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The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1 rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1 rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1 , which encodes a subunit of the MBF transcription complex, also rescues eco1 rad61 cell growth defects. Elevated expression of SWI6 , however, which encodes the Mbp1 -binding partner of MBF, exacerbates eco1 rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2 , that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression. [ABSTRACT FROM AUTHOR]

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations—whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA–DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation. Synopsis: The loop extrusion hypothesis has shaped current thinking about chromatin organization by SMC complexes. This article experimentally tests the hypothesis, with a negative outcome, and puts forward transcription as an alternative, external motor force promoting chromatin loop formation. Yeast cohesin variants unable to perform in vitro DNA loop extrusion readily form chromatin loops in vivo. Transcription occupies key roles in chromatin loop formation and growth. In a unified model, cohesin sequentially captures two DNAs to establish sister chromatid cohesion at DNA replication forks and chromatin loops at places of transcription. Cohesin variants defective in DNA loop extrusion in vitro can still form chromatin loops in living yeast cells. [ABSTRACT FROM AUTHOR]

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin‐mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter‐TAD loop formation. Based on the reanalysis of Nano‐C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read‐through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter‐TAD loops is restricted though. We speculate that the DNA‐encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer–promoter loop formation and other genomic processes. [ABSTRACT FROM AUTHOR]

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer–promoter (E‐P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward‐ and outward‐oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD‐boundary‐crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long‐distance E‐P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA‐mediated TAD boundary function via R‐loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E‐P interactions is developmentally regulated. [ABSTRACT FROM AUTHOR]

Cohesin is a ring‐shaped complex that is loaded on DNA in two different conformations. In one conformation, it forms loops to organize the interphase genome; in the other, it topologically encircles sibling chromosomes to facilitate homologous recombination and to establish the cohesion that is required for orderly segregation during mitosis. How, and even if, these two loading conformation are related is unclear. Here, I propose that loop binding is a required first step for topological binding. This loop‐binding‐first model integrates the known information about the two loading mechanisms, explains genetic requirements for the two and explains how topological loading evolved from loop binding. [ABSTRACT FROM AUTHOR]

AbstractMyelodysplastic syndrome (MDS) is a heterogeneous myeloid neoplasm that is hallmarked by the acquisition of genetic events that disrupt normal trilineage hematopoiesis and results in bone marrow dysfunction. Somatic genes involving transcriptional regulation, signal transduction, DNA methylation, and chromatin modification are often implicated in disease pathogenesis. The cohesin complex, composed of SMC1, SMC3, RAD21, and either STAG1 or STAG2, has been identified as a recurrent mutational target with STAG2 mutations accounting for more than half of all cohesin mutations in myeloid malignancies. In the last decade, STAG2 cohesin biology has been of great interest given its role in transcriptional activation, association with poorer prognosis, and lack of mutation-specific therapies. This review discusses the clinical landscape of cohesin mutant myeloid malignancies, particularly STAG2 mutant MDS, including molecular features of STAG2 mutations, clinical implications of cohesin mutant neoplasms, and the current understanding of the pathophysiological function of STAG2 mutations in MDS. [ABSTRACT FROM AUTHOR]

Genomic data analysis has witnessed a surge in complexity and volume, primarily driven by the advent of high-throughput technologies. In particular, studying chromatin loops and structures has become pivotal in understanding gene regulation and genome organization. This systematic investigation explores the realm of specialized bioinformatics pipelines designed specifically for the analysis of chromatin loops and structures. Our investigation incorporates two protein (CTCF and Cohesin) factor-specific loop interaction datasets from six distinct pipelines, amassing a comprehensive collection of 36 diverse datasets. Through a meticulous review of existing literature, we offer a holistic perspective on the methodologies, tools and algorithms underpinning the analysis of this multifaceted genomic feature. We illuminate the vast array of approaches deployed, encompassing pivotal aspects such as data preparation pipeline, preprocessing, statistical features and modelling techniques. Beyond this, we rigorously assess the strengths and limitations inherent in these bioinformatics pipelines, shedding light on the interplay between data quality and the performance of deep learning models, ultimately advancing our comprehension of genomic intricacies. [ABSTRACT FROM AUTHOR]

