Dataset Information

ChromID reveals the proteome composition of key chromatin states in murine stem cells

ABSTRACT: Chromatin modifications instruct genome function through spatiotemporal recruitment of regulatory factors to the genome. However, how these modifications define the proteome composition at distinct chromatin states remains to be fully characterized. Here, we made use of natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for histone and DNA modifications. By stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localisation, genomicdistribution and histone-PTM-binding preference, we first demonstrate their applicability as selective chromatin binders in living cells. Finally, we exploit the binding specificity of eCRs to establish ChromID, a new method for chromatin-dependent proteome identification based on proximity biotinylation. We use ChromID to reveal the proteome at distinct chromatin states in mouse stem cells, and by using a synthetic dual-modification reader, we furthermore uncover the protein composition at bivalent promoters marked by H3K4me3 and H3K27me3. These results highlight the applicability of ChromID as novel method to obtaina detailed view of the protein interaction network determined by the chemical language on chromatin.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Stem Cell, Cell Culture

SUBMITTER: Tuncay Baubec

LAB HEAD: Tuncay Baubec

PROVIDER: PXD014483 | Pride | 2020-04-30

REPOSITORIES: Pride

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Publications

ChromID identifies the protein interactome at chromatin marks.

Villaseñor Rodrigo R Pfaendler Ramon R Ambrosi Christina C Butz Stefan S Giuliani Sara S Bryan Elana E Sheahan Thomas W TW Gable Annika L AL Schmolka Nina N Manzo Massimiliano M Wirz Joël J Feller Christian C von Mering Christian C Aebersold Ruedi R Voigt Philipp P Baubec Tuncay T

Nature biotechnology 20200302 6

Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably exp ...[more]

PMID: 32123383

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Project description:We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from âAAAâ to âDDDâ) to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody. We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBA (www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label âChIP-seq gradeâ. The NGS-QC team has setup a certification procedure for Antibodies dedicated to ChIP-seq applications based on (1)a highly reproducible pipeline for performing ChIP-seq assays including the use of an automated liquid handling platform (TECAN EVO75) for ChIP and sequencing library preparation steps and (2) the use of the NGS-QC Generator tool for providing a ChIP-seq quality grade and to evaluate other parameters like the Optimal sequencing depth, the degree of reproducibility among biological replicates and the comparison of the quality grades of the certified antibody with datasets retrieved in the public domain. This quality certification structure makes possible to assign a measured Quality Stamp to ChIP-seq grade Antibodies.

Dataset Information

ChromID reveals the proteome composition of key chromatin states in murine stem cells

Publications

ChromID identifies the protein interactome at chromatin marks.

Similar Datasets

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