Figure 5.
Conserved mechanism of Mi-2β regulation in keratinocytes.(A) Overlap of genes associated with Mi-2β enrichment peaks in mouse and human keratinocytes is shown. A highly significant P value < 2.77 × 10−68 for the overlap between the two gene sets was calculated by Fisher’s exact test. (B) Pathway analyses of genes associated with Mi-2β enrichment peaks in both mouse and human keratinocytes. Select GO pathways and associated P values (−log10) with fold enrichment are shown. (C) Genome distribution of Mi-2β peaks in mouse and human keratinocytes reveals a similar association with annotated genomic elements. (D) Bar graph depicting the frequency and number of significantly (|log2 fold change| ≥1, FDR <0.05) up-regulated genes after TS (6 or 24 h) that are also associated with Mi-2β enrichment peaks in mouse (left) and human (right) keratinocytes. The frequency of association with Mi-2β peaks was significantly higher for the subset of genes commonly up-regulated at both 6 and 24 h after TS and in Mi2Δ keratinocytes (shared) as determined by Fisher’s exact test (*, P < 0.0001). (E) Experimental design for knockdown of Mi-2β in primary NHKs by lentiviral shRNA transduction and further studies on transcriptome (RNA-seq), chromatin, and TF landscapes (ChIP-seq). (F) Heatmap of K-means clustering analysis of ChIP-seq data for Mi-2β, histone modifications (H3K27Ac and H3K4me3), and RNApII from CTR and Mi2KD human primary keratinocytes, centered on Mi-2β enrichment peaks from the same cells. C1, C2, and C5 were marked by H3K4me3, H3K27Ac, and RNApII and identified as promoters or promoter-proximal regions. C3 associated with weak H3K27Ac and RNApII signal but not with H3K4me3 and was identified as a cluster of poised enhancers. The upward arrow indicates an increase in H3K27Ac upon Mi2KD. (G) An increase in enrichment for H3K27Ac and RNApII, detected upon Mi2KD in C2 and C3, is shown as histograms of read densities (read count per million mapped reads) plotted over the C2 promoter and C3 enhancer clusters. No change in the high level of H3K4me3 detected at the C2 promoters was seen upon Mi2KD. (H) De novo motif discovery for potential TFs associated with Mi-2β peaks in C3 and P values for discovery are shown. (I) Pathway analyses of up-regulated genes after Mi2KD in NHKs that are associated with C3 enhancers. Select GO pathways and associated P values (−log10) with fold enrichment are shown. ChIP-seq datasets were generated from primary NHKs expanded in culture and infected with shRNA CTR or shRNA for CHD4 (Mi-2β). One of two representative ChIP-seq experiments is shown.

Conserved mechanism of Mi-2 β regulation in keratinocytes. (A) Overlap of genes associated with Mi-2β enrichment peaks in mouse and human keratinocytes is shown. A highly significant P value < 2.77 × 10−68 for the overlap between the two gene sets was calculated by Fisher’s exact test. (B) Pathway analyses of genes associated with Mi-2β enrichment peaks in both mouse and human keratinocytes. Select GO pathways and associated P values (−log10) with fold enrichment are shown. (C) Genome distribution of Mi-2β peaks in mouse and human keratinocytes reveals a similar association with annotated genomic elements. (D) Bar graph depicting the frequency and number of significantly (|log2 fold change| ≥1, FDR <0.05) up-regulated genes after TS (6 or 24 h) that are also associated with Mi-2β enrichment peaks in mouse (left) and human (right) keratinocytes. The frequency of association with Mi-2β peaks was significantly higher for the subset of genes commonly up-regulated at both 6 and 24 h after TS and in Mi2Δ keratinocytes (shared) as determined by Fisher’s exact test (*, P < 0.0001). (E) Experimental design for knockdown of Mi-2β in primary NHKs by lentiviral shRNA transduction and further studies on transcriptome (RNA-seq), chromatin, and TF landscapes (ChIP-seq). (F) Heatmap of K-means clustering analysis of ChIP-seq data for Mi-2β, histone modifications (H3K27Ac and H3K4me3), and RNApII from CTR and Mi2KD human primary keratinocytes, centered on Mi-2β enrichment peaks from the same cells. C1, C2, and C5 were marked by H3K4me3, H3K27Ac, and RNApII and identified as promoters or promoter-proximal regions. C3 associated with weak H3K27Ac and RNApII signal but not with H3K4me3 and was identified as a cluster of poised enhancers. The upward arrow indicates an increase in H3K27Ac upon Mi2KD. (G) An increase in enrichment for H3K27Ac and RNApII, detected upon Mi2KD in C2 and C3, is shown as histograms of read densities (read count per million mapped reads) plotted over the C2 promoter and C3 enhancer clusters. No change in the high level of H3K4me3 detected at the C2 promoters was seen upon Mi2KD. (H) De novo motif discovery for potential TFs associated with Mi-2β peaks in C3 and P values for discovery are shown. (I) Pathway analyses of up-regulated genes after Mi2KD in NHKs that are associated with C3 enhancers. Select GO pathways and associated P values (−log10) with fold enrichment are shown. ChIP-seq datasets were generated from primary NHKs expanded in culture and infected with shRNA CTR or shRNA for CHD4 (Mi-2β). One of two representative ChIP-seq experiments is shown.

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