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Crosslinking MS to elucidate the Architecture of the Nucleosome Remodelling and Deacetylase (NuRD) complex

ABSTRACT: The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to detailed analysis because of its low abundance and resistance to recombinant production. In combination with other techniques, crosslinking mass spectrometry was used to elucidate the structure of the Nucleosome Remodelling and Deacetylase (NuRD) complex. Natively-purified NuRD, and four recombinantly-expressed NuRD subcomplexes, and a PWWP2A-MTA-HDAC-RBBP alternative ‘NuRD-like’ complex were subjected to crosslinking with DSS, ADH, BS3 and DMTMM.

INSTRUMENT(S): Q Exactive Plus, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Jason Low

LAB HEAD: Joel Mackay

PROVIDER: PXD010111 | Pride | 2021-01-08

REPOSITORIES: Pride

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The Nucleosome Remodeling and Deacetylase Complex Has an Asymmetric, Dynamic, and Modular Architecture.

Low Jason K K JKK Silva Ana P G APG Sharifi Tabar Mehdi M Torrado Mario M Webb Sarah R SR Parker Benjamin L BL Sana Maryam M Smits Callum C Schmidberger Jason W JW Brillault Lou L Jackman Matthew J MJ Williams David C DC Blobel Gerd A GA Hake Sandra B SB Shepherd Nicholas E NE Landsberg Michael J MJ Mackay Joel P JP

Cell reports 20201201 9

The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) ...[more]

PMID: 33264611

Similar Datasets

Stoichiometry of Nucleosome Remodelling and Deacetylase (NuRD) complex

Project description:The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to detailed analysis because of its low abundance and resistance to recombinant production. As part of a larger project, absolute quantification using isotopically-labelled AQUA peptides was performed on natively-purified NuRD and six recombinantly-expressed NuRD subcomplexes. This is the first time the NuRD complex has been quantified using the AQUA strategy.

2021-01-08 | PXD010110 | Pride

The Bloom’s syndrome helicase BLM functions as a homodimer within the Bloom’s complex

Project description:Homologous recombination (HR) is an essential and highly regulated cellular process. Unintended HR in somatic cells is potentially deleterious; it can result in translocations and/or somatic cell loss of heterozygosity. This is suppressed by the BLM protein in combination with topoisomerase III, RMI1 and RMI2 in a hetero-tetrameric complex (the ‘Bloom’s complex’). To understand how the activities of BLM helicase and topoisomerase III are coupled, we purified the four-subunit complex. Chemical crosslinking and mass spectrometry revealed a unique architecture that couples helicase and topoisomerase domains.

2022-01-17 | PXD011078 | Pride

Unique Protein Interaction Networks Define The Chromatin Remodeling Module of The NuRD Complex

Project description:The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1, and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodeling module of the Nucleosome Remodeling and Deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential in defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS) we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodeling activity into both the ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the Nuclear Receptor Co-Repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

2023-03-11 | PXD025263 | Pride

A variant NuRD complex containing PWWP2A/B excludes MBD2/3 and directs HDAC activity to regulate histone acetylation and RNA Pol II dynamics

Project description:Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/β, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3-containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.

2018-07-20 | GSE112113 | GEO

Nascent transcription of E14, PWWP2AKO, and PWWP2A/B DKO

2018-07-20 | GSE112112 | GEO

Chd4 ensures stem cell lineage fidelity during skeletal muscle regeneration

Project description:The chromatin remodeler Chd4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells.

2021-09-03 | GSE179683 | GEO

Comparison of the composition and interaction partners of the Nucleosome Remodeling and Deacetylase (NuRD) complex in cancer cells

Project description:The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for normal development in metazoans and has direct implications in a range of human diseases. Amongst all classes of chromatin remodeling complexes, the NuRD complex has been the most recalcitrant to analysis. This is, in part, due to difficulties with producing the intact complex recombinantly. Using affinity-purification followed by mass spectrometry, we determined the stoichiometry of the NuRD complex from three cancer cell lines representing three different human tissues. We also analysed the data to determine the interactome of the NuRD complex and identified potential NuRD interactors.

2019-03-13 | PXD012061 | Pride

PWWP2A variant NuRD complex represses intragenic spurious transcription initiation

Project description:H3 trimethylation at K36 deposited over the body of actively transcribed gene, is recognized by readers to modulate the epigenetic states, such as DNA methylation and histone deacetylation, to ensure the proper gene transcription. PWWP2A was identified as the reading component of variant NuRD complex to deacetylate gene bodies in mammalian cells. However, whether this deacetylation affects intragenic transcription is not investigated. Here, we performed CAGE-seq to profile transcription initiation in PWWP2A/B double knockout and fount that intragenic spurious transcription initiations at pre-existing low-level initiation from highly expressed genes were enhanced, accompanying with elevated acetylation level at H3K9 and H3K27, as well as nascent transcription. Additionally, the intragenic spurious transcriptions in PWWP2A/B loss are distinct with those in DNMT3B loss which is prone to produce de novo initiation and lack of PWWP2A/B-related chromatin changes. Collectively, PWWP2A/B-NuRD complex deacetylates active gene bodies to suppress intragenic spurious transcription in mammals.

2020-09-15 | GSE148382 | GEO

A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes

Project description:Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/β, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3-containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.

2018-07-19 | PXD010445 | Pride

ChIP-seq of NuRD components and pluripotency factors in wild-type, Mbd3 and Sall4/1 mutant embryonic stem cells

Project description:Members of the Nucleosome Remodeling and Deacetylase (NuRD) complex Mbd3 and Mi2beta (Chd4) along with pluripotency regulators Nanog, Oct4, Klf4 and Esrrb were profiled for chromatin association by ChIP-seq in mouse embryonic stem cells mutant for Mbd3, Sall4 and/or the related Sall1 pluripotency-associated factors.

2016-08-09 | E-MTAB-4565 | biostudies-arrayexpress

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