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A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

ABSTRACT: Condensin complexes compact and disentangle chromosomes in preparation for cell division. Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI/SNF family of chromatin remodelling complexes in an NCAPH2-independent manner. This cross-reactivity with an abundant chromatin-associated factor is likely to affect the interpretation of protein and chromatin immunoprecipitation experiments that make use of this antibody probe.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Jimi Wills

LAB HEAD: Andrew J Wood

PROVIDER: PXD022664 | Pride | 2020-12-14

REPOSITORIES: Pride

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			Action	DRS
	02_104WT-E14-IgG-03.raw	Raw
	03_105WT-E14-IgG-04.raw	Raw
	04_106WT-E14-H2-02.raw	Raw
	05_107WT-E14-H2-03.raw	Raw
	05_E14-IgG.raw	Raw

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Similar Datasets

A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI-SNF complex component

Project description:Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI-SNF family of chromatin remodelling complexes in an NCAPH2-independent manner.

2020-12-14 | PXD022620 | Pride

Condensin association with chromosomes depends on a DNA binding domain formed by its HEAT-repeat subunits

Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensinM-bM-^@M-^Ys HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain. Analysis of condensin binding genomewide in a wild type and a condensin mutant

2014-05-16 | E-GEOD-55948 | biostudies-arrayexpress

Condensin association with chromosomes depends on a DNA binding domain formed by its HEAT-repeat subunits

Project description:Condensin protein complexes play central roles in the three-dimensional organization of chromosomes during mitotic and meiotic cell divisions. How condensin interacts with its chromatin substrates to promote sister chromatid decatenation and segregation is largely unknown. Previous work suggested that condensin, in addition to encircling chromatin fibers topologically within the large ring-shaped structure formed by its structural maintenance of chromosomes (SMC) and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA helices formed by condensin’s HEAT-repeat subunits. Using detailed mapping data of the interfaces between the HEAT-repeat and the kleisin subunits, we generated mutant complexes that lack the Ycg1/CAP-G HEAT-repeat subunit. These tetrameric condensin complexes fail to associate stably with chromosomes in yeast and human cells. We suggest that condensin controls chromosome architecture by stabilizing chromatin loops of chromatin fibers through interaction with its unconventional HEAT-repeat DNA binding domain.

2014-05-16 | GSE55948 | GEO

Condensin II inactivation in interphase does not affect chromatin folding or gene expression

Project description:Condensin complexes have been proposed to play a prominent role in interphase chromatin organization and control of gene expression. Here, we report that the deletion of the central condensin II kleisin subunit Ncaph2 in differentiated mouse hepatocytes does not lead to significant changes in chromosome organization or in gene expression. Both observations challenge current views that implicate condensin in interphase chromosomal domain formation and in enhancer-promoter interactions. Instead, we suggest that the previously reported effects of condensin perturbation may result from their structural role during mitosis, which might indirectly impact the re-establishment of interphase chromosomal architecture after cell division.

2019-05-05 | GSE122157 | GEO

Interaction mapping of human condensin I and condensin II complexes using DSBU-cross-linking and mass spectrometry

Project description:Human condensin I and condensin II complexes play essential roles in chromatin condensation during the cell division process. Both complexes use ATP as the energy source to pack extended chromatin into the short form of chromosome. In order to understand the mechanism of their function, we performed cross-linking mass spectrometry (XL-MS) analyses of both complexes and mapped the inter-molecular and intra-molecular interactions of the subunits.

2020-05-25 | PXD017531 | Pride

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

Project description:The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesin’s loop extrusion during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells derived from patients affected by microcephaly caused by mutations in the MCPH1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin II’s NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1’s ability to block condensin II’s association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both cohesin and condensin must be tightly regulated to adjust the structure of chromatids for their successful segregation.

2021-11-19 | GSE188988 | GEO

3D topology of the SMC2/SMC4 sub-complex from chicken condensin I revealed by cross-linking and molecular modelling

Project description:SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organisation. SMC-containing complexes are large (>650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here we have used amino acid-selective cross-linking and mass spectrometry (CLMS) combined with structure prediction to develop a full-length molecular draft 3D-structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularity. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.

2015-07-13 | PXD001835 | Pride

Fission yeast condensin contributes to interphase chromatin organization and prevents transcription-coupled DNA damage

Project description:Background: Structural maintenance of chromosomes (SMC) complexes are central organizers of chromatin architecture throughout the cell cycle. The SMC family member condensin is best known for establishing long-range chromatin interactions in mitosis. These compact chromatin and create mechanically stable chromosomes. How condensin contributes to chromatin organization in interphase is less well understood. Results: Here, we use efficient conditional depletion of fission yeast condensin to determine its contribution to interphase chromatin organization. We deplete condensin in G2 arrested cells to preempt confounding effects from cell cycle progression without condensin. Genome-wide chromatin interaction mapping, using Hi-C, reveals condensin-mediated chromatin interactions in interphase that are qualitatively similar to those observed in mitosis, but quantitatively far less prevalent. Despite its low abundance, chromatin mobility tracking shows that condensin markedly confines interphase chromatin movements. Without condensin, chromatin behaves as an unconstrained Rouse polymer with excluded volume, while condensin constrains its mobility. Unexpectedly, we find that condensin is required during interphase to prevent ongoing transcription from eliciting a DNA damage response. Conclusions: In addition to establishing mitotic chromosome architecture, condensin-mediated long-range chromatin interactions contribute to shaping chromatin organization in interphase. The resulting structure confines chromatin mobility and protects the genome from transcription-induced DNA damage. This adds to the important roles of condensin in maintaining chromosome stability.

2020-09-14 | GSE143338 | GEO

MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

Project description:MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface

| PRJNA780949 | ENA

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation

Project description:The experiment was performed to assess the importance of nucleosome eviction for the binding of condensin to chromatin during mitosis. The condensin complex associates with chromatin during mitosis to ensure chromosome condensation and accurate segregation, but how this binding is achieved remains poorly understood. Our study indicates that transcription co-activators Gcn5 and Mst2 assist condensin binding during mitosis by evicting nucleosomes. To reach this conclusion, we mapped nucleosomes, during mitosis, in wild-type and gcn5mst2 mutant strains. This experiment allowed us (1) to identify nucleosome-depleted regions (ndrs) during mitosis and to show that condensin tends to colocalize with ndr at the 3ends of genes, and (2) to confirm the importance of nucleosome eviction from ndrs by gcn5 and mst2, during mitosis, for the binding of condensin to chromatin.this experiment determines the patterns of nucleosomes in wild type and mutant fission yeast cells arrested in early mitosis. We analysed wild-type cells, cells lacking Gcn5 histone acetylatransferase (gcn5D) and cells lacking both Gcn5 and Mst2 histone acetyltransferases (Gcn5Dmst2D). All cells used in this study expressed a GFP-tagged version of condensin (Cnd2-GFP) and were blocked in early mitosis at 19C by the nda3-KM311 mutation. Mitotic indexes were determined by scoring the accumulation of Cnd2-GFP in the nucleus. Mitotic cells were collected and chromatin was digested by increasing amount of MNase to produce mononucleosomes. Mononucleosomal DNA was purified on an agarose gel and sequence on an Illumina Nextseq 500 apparatus. Three replicates of wild type, gcn5D and gcn5Dmst2D were analysed. Nucleosome patterns were determined in wild-type cells, cells lacking Gcn5 and cells lacking both Gcn5 and Mst2.

2016-04-18 | E-MTAB-4620 | biostudies-arrayexpress

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