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Comprehensive cross-linking mass spectrometry reveals molecular insights into the between peripheral and core NuRD complex subunits

ABSTRACT: Affinity purifications of CHD4 and CDK2AP2 to identify their interactors. The C-terminally tagged CHD4 has a C-terminal truncation to test whether this domain is required for the interaction with other NuRD subunits. GFP-purifications of both N- and C-terminally tagged CDK2AP2 were performed.

INSTRUMENT(S): LTQ FT, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Cornelia Spruijt

LAB HEAD: Michiel Vermeulen

PROVIDER: PXD017378 | Pride | 2020-12-07

REPOSITORIES: Pride

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Comprehensive cross-linking mass spectrometry reveals molecular insights into the between peripheral and core NuRD complex subunits

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CTCF loss has limited effects on genome architecture in Drosophila despite critical regulatory functions.

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Small RNA loading licenses Argonaute for assembly into a transcriptional silencing complex

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H4K20me2 SILAC Nucleosome Affinity Purification

Project description:Goal of this project was the identification of chromatin interacting proteins whose binding is differentially regulated by di-methylation of lysine 20 on histone H4 (H4K20me2). To achieve this unodified and H4K20me2-modified histone H4 were generated by native chemical ligation and assembled into recombinant di-nucleosomes. The di-nucleosomes were immobilized on streptavidin-coated beads via the biotinylated di-nucleosomal DNA and used for nucleosome affinity purifications to identify proteins regulated by H4K20me2 from SILAC-labelled HeLaS3 nuclear extracts (Arg10 and Lys8). SILAC affinity purifications were carried out in "forward" (heavy extract on modified nucleosome and light extract on unmodified nucleosome) and "reverse" (light extract on modified nucleosome and heavy extract on unmodified nucleosome) label-swap experiments and protein abundances were quantified by MaxQuant.

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Determining DNA-protein binding affinities and specificities from crude lysates using a combined SILAC/TMT labeling strategy

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