Genome-wide DNA methylation analysis in cohesin mutant human cell lines

The cohesin complex has recently been shown to be a key regulator of eukaryotic gene expression, although the mechanisms by which it exerts its effects are poorly understood. We have undertaken a genome-wide analysis of DNA methylation in cohesin-deficient cell lines from probands with Cornelia de Lange syndrome (CdLS). Heterozygous mutations in NIPBL, SMC1A and SMC3 genes account for ∼65% of individuals with CdLS. SMC1A and SMC3 are subunits of the cohesin complex that controls sister chromatid cohesion, whereas NIPBL facilitates cohesin loading and unloading. We have examined the methylation status of 27 578 CpG dinucleotides in 72 CdLS and control samples. We have documented the DNA methylation pattern in human lymphoblastoid cell lines (LCLs) as well as identified specific differential DNA methylation in CdLS. Subgroups of CdLS probands and controls can be classified using selected CpG loci. The X chromosome was also found to have a unique DNA methylation pattern in CdLS. Cohesin preferentially binds to hypo-methylated DNA in control LCLs, whereas the differential DNA methylation alters cohesin binding in CdLS. Our results suggest that in addition to DNA methylation multiple mechanisms may be involved in transcriptional regulation in human cells and in the resultant gene misexpression in CdLS

The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress▿

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1

Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors

Abstract Cohesin regulates gene expression through context-specific chromatin folding mechanisms such as enhancer–promoter looping and topologically associating domain (TAD) formation by cooperating with factors such as cohesin loaders and the insulation factor CTCF. We developed a computational workflow to explore how three-dimensional (3D) structure and gene expression are regulated collectively or individually by cohesin and related factors. The main component is CustardPy, by which multi-omics datasets are compared systematically. To validate our methodology, we generated 3D genome, transcriptome, and epigenome data before and after depletion of cohesin and related factors and compared the effects of depletion. We observed diverse effects on the 3D genome and transcriptome, and gene expression changes were correlated with the splitting of TADs caused by cohesin loss. We also observed variations in long-range interactions across TADs, which correlated with their epigenomic states. These computational tools and datasets will be valuable for 3D genome and epigenome studies

Transcription-driven cohesin repositioning rewires chromatin loops in cellular senescence

Senescence is a phenotypic state of stable proliferative arrest, typically occurring in lineage-committed cells and triggered by various stimuli. It is generally accompanied by activation of a secretory program (senescence-associated secretory phenotype, SASP), which modulates both local (tissue microenvironment) and systemic (ageing) homeostasis 1,2 . Enhancer-promoter interactions play a key role in gene regulation 3–5 , facilitated by chromatin loops, mostly formed via CCCTC binding factor (CTCF) and cohesin tethering 6–8 . The three-dimensional chromatin structure of senescent cells has been characterised 9–11 mostly in terms of macro-domain structures, but its relevance in gene expression remains elusive. Here, we use Hi-C and capture Hi-C 12,13 to show that oncogenic HRAS-induced senescence (RIS) in human diploid fibroblasts (HDFs) is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks at the 3’ end of a subset of active genes, reminiscent of the transcription-driven ‘cohesin islands’ recently discovered in mouse embryonic fibroblasts deficient in both CTCF and the cohesin release factor Wings apart-like (Wapl) 14 . RIS de novo cohesin peaks are also transcription-dependent and enriched for SASP genes, as exemplified by IL1B , where de novo cohesin binding is involved in new loop formation. Cytokine induction is associated with similar cohesin islands appearance and enhancer-promoter rewiring during the terminal differentiation of monocytes to macrophages 15 , but not upon acute TNFα treatment of HDFs 16 . These results suggest that RIS represents a fate-determined process in which gene expression is regulated beyond the cell type specific 3D chromatin framework, in part through cohesin redistribution

Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL) facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS). Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor