Polycomb Protein EED is Required for Silencing of Pluripotency Genes upon ESC Differentiation
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Eed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation.
KeywordsPluripotency Stem cells ES cell differentiation Eed Polycomb repressive Complex (PRC) 2 Oct4 Silencing
We thank Dr. A. Wutz for providing Eed KO ESCs, Heike Wecklein for excellent technical assistance, Jessica Schmitt for helping with pictures, Kristian Helin’s lab for the pCMVHA EED wt plasmid (Addgene). This work was supported by the DFG-funded GRK 1048 (AM) and SPP1356: Pluripotency and cellular reprogramming (AM, MZ). This work was supported in part by the Graduate School of Life Sciences, University of Würzburg.
NO, VH performed experiments; NO, MB, AM designed experiments, analyzed data, wrote paper; NO, QL, MZ and PC performed microarray and bioinformatic analyses.
The microarray data sets are available from the GEO (Gene Expression Omnibus) website under accession number GSE49305.
Conflict of interest
The authors declare no potential conflicts of interest.
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