Background: The STAG1 gene encodes a component of the cohesin complex, involved in chromosome segregation and DNA repair. Variants in genes of the cohesin complex determine clinical conditions characterized by facial dysmorphisms, upper limb anomalies, intellectual disability, and other neurological deficits. However, to date, the STAG1-related clinical phenotype has been poorly investigated (around 20 cases reported). Methods and Results: We report, for the first time, two twins affected by a syndromic neurodevelopmental disorder associated with a de novo variant in the STAG1 gene. Although both the twins showed a neurodevelopmental delay, one of them showed a more severe phenotype with greater behavioral problems, speech defects and limb apraxia. CGH array showed a 15q13.3 microduplication, inherited from an unaffected mother. Conclusions: We found different degrees of behavioral, speech and cognitive impairment in two twins affected by a neurodevelopmental disorder associated with a STAG1 variant. These findings highlight the variability of the STAG1-associated phenotype or a probable role of associated variants (like the discovered 15q13.3 microduplication) in modulating the clinical features. [ABSTRACT FROM AUTHOR]

Structural Maintenance of Chromosomes (SMC) complexes are an evolutionary conserved protein family. In most eukaryotes, three SMC complexes have been characterized, as follows: cohesin, condensin, and SMC5/6 complexes. These complexes are involved in a plethora of functions, and defects in SMC genes can lead to an increased risk of chromosomal abnormalities, infertility, and cancer. To investigate the evolution of SMC complex genes in mammals, we analyzed their selective patterns in an extended phylogeny. Signals of positive selection were identified for condensin NCAPG, for two SMC5/6 complex genes (SMC5 and NSMCE4A), and for all cohesin genes with almost exclusive meiotic expression (RAD21L1, REC8, SMC1B, and STAG3). For the latter, evolutionary rates correlate with expression during female meiosis, and most positively selected sites fall in intrinsically disordered regions (IDRs). Our results support growing evidence that IDRs are fast evolving, and that they most likely contribute to adaptation through modulation of phase separation. We suggest that the natural selection signals identified in SMC complexes may be the result of different selective pressures: a host-pathogen arms race in the condensin and SMC5/6 complexes, and an intragenomic conflict for meiotic cohesin genes that is similar to that described for centromeres and telomeres. [ABSTRACT FROM AUTHOR]

In recent years, various technologies have emerged for the imaging of chromatin loci in living cells via catalytically inactive Cas9 (dCas9). These technologies facilitate a deeper understanding of the mechanisms behind the chromatin dynamics and provide valuable kinetic data that could not have previously been obtained via FISH applied to fixed cells. However, such technologies are relatively complicated, as they involve the expression of several chimeric proteins as well as sgRNAs targeting the visualized loci, a process that entails many technical subtleties. Therefore, the effectiveness in visualizing a specific target locus may be quite low. In this study, we directly compared two versions of a previously published CRISPR-Sirius method based on the use of sgRNAs containing eight MS2 or PP7 stem loops and the expression of MCP or PCP fused to fluorescent proteins. We assessed the visualization efficiency for several unique genomic loci by comparing the two approaches in delivering sgRNA genes (transient transfection and lentiviral transduction), as well as two CRISPR-Sirius versions (with PCP and with MCP). The efficiency of visualization varied among the loci, and not all loci could be visualized. However, the MCP-sfGFP version provided more efficient visualization in terms of the number of cells with signals than PCP-sfGFP for all tested loci. We also showed that lentiviral transduction was more efficient in locus imaging than transient transfection for both CRISPR-Sirius systems. Most of the target loci in our study were located at the borders of topologically associating domains, and we defined a set of TAD borders that could be effectively visualized using the MCP-sfGFP version of the CRISPR-Sirius system. Altogether, our study validates the use of the CRISPR-Sirius technology for live-cell visualization and highlights various technical details that should be considered when using this method. [ABSTRACT FROM AUTHOR]

The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation. [ABSTRACT FROM AUTHOR]

Two different models have been proposed to explain how the endpoints of chromatin looped domains ('TADs') in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the 'loop-extrusion' and the 'boundary-pairing' models. Our findings are incompatible with the loop-extrusion model, and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion. [ABSTRACT FROM AUTHOR]

