Dataset Information

Quantitative subcellular proteomics using SILAC reveals enhanced metabolic buffering in ground state pluripotency

ABSTRACT: The ground state of pluripotency is defined as a minimal unrestricted state as present in the Inner Cell Mass (ICM). Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors (‘2i’) reflect this state, whereas ESCs grown in the presence of serum (‘serum’) share more similarities with post implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chromatin-associated proteins. Therefore, quantitative information on the (sub)cellular proteome is essential to gain insight in the molecular mechanisms driving different pluripotent states. Here, we describe a full SILAC workflow and quality controls for proteomic comparison of 2i and serum ESCs. We demonstrate that this workflow is applicable for subcellular proteomics of the cytoplasm, nuclear and chromatin. The obtained quantitative information revealed increased levels of naïve pluripotency factors on the chromatin of 2i ESCs. Further, we demonstrate that these pluripotent states are supported by distinct metabolic programs, which include upregulation of free radical buffering by the glutathione pathway in 2i ESCs. Through induction of intracellular radicals, we show that the altered metabolic environment renders 2i ESCs less sensitive to oxidative stress. Altogether, this work provides novel insights into the proteome landscape underlying ground state pluripotency.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Guido van Mierlo

LAB HEAD: Hendrik Marks

PROVIDER: PXD009217 | Pride | 2018-11-07

REPOSITORIES: Pride

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Quantitative subcellular proteomics using SILAC reveals enhanced metabolic buffering in the pluripotent ground state.

van Mierlo Guido G Wester Roelof A RA Marks Hendrik H

Stem cell research 20180924

The ground state of pluripotency is defined as a minimal unrestricted epigenetic state as present in the Inner Cell Mass. Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors ("2i ESCs") have been postulated to reflect ground-state pluripotency, whereas ESCs grown in the presence of serum ("serum ESCs") share more similarities with post-implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chrom ...[more]

PMID: 30352361

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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

Quantitative subcellular proteomics using SILAC reveals enhanced metabolic buffering in ground state pluripotency

Publications

Quantitative subcellular proteomics using SILAC reveals enhanced metabolic buffering in the pluripotent ground state.

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