(A) Proteins interacting with bfEZH2, bfEED or bfEZH1 were pulled down by FLAG and biotin-streptavidin (SA) mediated tandem coIP and subsequently identified by Mass Spec (MS) sequencing. Y-axis shows relative ratio of peptides representing an identified protein (x–axis) in total number of peptides identified by MS. Error bars represent standard deviations of ratios of peptides identified by MS in independent pull-down experiments. Neither bfEZH2 nor bfEZH1 pulls down each other (indicated by asterisks).
(B) One-step SA coIP and Western blotting of J1 ES cells expressing birA alone or with any of bfEZH2, bfEZH1 and bfEED. “I” represents inputs and “P” represents coIP’ed fractions.
(C) bfEZH1 is coIP’ed with endogenous EZH2.
(D) SA coIP and Western blot analysis of Ezh2^−/− ES cells expressing bfEZH1, bfEED or bfEZH2.
(E) Colocalization of bfEZH1 with bfEZH2 and bfEED at promoters of PcG targets shown by bioChIP-qPCR analysis in J1 ES cells.
(F–G) The binding of bfEZH1 (F) and bfEED (G) to PcG target loci in Ezh2^−/− ES cells.

(A–B) GSEA profiles of three subsets of H3K27me3 targets by comparing undifferentiated Eed^−/−(A) or Ezh2^−/− ES cells (B) to wild-type (WT) ES cells. Panel I, WT^H3K27me3 – day 6 up genes; Panel II, Ezh2^{−/−, H3K27me3+} – day 6 up genes; Panel III, Ezh2^{−/−, H3K27me3−} – day 6 up genes. All three subsets show positive normalized enrichment scores (NES) in Eed^−/− ES cells but negative NES in Ezh2^−/− ES cells, suggesting derepression in Eed^−/− ES cells but repression in Ezh2^−/− ES cells. (C) Ezh2 transcript is reduced ~ 1000 fold in Ezh2^−/− cells compared to that in WT and Eed^−/− cells. (D) Fold changes of gene expression in mutants versus wild-type cells.

(A) Heat map showing impaired activation of known mesodermal markers in Ezh2^−/− and Eed^−/−cells as compared to wild-type (WT) cells.
(B) RT-qPCR analysis of T expression along ES cell differentiation by LIF withdrawal.
(C–D) GSEA profiles of ME-high (panel I) and ES-high (panel II) genes by comparing day-6 differentiated Eed^−/− (C) or Ezh2^−/− (D) cells to wild-type cells.
(E) Heat map of ME-high genes in wild-type and mutant ES cells.
(F) RT-qPCR analysis of pluripotency markers along ES cell differentiation.

(A) Targeting strategy to generate Ezh2 conditional and knockout alleles. Exon number is indicated above each exon shown in boxes. Lines represent introns.
(B–C) Western blot analysis of nuclear extracts (B) and histones (C) isolated from wild-type (J1 and CJ7) and mutant (Ezh2^−/− and Eed^−/−) ES cells.
(D) ChIP-qPCR analysis of H3K27me3, EZH2 and SUZ12 binding at promoters of PcG target genes in wild-type (WT), Ezh2^−/− and Eed^−/− ES cells. The y-axis shows relative enrichment of ChIP’ed DNA versus the inputs. Actb promoter and an intergenetic region (Cs) serve as negative controls.