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation during meiosis requires the maintenance of sister chromatid cohesion, initially established during premeiotic S phase. In human oocytes, DNA replication and cohesion establishment occur decades before chromosome segregation and deterioration of meiotic cohesion is one factor that leads to increased segregation errors as women age. Our previous work led us to propose that a cohesion rejuvenation program operates to establish new cohesive linkages during meiotic prophase in Drosophila oocytes and depends on the cohesin loader Nipped-B and the cohesion establishment factor Eco. In support of this model, we recently demonstrated that chromosome-associated cohesin turns over extensively during meiotic prophase and failure to load cohesin onto chromosomes after premeiotic S phase results in arm cohesion defects in Drosophila oocytes. To identify proteins required for prophase cohesion rejuvenation but not S phase establishment, we conducted a Gal4-UAS inducible RNAi screen that utilized two distinct germline drivers. Using this strategy, we identified 29 gene products for which hairpin expression during meiotic prophase, but not premeiotic S phase, significantly increased segregation errors. Prophase knockdown of Brahma or Pumilio, two positives with functional links to the cohesin loader, caused a significant elevation in the missegregation of recombinant homologs, a phenotype consistent with premature loss of arm cohesion. Moreover, fluorescence in situ hybridization confirmed that Brahma, Pumilio, and Nipped-B are required during meiotic prophase for the maintenance of arm cohesion. Our data support the model that Brahma and Pumilio regulate Nipped-B-dependent cohesin loading during rejuvenation. Future analyses will better define the mechanism(s) that govern meiotic cohesion rejuvenation and whether additional prophase-specific positives function in this process. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes are organized into chromatin domains through long-range chromatin interactions which are mediated by the binding of architectural proteins, such as CTCF and cohesin, and histone modifications. Based on the published Hi-C and ChIP-seq datasets in human monocyte-derived macrophages, we identified 206 and 127 differential chromatin interactions (DCIs) that were not located within transcription readthrough regions in influenza A virus- and interferon β-treated cells, respectively, and found that the binding positions of CTCF and RAD21 within more than half of the DCI sites did not change. However, five histone modifications, H3K4me3, H3K27ac, H3K36me3, H3K9me3, and H3K27me3, showed significantly more dramatic changes than CTCF and RAD21 within the DCI sites. For H3K4me3, H3K27ac, H3K36me3, and H3K27me3, significantly more dramatic changes were observed outside than within the DCI sites. We further applied a motif scanning approach to discover proteins that might correlate with changes in histone modifications and chromatin interactions and found that PRDM9, ZNF384, and STAT2 frequently bound to DNA sequences corresponding to 1 kb genomic intervals with gains or losses of a histone modification within the DCI sites. This study explores the dynamic regulation of chromatin interactions and extends the current knowledge of the relationship between histone modifications and chromatin interactions. [ABSTRACT FROM AUTHOR]

We show here that in the fungus Sordaria macrospora, the meiosis-specific HORMA-domain protein Hop1 is not essential for the basic early events of chromosome axis development, recombination initiation, or recombination-mediated homolog coalignment/pairing. In striking contrast, Hop1 plays a critical role at the leptotene/zygotene transition which is defined by transition from pairing to synaptonemal complex (SC) formation. During this transition, Hop1 is required for maintenance of normal axis structure, formation of SC from telomere to telomere, and development of recombination foci. These hop1Δ mutant defects are DSB dependent and require Sme4/Zip1-mediated progression of the interhomolog interaction program, potentially via a pre-SC role. The same phenotype occurs not only in hop1Δ but also in absence of the cohesin Rec8 and in spo76-1, a non-null mutant of cohesin-associated Spo76/Pds5. Thus, Hop1 and cohesins collaborate at this crucial step of meiotic prophase. In addition, analysis of 4 non-null mutants that lack this transition defect reveals that Hop1 also plays important roles in modulation of axis length, homolog-axis juxtaposition, interlock resolution, and spreading of the crossover interference signal. Finally, unexpected variations in crossover density point to the existence of effects that both enhance and limit crossover formation. Links to previously described roles of the protein in other organisms are discussed. Hop1 has been previously shown to display diverse meiotic functions in different organisms. This study identifies and characterizes the functions of Hop1 in the fungus Sordaria macrospora and shows that it plays a crucial role during the leptotene/zygotene transition and formation of the synaptonemal complex during meiotic prophase. [ABSTRACT FROM AUTHOR]

The cohesin complex is a ring-shaped protein structure involved in DNA repair and chromosomal segregation. Studies have showed that genomic alterations in the cohesin complex members are among the initial occurrences in the development of acute myeloid leukemia (AML). STAG2 is the most commonly mutated and best-studied member of the cohesin complex in AML and mutations in this gene have been associated with adverse outcomes and are diagnostically relevant. However, the exact role of mutations in other members of the cohesin complex in the development of myeloid neoplasia is controversial. In this single institution study, we retrospectively reviewed data from the molecular profiles of 1,381 AML patients and identified 14 patients with mutations in RAD21, another member of the cohesin complex. We evaluated the frequency, mutational profile, clinico-pathologic features, and prognostic impact of RAD21 in this cohort. This study showed that RAD21-mutated AML often associates with monocytic differentiation, CD7 expression, co-existing mutations in epigenetic regulators, a normal karyotype, and poor prognosis. Our findings provide additional insights into the morphologic, immunophenotypic, and genomic profile of RAD21 mutation-positive AML and suggest that RAD21 mutations should be evaluated for independent prognostic significance in AML. [ABSTRACT FROM AUTHOR]

