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RNA Seq analysis of SCC2 mutations R787G and G1242V at 3 hours following SCC2 depletion in Saccharomyces cerevisiae

ABSTRACT: The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses RNA-Seq analysis to study the effect of these mutations on gene expression.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE201191 | GEO | 2022/05/25

REPOSITORIES: GEO

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RNA Seq analysis of SCC2 mutations R787G and G1242V at 9 hours following SCC2 depletion in Saccharomyces cerevisiae

Project description:The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses RNA-Seq analysis to study the effect of these mutations on gene expression.

2022-05-25 | GSE201192 | GEO

ChIP Seq Analysis of SCC2 in the Presence or Absence of MED14 in Saccharomyces cerevisiae

Project description:The ring-like cohesin complex plays an essential role in chromosome segregation, organization, and double-strand break repair through its ability to bring two DNA double helices together. Scc2 (NIPBL in humans) together with Scc4 function as the loader of cohesin onto chromosomes. Chromatin adapters such as the RSC complex facilitate localization of the Scc2-Scc4 cohesin loader. Here we identify a broad range of Scc2- chromatin protein interactions that are evolutionarily conserved and reveal a role for one complex, Mediator, in recruitment of the cohesin loader. We identified budding yeast Med14, a subunit of the Mediator complex, as a high copy suppressor of poor growth in Scc2 mutant strains. Physical and genetic interactions between Scc2 and Mediator are functionally substantiated in direct recruitment and cohesion assays. Depletion of Med14 results in defective sister chromatid cohesion and decreased binding of Scc2 at RNA Pol II transcribed genes. Previous work has suggested that Mediator, Nipbl, and cohesin connect enhancers and promoters of active mammalian genes. Our studies suggest an evolutionarily conserved fundamental role for Mediator in direct recruitment of Scc2 to RNA pol II transcribed genes. We identified two mutations in the evolutionarily conserved HEAT domain of SCC2 that result in significantly reduced growth, scc2R787G and scc2G1242V. This experiment uses ChIP Seq to examine global localization of Scc2 in the presence or absence of MED14.

2022-05-25 | GSE201193 | GEO

Functional crosstalk between the cohesin loader and chromatin remodelers

Project description:The cohesin complex has crucial roles in many structural and functional aspects of chromosomes including sister chromatid cohesion, genome organization, gene transcription and DNA repair. Cohesin recruitment onto chromosomes requires nucleosome free DNA and a specialized cohesin loader complex comprised of the Scc2 and Scc4 subunits. The cohesin loader, in addition to stimulating cohesin ATP hydrolysis and facilitating topological loading onto DNA, leads cohesin to chromatin receptors such as the RSC chromatin remodeling complex. Here, we explore the cohesin loader-RSC interaction and show that its Scc4 subunit contacts a conserved RSC ATPase motor module. The cohesin loader enhances RSC chromatin remodeling activity in vitro, as well as promoter nucleosome eviction in vivo. These findings provide insight into how the cohesin loader recognizes, as well as influences, the chromatin landscape, with implications for our understanding of human developmental disorders including Cornelia de Lange and Coffin-Siris syndromes.

2022-11-29 | PXD033446 | Pride

Structure of the Cohesin Loader Scc2-Scc4 and its Binding to a Folded Cohesin

Project description:The functions of cohesin are central to genome integrity, chromosome organization, and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex, however, little is known about how it stimulates the cohesin loading activity. Here we determine the large “hook” structure of Scc2 responsible for catalyzing cohesin loading. We identify key Scc2 surfaces that are crucial for DNA binding and for cohesin loading in vivo. Using previously determined structures and modeling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, our crosslinking and electron microscopy analyses reveal that Scc2-Scc4 utilizes its modular structure to make multiple contacts with a folded cohesin at an interface created by the cohesin head-hinge interaction.

2017-01-11 | PXD004692 | Pride

Disruption of Nipbl/Scc2 in Cornelia de Lange Syndrome provokes cohesin genome-wide redistribution with an impact in the transcriptome

Project description:Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, Nipbl/Scc2 were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanism driving the CdLS phenotypes are not understood. Apart from its canonical role in sister chromatid cohesion, cohesin has also been involved in the regulation of the spatial organization of the genome. Here, we investigated the transcriptome of CdLS-derived primary fibroblasts (gene expression microarray data included in the manuscript as an excel file) and observed the downregulation of genes involved in development and system skeletal organization providing a link to the developmental alterations and limb abnormalities characteristics of the CdLS patients. Genome-wide distribution studies demonstrated a global reduction of Nipbl at the Nipbl-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at Nipbl-occupied sites at CpG islands probably due to reduced cohesin translocation along chromosomes and fewer cohesin peaks colocalize with CTCF.

2021-05-27 | GSE145966 | GEO

Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP association

Project description:The ring-shaped cohesin complex is the key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is also hindered by Smc3 acetylation. However, the molecular mechanism behind this inhibition remains mysterious. Here we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3’s head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled-coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.

2023-08-16 | GSE217833 | GEO

The Scc2NIPBL/Scc4MAU2 complex acts in sister chromatid cohesion and transcriptional regulation by maintaining nucleosome-free regions [ChIP-seq]

Project description:The Scc2/Scc4 complex binds to broad nucleosome-free regions in the promoters of highly expressed genes. The cohesin loader is recruited to these sites by the RSC chromatin remodeling complex ChIp- Sequencing analysis of the distribution of Scc2 and Scc4 on budding yeast chromosomes.An input sample was analysed as a control.

2014-08-01 | E-GEOD-56993 | biostudies-arrayexpress

A Role for Chromatin Remodeling in Cohesin Loading onto Chromosomes

Project description:The RSC chromatin remodeling complex recruits the cohesin loader Scc2-Scc4 by a direct protein interaction. Nucleosome-free DNA is the substrate for the cohesin loading reaction.

2019-03-19 | GSE117881 | GEO

Inactivation of the Budding Yeast Cohesin Loader Scc2 alters Gene Expression both Globally and in Response to a Single DNA Double Strand Break

Project description:Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2, but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding. We investigated the gene transcription profiles using GeneChip Yeast Genome 2.0 Array in WT and Scc2 deficient cells in the absence and presence of DNA damage.

2014-10-16 | E-GEOD-61530 | biostudies-arrayexpress

Inactivation of the Budding Yeast Cohesin Loader Scc2 alters Gene Expression both Globally and in Response to a Single DNA Double Strand Break [ChIP-seq]

Project description:Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2, but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding. We investigated Scc1 FLAG tagged binding in WT ans Scc2-4 mutant in absence and presence of a single DNA DSB on Chr. VI

2014-10-21 | E-GEOD-62493 | biostudies-arrayexpress

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