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High-throughput Oligopaint screen identifies druggable 3D genome regulators

ABSTRACT: Although the molecular rules governing chromatin folding are being quickly elucidated, relatively few proteins regulating genome organization have been identified. To address this, we developed HiDRO (high-throughput labeling of DNA or RNA with optimized Oligopaint probes). Combined with automated image analysis, we used HiDRO to conduct a novel FISH-based screen of ~3,000 druggable genes that alter inter-TAD interactions. We isolated several enzymes that alter the integrity of architectural boundaries, including our top hit Glycogen synthase kinase alpha (GSK3A). We demonstrated using chemical and genetic inhibition that this ubiquitously expressed kinase enhances chromatin looping by regulating the chromatin binding of cohesin regulators. Finally, our data demonstrate that a GSK3A inhibitor can rescue both chromatin misfolding and gene misexpression at a sensitive locus after partial cohesin loss. These results illustrate a noncanonical role for GSK3A in cohesin regulation and highlight the broader utility of HiDRO-based screening to identify novel genome organizing factors.

ORGANISM(S): Homo sapiens

PROVIDER: GSE199607 | GEO | 2023/05/05

REPOSITORIES: GEO

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Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression

Project description:The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of ~2,000 genes, with only ~30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to either more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events.

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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 | PXD036004 | Pride

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

2023-06-05 | PXD028860 | Pride

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

2023-06-05 | PXD035893 | Pride

THE SPATIAL REGULATION OF CONDENSIN ACTIVITY IN CHROMOSOME CONDENSATION

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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

Dose-dependent role of the cohesin complex in normal and malignant hematopoiesis [ATAC-Seq]

Project description:Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation, however cohesin-mutant leukemias do not show genomic instability. We hypothesized reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Bi-allelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation, and revealed an absolute requirement for cohesin in hematopoietic stem cell function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose-dependency for cohesin in regulating chromatin structure and hematopoietic stem cell function. ATAC-seq in murine c-kit+ cells for the following genotypes: Smc3 fl/+, Smc3 del/+, Flt3-ITD, Smc3 fl/del Flt3-ITD

2015-09-21 | E-GEOD-73215 | biostudies-arrayexpress

Cohesin-dependent regulation of dynamic gene expression during differentiation is lost in Cohesin-mutated myeloid malignancies

Project description:The Cohesin complex critically controls interactions between proximal and distal cis-regulatory elements, while Cohesin disruption causes improper gene activation. This process associates with biased hematopoiesis, and Cohesin mutations are common in myeloid neoplasia. In this study, we initially explore Cohesin-associated dynamics and regulation of hematopoietic stem cell renewal and erythropoiesis. Cohesin binding to chromatin increases during erythroid differentiation at active promoters and enhancers only, with pre-binding of Etv6 predicting the subsequent dynamics of Cohesin binding at these elements. Etv6 also binds with the Cohesin complex at chromatin during haematopoiesis. Perturbation of Cohesin members does not alter hematopoietic self-renewal, but severely impairs erythroid differentiation. As in normal erythroid differentiation, the strongest impairment of gene activation in Cohesin-perturbed erythropoiesis occurs at Etv6-pre-bound loci. Taken together these findings propose a mechanism for how the Cohesin complex regulates the induction of gene expression during differentiation and how this may be perturbed in myeloid malignancies.

2019-04-09 | GSE129478 | GEO

SCC1 interaction proteomics in Saccharomyces cerevisiae

Project description:FACT mediates cohesin function on chromatin Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion and the organisation of higher-order structures during interphase and mitosis. The recruitment and mobility of cohesin complexes on DNA are restricted by nucleosomes. Here we show that cohesin role in chromosome organization requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin dependent compartmentalization of chromosomes into loop domains.

2019-10-03 | PXD014896 | Pride

Genome-wide control of RNA polymerase II activity by cohesin (tiling)

Project description:Cohesin is a well-known mediator of sister chromatid cohesion, but it also influences gene expression and development. These non-canonical roles of cohesin are not well understood, but are vital: gene expression and development are altered by modest changes in cohesin function that do not disrupt chromatid cohesion. To clarify cohesinM-bM-^@M-^Ys roles in transcription, we measured how cohesin controls RNA polymerase II (Pol II) activity by genome-wide chromatin immunoprecipitation and precision global run-on sequencing. On average, cohesin-binding genes have more transcriptionally active Pol II and promoter-proximal Pol II pausing than non-binding genes, and are more efficient, producing higher steady state levels of mRNA per transcribing Pol II complex. Cohesin depletion frequently increases pausing at cohesin-binding genes, indicating that cohesin often facilitates transition of paused Pol II to elongation. In many cases this likely reflects a role for cohesin in transcriptional enhancer function. Strikingly, more than 95% of predicted extragenic enhancers bind cohesin, and cohesin depletion can reduce their association with Pol II, indicating that cohesin facilitates enhancer-promoter contact. Cohesin directly promotes transcription of the myc gene, and cohesin depletion reduces Pol II activity at most Myc target genes. The multiple transcriptional roles of cohesin revealed by these studies likely underlie the growth and developmental deficits caused by minor changes in cohesin activity. We performed ChIP-chip of Rpb3 (representing total Pol II), Ser2P-Pol II (representing elongating Pol II), and Cdk12 and CycT Pol II kinase components in Mock RNAi-treated and cohesin subunit Rad21 RNAi-treated ML-DmBG3-c2 cells, which revealed that cohesin depletion has a variety of effects on Pol II occupancy and modification, as well as on occupancy of Pol II kinases.

2013-04-24 | E-GEOD-42360 | biostudies-arrayexpress

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