Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Catalytically inactive Ring1B maintains near wildtype levels of gene expression in mESCs

ABSTRACT: This experiment was designed to determine the extent of gene misregulation in mESCs containing catalytically dead Ring1B in comparison to mESCs lacking Ring1B. Polyadenylated mRNA was prepared from wildtype mESCs (WT), mESCs deficient for Ring1B (KO) and mESCs cells containing catalytically dead Ring1B (I53A). Each sample is represented by 3 biological replicate hybridisations.

ORGANISM(S): Mus musculus

SUBMITTER: Robert Illingworth

PROVIDER: E-GEOD-69824 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development.

Illingworth Robert S RS Moffat Michael M Mann Abigail R AR Read David D Hunter Chris J CJ Pradeepa Madapura M MM Adams Ian R IR Bickmore Wendy A WA

Genes & development 20150901 18

Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other' ...[more]

PMID: 26385961

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Project description:Serum-to-2i interconversion of mouse Embryonic Stem Cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary but not sufficient to establish these interactions, as confirmed by Capture Hi-C on Eed-/- serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation. To study dynamics in chromatin architecture and to characterize long-range interaction, we performed Hi-C using DpnII as the restriction enzyme, potentially reaching a genome-wide coverage at a less than 1Kb resolution. We subsequently performed enrichment of interaction by a target capture similar to the exome sequencing approach. We enriched for DNaseI hyper-sensitive sites (DHSâs) in chromatin from mESCs. Probes were designed against the union of all DHSâs of Serum and 2i mESCs. Capture Hi-C reveals Extremely Long-Range Interactions (ELRI) in Serum but not in 2i ESCs. We observed H3K27me3 as a prominent characteristic, but not exclusive feature of ELRI loci in Serum mESCs. To further elucidate the involvement of constituents of PRC1 and PRC2 in ELRI, we performed ChIP-seq experiment on Suz12 and Ring1B during serum-to-2i transition. In addition, RNA-seq was performed to compare the expression levels of genes.

Dataset Information

Catalytically inactive Ring1B maintains near wildtype levels of gene expression in mESCs

Publications

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development.

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