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Identification of the protein interaction network of Stag proteins

ABSTRACT: This study involves the identification of specific Stag1 interactors with and without Cohesin loss. Stag1 was immunoprecipiated from human HCT116 cells, under 4 treatments - untreated (UTR), Non-specific siRNA treatment (SiCON), SA1 siRNA treatment (SiSA1) and Auxin treatment for Cohesin loss (IAA). An unspecific IgG pulldown was also conducted to assess for unspecific protein binding to the bead system (Mock). Significant proteins were determined by label-free quantification and statistical analysis using MaxQuant and MSstats.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Colon Cancer

SUBMITTER: Silvia Surinova

LAB HEAD: Silvia Surinova

PROVIDER: PXD024354 | Pride | 2023-04-18

REPOSITORIES: Pride

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Identification of the protein interaction network of STAG2 protein isoforms with inclusion and exclusion of exon32

Project description:This study involves the identification of specific interactors for STAG2ex32+ and STAG2ex32delta. Plasmids expressing YFP-STAG2 isoforms were transfected into HCT116OsTIR cells using untransfected cells as mock control. 40h post transfection, whole cell lysates were extracted and STAG2 isoforms were immunoprecipiated using GFP-TRAP. An unspecific GFP-TRAP pulldown was also conducted using lysates from untransfected cells to assess for unspecific protein binding to the beads (Mock). Significant proteins were determined by label-free quantification and statistical analysis using MaxQuant and MSstats.

2023-05-04 | PXD041975 | Pride

Absolute quantification of cohesin and its regulators in human cells

Project description:The organisation of mammalian genomes into loops and topologically associated domains (TADs) regulates gene expression. The formation and maintenance of loops and TADs depends on the protein complex cohesin, which also holds replicated sister chromatids together from S phase until mitosis. To understand how cohesin functions, it is essential to know how many cohesin complexes and regulators exist in a typical cell and how they are distributed genome-wide. Here, we have used quantitative mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching to measure the absolute number and dynamics of core subunits and regulators of the cohesin complex in human cells. We find that 60000 – 134000 cohesin complexes are bound to chromatin per G1 HeLa cell, suggesting that cohesin most likely does not occupy all its binding sites concurrently. We incorporate our data into mathematical models and discuss implications for how cohesin might contribute to genome organisation and sister chromatid cohesion.

2019-11-13 | PXD012712 | Pride

Multiomic analysis of Cohesin reveals that ZBTB transcription factors contribute to chromatin interactions

Project description:One bottleneck in understanding the principles of 3D chromatin structures is caused by the paucity of known regulators. Cohesin is essential for 3D chromatin organization, and its interacting partners are candidate regulators. Here, we performed proteomic profiling of the Cohesin in chromatin and identified transcription factors, RNA-binding proteins, and chromatin regulators associated with Cohesin. Acute protein degradation followed by time-series genomic binding quantitation and BAT Hi-C analysis were conducted, and the results showed that the transcription factor ZBTB21 contributes to Cohesin chromatin binding, 3D chromatin interactions and transcriptional repression. Strikingly, multiomic analyses revealed that the other four ZBTB factors interacted with Cohesin, and double degradation of ZBTB21 and ZBTB7B led to a further decrease in Cohesin chromatin occupancy. We propose that multiple ZBTB transcription factors orchestrate the chromatin binding of Cohesin to regulate chromatin interactions, and we provide a catalog of many additional proteins associated with Cohesin that warrant further investigation.

