSSs was calculated and divided into 10 quantiles (0–10% has lowest 10% of paused ratio, 90–100% has highest 10% of paused ratio). The percentages of transcripts nearest to Gro binding sites that fall into each quantile were calculated. B) Venn diagram illustrating the overlap between Gro and GAGA Factor (GAF) binding. The GAF peaks were derived from ChIP-seq data generated as part of the modENCODE project ([24]; http://www.modencode.org). C) Profile plot of total RNAP II (using anti-Rbp3 antibody) and D) elongation competent RNAP II (using anti-Ser2-P antibody) across the E(spl)mβ-HLH locus in Kc167 in untreated (black) and gro RNAi treated (red) cells. Profiles were taken from the average normalized counts of 100 bp fragments from an analysis in edgeR [68].