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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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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

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

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

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

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

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

Genome-wide binding of condensin II and TFIIIC upon respective knockdown of a TFIIIC and condensin II subunit

Project description:Condensin complexes are highly conserved for chromosome compaction to ensure their faithful segregation in mitosis. However, little is known about the role of condensin complexes in interphase. Condensins exists in two complexes, condensins I and II, in higher eukayotic cells. During interphase, condensin II is predominantly localized in the nucleus throughout the cell cycle, whereas condensin I is localized at the cytoplasm in interphase. The distinct localization patterns suggest that condensin II, but not condensin I, may contribute to genome organization in interphase. Our results suggest that condensin II is associated with TFIIIC complex in vivo. The aim of these experiments is to unravel the dependency of each other on binding to chromatin.

2017-06-01 | GSE80034 | GEO

Transcription-associated Loading and Translocation of Condensin II on Chromosome Arms in Embryonic Stem Cells (flavopiridol ChIP-Seq)

Project description:Condensin molecules are loaded onto the genome to mediate essential changes in chromosome condensation during mitosis, but it is not clear why there are two forms of vertebrate condensin that become differentially distributed on chromosomes. We report here that condensin II, the form of condensin present in the nucleus throughout the cell cycle, functions specifically at active genes. Condensin II is loaded at transcriptionally active promoters in embryonic stem cells (ESCs), migrates through these genes in a transcription-dependent fashion and accumulates in transcription termination regions. Unlike cohesin, which is also loaded at active promoters, condensin II has little influence on transcription. We conclude that condensin II is loaded and distributed across actively transcribed chromatin and thus serves to specifically condense this euchromatic portion of chromosomes during the cell division cycle. ChIP-Seq data for Condensin II and Cohesin in v6.5 ESCs treated or not with the RNA polymerase II elongation inhibitor flavopiridol.

2012-02-09 | E-GEOD-30919 | biostudies-arrayexpress

RNA Pol II transcription antagonises mitotic chromosome assembly and segregation by condensin

Project description:Condensin drives mitotic chromosome assembly by folding chromatin into loops and is enriched in the vicinity of highly expressed genes, but the significance of such proximity with respect to condensin activity has remained unclear. Here, by modulating the occupancy of RNA Pol II in vivo, we show that transcription plays no role in the steady state association of condensin with DNA. Rather, transcription stalls and even displaces condensin, hindering its ability to fold chromatin and to support chromosome segregation. Our results highlight a key aspect of the integrated functioning of condensin and suggest that a tight control of transcription underlies mitotic chromosome assembly.

2024-02-03 | GSE236395 | GEO

Condensin II complex mediates interactions of histone gene loci

Project description:Condensin complexes are known for their importance in chromosome condensation during mitosis. However, condensin II binds to the geome during interphase as well. The role of condensin II in genome organization in mammalian interphase nuclei is not characterized. Since condensin II binds to the promoters of the transcriptionally active promoters in interphase genome, the aim of this study is to uncover its role in mediating chromatin interactions between active gene promoters such as histone gene loci.

2017-06-01 | GSE80219 | GEO

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