Polycomb Repressive Complex 2 Regulates Lineage Fidelity during Embryonic Stem Cell Differentiation
(A) At top, a diagram of the structure of the wild-type (wt) Suz12 gene. Below, the proteins encoded by the two mutant alleles used here (SUZ12GT and SUZ12Δ) and the wt allele are shown to scale, and domains of interest are noted on wt SUZ12. (B) X-gal staining was performed on wt ESCs (upper left) and Suz12GT ESCs (upper right) expressing either a scrambled control hairpin, a hairpin targeted to LacZ (encoding β-galactosidase) (lower left), or a hairpin targeted to the 5′ end of Suz12 (lower right). (C) Immunoprecipitation of EED was performed in wt, Suz12GT, Suz12Δ, and Eednull ESCs. The samples, including 3% input, were subjected to SDS-PAGE. EZH2 immunoblot was performed as indicated by the labeled band (left). EZH2 degradation product is marked by an asterisk (*). (D) ChIP-qPCR for H3K27me3 was performed on wt and Suz12GT ESCs expressing hairpins: scr (scrambled control), Ezh2-kd (targeted to Ezh2), and Ezh1-kd (targeted to Ezh1). All genes tested except Oct4 are PRC2 target genes. Error bars show standard deviation of three technical replicates. In (E) and (F), ChIP-seq for H3K27me3 was performed on wt, Suz12GT, Suz12Δ, and Eednull ESCs. ChIP-seq datasets are normalized to the total mapped reads. (E) A metagene analysis of H3K27me3 ChIP-seq enrichment is shown across the average of all PRC2 target genes +/− 2 kb relative to the TSS for wt, Suz12GT, Suz12Δ, and Eednull ESCs, as well as input. (F) H3K27me3 ChIP-seq tracks in ESCs. Representative examples of PRC2 target promoters (Gata6 and Bmp2) showing H3K27me3 levels in Suz12GT, Suz12Δ, and Eednull ESCs.