Fig. 8

The reduction of H3K9me2/3 after Hira or Ubn2 depletion. a Heatmap of RNA-Seq expression Z-scores computed for genes that are differentially expressed between WT ESCs and H3.3 knockout ESCs. The left side shows the genes regulated by Hira and Ubn2 respectively. The upregulated and downregulated genes are represented with red and blue colors, respectively. Each column corresponds to a sample and each row corresponds to a specific gene. b ChIP-seq enrichment of H3.3 around the center of IAPLTR2b or RLTR10D locus in WT ESCs (blue) and Hira^−/− ESCs (green). The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. The published ChIP-seq data is from GEO: GSE117034. c ChIP-seq enrichment of H3.3 around the center of MMERGLN_LTR or MT2 locus in WT ESCs (blue) and Ubn2^−/− ESCs (red). The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. The data of ChIP-seq are available at GEO: GSE117034. d Western blot analysis of H3, β-Tubulin, H3K9me2 and H3K9me3 protein levels in ESCs transfected with shRNAs against target genes (Hira, Ubn2, and Ubn1) or control shRNA respectively. β-Tubulin was included as a loading control. e The relative protein level of H3K9me2 and H3K9me3 was obtained according to western blot band in (d). Gray scanning analysis was normalized to that of β-Tubulin. Data are represented as mean ± s.e.m. (n = 3 independent experiments). ns: non-significant, *p < 0.05; ***p < 0.001 in Student’s t test. f Schematic of Hira and Ubn2 function in the repression of retrotransposons. In WT ESCs, Hira and Ubn2 mediate the placement of H3.3 and H3K9 methylation, and thus repress the expression of retrotransposons. In the absence of Ubn2 or Hira, retrotransposon-associated H3.3 and H3K9me2/3 reduce, and thus activate the expression of retrotransposons