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Fig. 4 | Genome Biology

Fig. 4

From: ncHMR detector: a computational framework to systematically reveal non-classical functions of histone modification regulators

Fig. 4

Non-classical function of CBX7 for the maintenance of pluripotency. a Heatmap showing CBX7, CBX7’s classical substrates H3K27me3 and H3K9me3, predicted cofactor NANOG, and H3K27ac enrichment around CBX7 ChIP-seq peak centers. Rows represent CBX7 binding sites and are ranked by normalized H3K27me3 signals. The colors indicate the normalized ChIP-seq enrichment level and the values are scaled by row. CBX7, NANOG, H3K27me3, H3K9me3, and H3K27ac ChIP-seq data were obtained from GSE64008, GSE90893, GSE58023, GSE90895, and GSE67867. b Stacked bar plot showing the percentages of classical sites, non-classical sites, and the whole genome that reside in promoter, gene body, and intergenic regions. The promoter is defined as ± 3 kb around TSS of the gene. c Real-time qPCR analysis for the expression of CBX7 and NANOG in wild type, Cbx7-knockout, and Cbx7-knockdown mESCs. Error bars represent the standard deviation for triplicate experiments, and unpaired t test with Welch’s correction was used to calculate the statistical significance for comparison (*p value < 0.05, **p value < 0.01, ***p value < 0.001, and n.s represents non-significant). d Western blot analysis of CBX7 and NANOG level in wild type, Cbx7-knockout, and Cbx7-knockdown mESCs. Tubulin was used as a loading control. Blots were cut before antibody application. Gel images for Western blot are shown in Additional file 1: Fig. S6a. e The UCSC genome browser view of CBX7, H3K27me3, H3K9me3, NANOG, and H3K27ac enrichment at a previously reported enhancer of Nanog [37]. Enhancer loci are shaded in purple and signals represent ChIP-seq RPM. f Immunofluorescence staining for NANOG (red), OCT4 (green), DAPI (blue), and merged images in wild type, Cbx7-knockout, and Cbx7-knockdown mESCs. g The fluorescence intensity of NANOG for about 300 nuclei in wild type, Cbx7-knockout, and Cbx7-knockdown mESCs. Unpaired t test with Welch’s correction was used to calculate statistical significance for comparison

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