The CRL4-DCAF15 E3 ubiquitin ligase complex is targeted by the aryl-sulfonamide molecular glues, leading to neo-substrate recruitment, ubiquitination, and proteasomal degradation. However, the physiological function of DCAF15 remains unknown. Using a domain-focused genetic screening approach, we reveal DCAF15 as an acute myeloid leukemia (AML)-biased dependency. Loss of DCAF15 results in suppression of AML through compromised replication fork integrity and consequent accumulation of DNA damage. Accordingly, DCAF15 loss sensitizes AML to replication stress-inducing therapeutics. Mechanistically, we discover that DCAF15 directly interacts with the SMC1A protein of the cohesin complex and destabilizes the cohesin regulatory factors PDS5A and CDCA5. Loss of PDS5A and CDCA5 removal precludes cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication, and apoptosis. Collectively, our findings uncover an endogenous, cell autonomous function of DCAF15 in sustaining AML proliferation through post-translational control of cohesin dynamics. The DCAF15 E3 ubiquitin ligase is targeted by aryl-sulfonamide molecular glues leading to neo-substrate proteasomal degradation. Here, the authors reveal DCAF15 as a cell autonomous acute myeloid leukemia dependency sustaining proliferation through control of cohesin complex recycling dynamics. [ABSTRACT FROM AUTHOR]

The 16-subunit Constitutive Centromere-associated Network (CCAN)-based inner kinetochore is well-known for connecting centromeric chromatin to the spindle-binding outer kinetochore. Here, we report a non-canonical role for the inner kinetochore in directly regulating sister-chromatid cohesion at centromeres. We provide biochemical, X-ray crystal structure, and intracellular ectopic localization evidence that the inner kinetochore directly binds cohesin, a ring-shaped multi-subunit complex that holds sister chromatids together from S-phase until anaphase onset. This interaction is mediated by binding of the 5-subunit CENP-OPQUR sub-complex of CCAN to the Scc1-SA2 sub-complex of cohesin. Mutation in the CENP-U subunit of the CENP-OPQUR complex that abolishes its binding to the composite interface between Scc1 and SA2 weakens centromeric cohesion, leading to premature separation of sister chromatids during delayed metaphase. We further show that CENP-U competes with the cohesin release factor Wapl for binding the interface of Scc1-SA2, and that the cohesion-protecting role for CENP-U can be bypassed by depleting Wapl. Taken together, this study reveals an inner kinetochore-bound pool of cohesin, which strengthens centromeric sister-chromatid cohesion to resist metaphase spindle pulling forces. Synopsis: The inner kinetochore is well-known for connecting centromeric chromatin to the microtubule-binding outer kinetochore. This work reveals a non-canonical role for the inner kinetochore in binding cohesin complexes, and strengthening centromeric cohesion to resist metaphase spindle pulling forces. The CENP-OPQUR complex of the constitutive inner kinetochore interacts with the cohesin complex. Mutation of the CENP-U motif that interacts with the composite interface between the Scc1 and SA2 subunits of the cohesin complex weakens centromeric cohesion, leading to a defect in maintaining metaphase sister-chromatid cohesion. CENP-U competes with the cohesin release factor Wapl for binding to the Scc1-SA2 interface, thereby antagonizing Wapl-mediated cohesin removal from metaphase centromeres. CENP-U and Sgo1 additively contribute to the strength of centromeric cohesion. CENP-U competes with the release factor Wapl for Scc1-SA2 binding, providing a localized Sgo1-independent cohesin protection mechanism. [ABSTRACT FROM AUTHOR]

Cohesin is a multi-subunit protein that plays a pivotal role in holding sister chromatids together during cell division. Sister chromatid cohesion 3 (SCC3), constituents of cohesin complex, is highly conserved from yeast to mammals. Since the deletion of individual cohesin subunit always causes lethality, it is difficult to dissect its biological function in both mitosis and meiosis. Here, we obtained scc3 weak mutants using CRISPR-Cas9 system to explore its function during rice mitosis and meiosis. The scc3 weak mutants displayed obvious vegetative defects and complete sterility, underscoring the essential roles of SCC3 in both mitosis and meiosis. SCC3 is localized on chromatin from interphase to prometaphase in mitosis. However, in meiosis, SCC3 acts as an axial element during early prophase I and subsequently situates onto centromeric regions following the disassembly of the synaptonemal complex. The loading of SCC3 onto meiotic chromosomes depends on REC8. scc3 shows severe defects in homologous pairing and synapsis. Consequently, SCC3 functions as an axial element that is essential for maintaining homologous chromosome pairing and synapsis during meiosis. [ABSTRACT FROM AUTHOR]