(A) The overlapping percentages of target genes identified by H3K27me3 and bfEZH1 ChIP-chip in wild-type (WT) and Ezh2^−/− mutant (Mut) ES cells. The total numbers of target genes (p < 1e-7) is highlighted in grey.
(B) Representative view of bfEZH1 and H3K27me3 occupancy at various loci in WT (shown in blue) and Ezh2^−/− ES cells (in purple) by Affymetrix Integrated Genome Browser. Arrows indicate transcription starts and directions. Yellow bars indicate regions retaining strong binding in Ezh2^−/− cells. Asterisks in red indicate regions losing ChIP peaks in Ezh2^−/− cells. qPCR primers used in Panel E and F were designed in the above marked regions.
(C–D) Comparisons of peak positions and widths of H3K27me3 (C) or bfEZH1 (D) ChIP-chip in Ezh2^−/− and wild-type ES cells. Panel I shows deviations of mid-points of corresponding ChIP peaks in Ezh2^−/− and wild-type cells. The distribution is centered at 0 and the deviations, by large part, are within 1 kb on both sides. Panel II shows the distribution of width differences of corresponding ChIP peaks in Ezh2^−/− and wild-type cells. The distribution is heavily skewed to the left, indicating less broad peaks in Ezh2^−/− cells than in wild-type cells in general.
(E–F) qPCR verification of H3K27me3 ChIP (E) in WT and Ezh2^−/− ES cells without exogenous bfEzh1 and bfEZH1 bioChIP (F) in cells expressing bfEzh1.
(G) Gene Ontology (GO) analysis of H3K27me3 target genes. GO terms with an enrichment score larger than 2 (i.e. one-fold above genome background) and a p-value less than 1e-6 are considered significantly enriched and indicated by asterisks. The WT^H3K27me3 gene set comprises of all H3K27me3 target genes. Ezh2^{−/−, H3K27me3+} or Ezh2^{−/−, H3K27me3−} genes represent a subset of H3K27me3 target genes which retain or lose this mark in Ezh2^−/− cells, respectively.

(A–C) Ezh1 RNAi in Ezh2^−/− ES cells. (A) RT-qPCR analysis of Ezh1 and marker genes. Relative expression was normalized to that in untreated Ezh2^−/− ES cells. (B) Histone analysis. (C) Reduced H3K27me3 binding at PRC2 targets in Ezh1 RNAi cells shown by ChIP-qPCR.
(D–E) HMTase assays. (D) FLAG-tagged EZH1, EZH2 or EZH2ΔSET was co-expressed with EED, SUZ12, RBBP4 and AEBP2 in Sf21 cells. (E) FLAG-tagged EZH1 or EZH2 was co-expressed with indicated PRC2 components in Sf21 cells. Protein complexes were pulled down by anti-FLAG agarose and assayed for HMTase activity. The upper panels show the autoradiography and the lower panels show equal addition of core histone substrates.

(A) A model in wild-type (WT) ES cells. Both EZH2 and EZH1 bind to EED and SUZ12, forming alternative but interacting PRC2 complexes containing HMTase activity. EZH2-mediated PRC2 is more abundant in ES cells and plays a major role in forming H3K27me3, which is required for ES cell identity and proper differentiation. Both EZH1 and EZH2 containing PRC2 complexes may mediate monomethylation on H3K27 in a non-targeted manner by either acting transiently on nucleosomes across the genome or on free H3 histones prior to nucleosomal assembly during DNA replication.
(B) In Ezh2^−/− ES cells, EZH1-PRC2 mediates monomethylation and residual trimethylation on H3K27. EED may regulate EZH1-PRC2 complex in an EZH2-dependant manner. EZH1 mediates H3K27me3 on a set of developmental genes and prevents derepression of PRC2 target genes from globe loss of the H3K27me3 mark in Ezh2^−/− ES cells. Hereby, EZH1 complements EZH2 in the maintenance of ES cell identity. However, EZH1-PRC2 cannot fully rescue defects in mesoendodermal development during Ezh2^−/− cell differentiation, suggesting non-redundant functions of these two PRC2 complexes in lineage commitment.
(C) Eed^−/− ES cells lack all levels of methylation on H3K27, indicating the essential role of EED in EZH1 or EZH2 mediated PRC2 complexes. Complete loss of H3K27me3 in the absence of Eed confounds ES cell identity by de-repressing a set of differentiation-regulated PRC2 target genes, and blocks the proper execution of pluripotent programs.