2023-06-05 | PXD035893 | Pride

Multiomic analysis of Cohesin reveals that ZBTB transcription factors contribute to chromatin interactions

2023-06-05 | PXD036004 | Pride

Multiomic analysis of Cohesin reveals that ZBTB transcription factors contribute to chromatin interactions

2023-06-05 | PXD028860 | Pride

Cohesin profiling at DSBs

Project description:Chromatin undergoes major remodeling around DNA double strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high resolution map of gammaH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit from the cohesin complex, previously characterized as required for repair by homologous recombination. We found that the recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs. In addition, we show that the cohesin complex, which was also recently proposed to be a key player in chromosome organisation and chromatin looping, controls gammaH2AX distribution within domains. Indeed, as we reported for transcription, cohesin binding antagonizes gammaH2AX spreading. Remarkably, depletion of cohesin leads to an increase of gammaH2AX at cohesin-bound genes (revelead by gammaH2AX mapping in upon SCC1 siRNA), associated with a decrease in their expression level after DSB induction. Thus our study identifies a novel role for the cohesin complex in protecting the genes located in gammaH2AX domains from both gammaH2AX spreading and transcriptional shut-down after DSB induction.

2012-01-01 | E-TABM-1164 | biostudies-arrayexpress

Control of Embryonic Stem Cell State by Mediator and Cohesin (Illumina ChIP-Seq data)

Project description:The key transcription factors that control the embryonic stem cell gene expression program have been identified, but how they function to implement this program is not well understood. While screening for genes essential for maintenance of ES cell state, we identified many components of the Mediator and Cohesin complexes. Mediator and Cohesin were found to physically and functionally connect the enhancers and core promoters of active genes. An ES cell Mediator complex was found to copurify with Cohesin and its loading factor Nipbl, and normal levels of these proteins were essential for expression of the genes they occupy and for maintenance of ES cell state. See associated publication.

2010-08-12 | E-GEOD-22562 | biostudies-arrayexpress

Small RNA sequencing in mouse cardiomyocytes after siRNA mediated knockdown of Srf

Project description:We performed two independent siRNA mediated knockdowns of Srf (Srf si1 & Srf si2) and an unspecific siRNA (siNon) in mouse cardiomyocytes HL-1 cells. Small RNAs were sequenced by Illumina/Solexa next-generation (single-end) sequencing technology. The sequence reads were mapped to the mouse reference genome (NCBI v37, mm9) using MicroRazerS. MicroRazerS searches for the longest possible prefix-match of each read, i.e. the longest possible contiguous match starting at the first base. Hence, it is robust to possible adapter sequence at the 3' end of a read and requires no adapter trimming. Small RNA-seq profiles of two siRNA mediated knockdowns of Srf and an unspecific siRNA in mouse cardiomyocytes

2011-01-03 | E-GEOD-19066 | biostudies-arrayexpress

Cohesin is required for expression of the estrogen receptor-alpha (ESR1) gene

Project description:In this study we show that depletion of the cohesin subunit SMC3 or the Mediator subunit MED12 significantly impairs the ERα-regulated transcriptome. Surprisingly, SMC3 depletion appears to elicit this effect indirectly by rapidly decreasing ESR1 transcription and ERα protein levels. Moreover, we provide evidence that both SMC3 and MED12 colocalize on the ESR1 gene and are mutually required for occupancy as well as for transcriptional elongation. Finally, we show that extended proteasome inhibition decreases the mRNA expression of cohesin subunits which accompanies a decrease in ESR1 mRNA and ERα protein levels as well as estrogen-regulated transcription. Conclusions: These results identify the ESR1 gene as a cohesin/Mediator-dependent gene and indicate that this regulation may potentially be exploited for the treatment of estrogen-dependent breast cancer. This set contains 18 microarray samples with triplicates for each condition. 3 control siRNA, 3 control estrogen stimulated, 3 siRNA Med12 knockdown, 3 siRNA Med12 knockdown estrogen stimulated, 3 siRNA Smc3 knockdown, 3 siRNA Smc3 knockdown estrogen stimulated.

2013-05-20 | E-GEOD-38252 | biostudies-arrayexpress

Control of Embryonic Stem Cell State by Mediator and Cohesin

Project description:This SuperSeries is composed of the following subset Series: GSE22556: Control of Embryonic Stem Cell State by Mediator and Cohesin (Agilent gene expression data) GSE22562: Control of Embryonic Stem Cell State by Mediator and Cohesin (Illumina ChIP-Seq data) Refer to individual Series

2010-08-11 | E-GEOD-22557 | biostudies-arrayexpress

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