Figure 7.

LSH is required for recruitment of G9a to promoters of pluripotency genes during differentiation. (A) Western blots display G9a, LSH, HDAC1, and DNMT3B protein levels in shScr and shLsh ES cells during differentiation to embryoid bodies (EB) at days 2, 4, and 6. (B) Fold down-regulation of pluripotency markers mRNA as detected by qRT-PCR in wild-type and mutant EBs at day 8 relative to ES cells. (C) mRNA expression ratios relative to GAPDH of pluripotency genes in shScr versus shLsh ES cells and EBs at day 8 of differentiation indicate that these genes have equal expression in shScr and shLsh ES cells, but higher expression in shLsh EB8 relative to shScr EB8. (D) mRNA expression ratios relative to GAPDH of pluripotency genes in wild-type versus Ehmt2^−/− (G9a-null) ES cells and EB at day 8. (E) ChIP for acetylated H3, H3K9me2, and G9a at promoters of pluripotency genes in shScr and shLsh EBs at day 8 of differentiation. Ndf is a control promoter of constitutively expressed Ndufa1 gene. (F) ChIP for acetylated H3, H3K9me2, and G9a at promoters of pluripotency genes in wild-type and Ehmt2^−/− EBs at day 8 of differentiation. In E and F, the scale on the left applies to H3ac and H3K9me2; the scale on the right applies to G9a. (G,H) ChIP enrichment profiles of acetylated H3, H3K9me2, LSH, and G9a at the promoter of the Dppa4 gene during differentiation of shScr and shLsh ES cells. The scale on the left applies to H3ac and H3K9me2; the scale on the right applies to LSH, G9a, and the nonspecific IgG.