Stem Cell Reviews and Reports

, Volume 11, Issue 1, pp 50–61 | Cite as

Polycomb Protein EED is Required for Silencing of Pluripotency Genes upon ESC Differentiation

  • Nadine Obier
  • Qiong Lin
  • Pierre Cauchy
  • Vroni Hornich
  • Martin Zenke
  • Matthias Becker
  • Albrecht M. Müller
Article

Abstract

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.

Keywords

Pluripotency Stem cells ES cell differentiation Eed Polycomb repressive Complex (PRC) 2 Oct4 Silencing 

Supplementary material

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ESM 1(XLSX 60 kb)
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Fig. S1

Analysis of EZH2 and H3K27me3 levels and apoptosis in Eed KO ESCs. (A) Representative Western blot analysis for expression of EZH2 and H3K27me3. (B) One million WT and Eed KO ESCs were seeded in ESC culture conditions and counted after 2 days. n=3 (C) Frequencies of apoptotic cells in ESC cultures and in day 3 and day 7 EBs. n=3 (D) Frequency of EB forming cells in Eed KO and WT ESC cultures. Single cell suspensions were prepared and graded numbers of cells were seeded into hanging drop cultures. Drops that developed no EBs 2 weeks post seeding were scored as negative and fractions of negative drops were plotted against seeded cell numbers. n = 3. Regression curves were calculated and the function was used to calculate the frequency of EB forming cells. (JPEG 3186 kb)

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Fig. S2

Validation of microarray expression data via qRT PCR analysis. (A) Shown are qRT PCR results for Oct4, Sox2, Nanog, Eed, Ezh2 and Ehmt2 in WT and Eed KO ESCs or d3 and d7 EB cells. n=3 (B) Expression values of the same genes as in (A) as obtained from microarray analysis. (JPEG 4217 kb)

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Fig. S3

Overexpression of a WT Eed transgene in Eed KO ESCs partially rescues the KO phenotype. (A) Western blot analysis for expression of EED and H3K27me3. Eed KO ESCs (J1) were transiently transfected with a CMV-driven Eed expression plasmid. Protein extracts from mock-, empty vector- or Eed vector-transfected cells were harvested and analyzed 1 and 3 days post transfection. Wildtype ESCs (R1) or MEF are shown. (B) qRT PCR for expression of core pluripotency genes at 1, 3, or 7 days of EB differentiation. (C) Representative micrographs of mock-transfected WT ESCs (J1), of empty vector-transfected or wildtype EED vector transfected Eed KO ESCs (J1) at day 3 and day 7 of differentiation are shown. A-C: n=2. (JPEG 8278 kb)

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Fig. S4

Knockdown of Oct4 in Eed KO ESCs does not rescue the KO phenotype. (A) qRT PCR analysis of Oct4, Nanog and Sox2 expression in WT (J1), scr siRNA- or Oct4 siRNA-transfected Eed KO ESCs (J1). ESCs were differentiated for 1, 3 or 7 days. RNAs were harvested and qRT PCRs were performed. (B) Representative micrographs of mock-transfected WT, scr or Oct4 siRNA transfected Eed KO ESCs at day 3 and day 7 of differentiation are shown. A, B: n=4. (JPEG 8038 kb)

12015_2014_9550_Fig9_ESM.jpg (4.6 mb)
Fig. S5

Correlation of H3K27me3 and gene expression changes and box-whiskers-plots of wildtype and EZH2 KO ESC expression changes. (A) Shown are global H3K27me3 intensity plots at promoter regions (+/− 1,5 kb TSS) of all genes ranked on highest to lowest H3K27me3 values (left). Aligned are gene expression differences between day 7 differentiated and undifferentiated WT (middle) and KO ESCs (right). Color codes are indicated at bottom of diagrams. (B) Previously published microarray data of Ezh2 KO ESCs (d0) or d6 EB cells [10] were used to extract gene expression values of 158 PRC2-dependent genes (as defined in Fig. 4b). Expression values are shown as box plots. (JPEG 4684 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Nadine Obier
    • 1
    • 3
  • Qiong Lin
    • 2
  • Pierre Cauchy
    • 3
  • Vroni Hornich
    • 1
  • Martin Zenke
    • 2
  • Matthias Becker
    • 1
  • Albrecht M. Müller
    • 1
  1. 1.Institute for Medical Radiation and Cell Research (MSZ) in the Center of Experimental and Molecular Medicine (ZEMM)University of WürzburgWürzburgGermany
  2. 2.Department of Cell Biology, Helmholtz Institute for Biomedical EngineeringRWTH Aachen UniversityAachenGermany
  3. 3.School of Cancer Sciences, Institute of Biomedical Research, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUK

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