Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Mapping Genome Occupancy in Embryonic Stem Cells

• Growth Conditions
• Human ES Cell Quality Control
• Technology and Protocols

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• Data Analysis

PRC2 is composed of three core subunits, Suz12, Eed and Ezh2, and has been shown to mediate histone H3K27 methylation at specific genes in vivo (Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Muller et al., 2002; Cao and Zhang, 2004; Kirmizis et al., 2004). To confirm that Suz12 is associated with active PRC2 at target genes, we used chromatin immunoprecipitation with antibodies against Eed and the histone H3K27me3 mark and analyzed the results with promoter microarrays. We found that Eed and the histone H3K27me3 mark co-occurred with Suz12 at most genes (Figure 2A; Supplemental Data, Figure S6), as shown for NeuroD1 in Figure 2B.

Genetic and biochemical studies at selected genes indicate that PRC2-mediated H3K27 methylation represses gene expression, but it has not been established if it acts as a repressor genome-wide. If genes occupied by Suz12 are repressed by PRC2, then transcripts from these genes should generally be present at lower levels in ES cells than in differentiated cell types. To test this idea, we compared the expression levels of PRC2-occupied genes in four different ES cell lines with the expression level of these genes in 79 differentiated human cell and tissue types (Sato et al., 2003; Abeyta et al., 2004; Su et al., 2004). We found that PRC2 occupied genes were generally underexpressed in ES cells relative to other cell types (Figure 2C). A small fraction of the genes occupied by PRC2 were overexpressed in ES cells (Figure 2C) and these tended to show limited Suz12 occupancy (Supplemental Data). These results are consistent with the model that PRC2-mediated histone H3K27 methylation promotes gene silencing at the majority of its target genes throughout the genome in ES cells.

Suz12 is associated with Eed, histone H3K27me3 modification and transcriptional repression in ES cells - Figure 2.
Co-occupation of gene promoters by Suz12, Eed and H3K27me3 - Figure S6.

Details of the methods used for expressions analysis can be found in the Supplemental Information.