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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 different subunits of the NuRD complex, followed by cross-linking mass spectrometry (xIP-MS) to identify interaction domains. Six subunits and an interactor of NuRD were cross-inked with either BS3 or ADH/DMTMM and digested with either trypsin or chymotrypsin, to generate a dense cross-link network.

INSTRUMENT(S): LTQ FT, LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

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

SUBMITTER: Cornelia Spruijt

LAB HEAD: Michiel Vermeulen

PROVIDER: PXD017244 | Pride | 2020-12-07

REPOSITORIES: Pride

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			Action	DRS
	20161111_Q1_SA_NS_CDK2AP1_XL.raw	Raw
	20161111_Q1_SA_NS_MBD3_XL.raw	Raw
	20161111_Q1_SA_NS_MTA1_XL.raw	Raw
	20161111_Q1_SA_NS_ZNF687_XL.raw	Raw
	20161229_Fusion1_SA_NS_CHD4_BS3_tryp_XL.raw	Raw

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

Project description: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.

2020-12-07 | PXD017378 | Pride

Crosslinking MS to elucidate the Architecture of the 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. 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.

2021-01-08 | PXD010111 | 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

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

RNAi profiling by array of Drosophila S2 cells with RNAi knock down of NURK subunits and TTK69 transcription factor

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2010-07-01 | E-TABM-1010 | biostudies-arrayexpress

Stoichiometry of MBD2 vs. MBD3-NuRD

Project description:To obtain a comprehensive and quantitative view on MBD2 vs MBD3-NuRD complex stoichiometry, we performed biotin co-immunoprecipitations in Mbd3 KO ES cells expressing either biotin-tagged MBD2a or MBD3a and identified known NuRD complex members using label-free mass spectrometry (Supplementary Fig. 8b-c). We then calculated the intensity-based absolute quantification (iBAQ) values of the most predominant and statistically significant MBD-interacting proteins in both cell lines, which can be used to estimate the relative abundance. While we observe very similar complex composition between MBD2a-NuRD and MBD3a-NuRD, peptides shared between SALL1-4 proteins show a preferred interaction with the MBD2a-NuRD complex.

2023-06-30 | PXD042407 | Pride

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Project description:Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that the physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Å resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network that communicates with the PDE6 catalytic domains. Molecular dynamics (MD) simulations of cone GAFab revealed asymmetry in the two GAFab subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases.

2022-02-15 | PXD020817 | Pride

Transcription profiling of rat animal model of extremity melanoma after treatment with ADH-1 in combination with melphalan

Project description:To identify patterns of change in gene expression that correlated with drug treatment (saline control, ADH-1 alone, melphalan alone and ADH-1 plus melphalan) in our animal model of regional therapy for extremity melanoma we evaluated gene expression using microarray genechips and a Pearson correlation analysis. Genes were ranked according to the degree to which expression correlated with systemic ADH-1 treatment alone or in combination with regional melphalan. Tissue samples were harvested at 4, 24 and 48 hours after treatment with melphalan. Specific cellular pathways associated with drug treatment were identified using gene set enrichment analysis (GSEA). Experiment Overall Design: Sample size was 2 rats for each drug treatment group at both the 4 and 24 hour time points. At the 48 hour time point sample size was 1 rat for each drug treatment group. ADH-1 treatment preceded melphalan by 1 hour. ADH-1 treatment was repeated at 24 and 48 hrs. Tissue was harvested for RNA analysis at 4, 24 and 48 hours after completion of isolated limb infusion with melphalan (and 1 hour after an ADH-1 dose).

2008-11-07 | E-GEOD-8615 | biostudies-arrayexpress

Effect of H3.3 depletion on NuRD subunit binding and GE

Project description:Genome-wide mapping of NuRD subunits, H3.3, and histone modifications in WT and H3.3 KD 3T3 MEFs. RNAseq data from WT and H3.3 KD cells.

2018-09-03 | GSE110382 | GEO

Enhancer Decommissioning by LSD1 During Embryonic Stem Cell Differentiation (expression)

Project description:Transcription factors and chromatin modifiers play important roles in programming and reprogramming of cellular states during development. Much is known about the role of these regulators in gene activation, but relatively little is known about the critical process of enhancer silencing during differentiation. Here we show that the H3K4/K9 histone demethylase LSD1 plays an essential role in decommissioning enhancers during differentiation of embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes critical for control of ESC state. However, LSD1 is not essential for maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to fully differentiate and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At enhancers, LSD1 is a component of the NuRD complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program upon differentiation, which is essential for complete shutdown of the ESC gene expression program and the transition to new cell states. This represents the expression part of the study.

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