Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Polycomb-like 2 associates with PRC2 and is required for mouse embryonic stem cell

ABSTRACT: Polycomb group (PcG) proteins are highly conserved epigenetic transcriptional repressors important for the control of numerous developmental gene expression programs and have recently been implicated in the modulation of embryonic stem cell (ESC) identity. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for novel regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a novel Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation and altered patterns of histone methylation. Through integration of global gene expression and promoter occupancy analyses of both PCL2 and PRC2 components EZH2 and SUZ12, we have predicted PCL2 target genes and formulated regulatory networks describing the role of PCL2 both in modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation. Cells were stably expressing Pcl2 shRNA or shRNA mismatch control sequences. Hybridizations of three biological replicates for both the control and Pcl2 shRNA clone were performed.

ORGANISM(S): Mus musculus

SUBMITTER: Gerard Cagney

PROVIDER: E-GEOD-16364 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Polycomb-like 2 associates with PRC2 and regulates transcriptional networks during mouse embryonic stem cell self-renewal and differentiation.

Walker Emily E Chang Wing Y WY Hunkapiller Julie J Cagney Gerard G Garcha Kamal K Torchia Joseph J Krogan Nevan J NJ Reiter Jeremy F JF Stanford William L WL

Cell stem cell 20100201 2

Polycomb group (PcG) proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs and have recently been implicated in modulating embryonic stem cell (ESC) fate. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC-fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 ...[more]

PMID: 20144788

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Project description:The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention in recent years, but reports have so far been confined to the polyadenylated fraction of RNA, while the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family, which is mainly comprised of fundamental components of the RNA translational machinery, the RNA splicing machinery, and RNA-RNA or RNA-DNA interacting complexes, has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in murine ESCs and ESC-derived Neural Progenitor Cells (NPCs) using a combination of whole genome tiling arrays and Next Generation Sequencing technologies. This strategy allowed for the transcriptional characterization of already well-defined NPA RNA subclasses in this unique biological context as well as the identification of many new members of these functional ncRNA classes. In addition, we describe a group of novel, conserved RNAs (snacRNAs: small, non-polyadenylated conserved), some of which are differentially expressed between ESC and NPCs, providing the first evidence of a novel group of potentially functional ncRNAs involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal snRNAs, which are non-polyadenylated, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that non-polyadenylated RNA, both known and novel, play important roles in embryonic stem cells. We extracted total RNA from undifferentiated ESCs and 7 day NPCs. Total RNA was divided into poly(A+) and poly(A-) enriched fractions using a modified version of Oligotex extraction (Qiagen). Briefly, after the hybridization of the total RNA to oligo(dT) beads, supernatants were saved rather than discarded and then subjected to isopropanol-ethanol extraction to obtain the Poly(A-) fraction. This submission represents RNA-Seq component of study.

Dataset Information

Polycomb-like 2 associates with PRC2 and is required for mouse embryonic stem cell

Publications

Polycomb-like 2 associates with PRC2 and regulates transcriptional networks during mouse embryonic stem cell self-renewal and differentiation.

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