Inactivating mutations of genes encoding the cohesin complex are common in a wide range of human cancers. STAG2 is the most commonly mutated subunit. Here we report the impact of stable correction of endogenous, naturally occurring STAG2 mutations on gene expression, 3D genome organization, chromatin loops, and Polycomb signaling in glioblastoma multiforme (GBM). In two GBM cell lines, correction of their STAG2 mutations significantly altered the expression of ∼10% of all expressed genes. Virtually all the most highly regulated genes were negatively regulated by STAG2 (i.e., expressed higher in STAG2-mutant cells), and one of them—HEPH—was regulated by STAG2 in uncultured GBM tumors as well. While STAG2 correction had little effect on large-scale features of 3D genome organization (A/B compartments, TADs), STAG2 correction did alter thousands of individual chromatin loops, some of which controlled the expression of adjacent genes. Loops specific to STAG2-mutant cells, which were regulated by STAG1-containing cohesin complexes, were very large, supporting prior findings that STAG1-containing cohesin complexes have greater loop extrusion processivity than STAG2-containing cohesin complexes and suggesting that long loops may be a general feature of STAG2-mutant cancers. Finally, STAG2 mutation activated Polycomb activity leading to increased H3K27me3 marks, identifying Polycomb signaling as a potential target for therapeutic intervention in STAG2-mutant GBM tumors. Together, these findings illuminate the landscape of STAG2-regulated genes, A/B compartments, chromatin loops, and pathways in GBM, providing important clues into the largely still unknown mechanism of STAG2 tumor suppression. [ABSTRACT FROM AUTHOR]

• A random forest model is proposed for cell-type-specific cohesin-mediated chromatin loops prediction. • Chromatin state is responsible for cell-type-specificity of chromatin loops. • Chromatin state combines with the binding of key factors can predict cell-type-specific loops accurately. Chromatin loop is of crucial importance for the regulation of gene transcription. Cohesin is a type of chromatin-associated protein that mediates the interaction of chromatin through the loop extrusion. Cohesin-mediated chromatin interactions have strong cell-type specificity, posing a challenge for predicting chromatin loops. Existing computational methods perform poorly in predicting cell-type-specific chromatin loops. To address this issue, we propose a random forest model to predict cell-type-specific cohesin-mediated chromatin loops based on chromatin states identified by ChromHMM and the occupancy of related factors. Our results show that chromatin state is responsible for cell-type-specificity of loops. Using only chromatin states as features, the model achieved high accuracy in predicting cell-type-specific loops between two cell types and can be applied to different cell types. Furthermore, when chromatin states are combined with the occurrence frequency of CTCF, RAD21, YY1, and H3K27ac ChIP-seq peaks, more accurate prediction can be achieved. Our feature extraction method provides novel insights into predicting cell-type-specific chromatin loops and reveals the relationship between chromatin state and chromatin loop formation. [ABSTRACT FROM AUTHOR]

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORIN's functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1Δ mutants display cohesion defects, which are partially alleviated by wpl1Δ. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants. Cohesin functions must be tightly regulated. Here Prusén Mota et al. show that the cohesin regulator SORORIN is conserved beyond the animal kingdom and they provide the first organismal in vivo evidence that SORORIN antagonizes WAPL. [ABSTRACT FROM AUTHOR]

In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets. Chromatin within interphase nuclei forms compacted nanoscale DNA domains of uniform size. By integrating theory and imaging, here the authors show how the interplay between transcription and epigenetic mechanisms determine this size, independent of cell type. [ABSTRACT FROM AUTHOR]

In the last few years, Micro-C has shown itself as an improved alternative to Hi-C. It replaced the restriction enzymes in Hi-C assays with micrococcal nuclease (MNase), resulting in capturing nucleosome resolution chromatin interactions. The signal-to-noise improvement of Micro-C allows it to detect more chromatin loops than high-resolution Hi-C. However, compared with massive Hi-C datasets available in the literature, there are only a limited number of Micro-C datasets. To take full advantage of these Hi-C datasets, we present HiC2MicroC, a computational method learning and then predicting Micro-C from Hi-C based on the denoising diffusion probabilistic models (DDPM). We trained our DDPM and other regression models in human foreskin fibroblast (HFFc6) cell line and evaluated these methods in six different cell types at 5-kb and 1-kb resolution. Our evaluations demonstrate that both HiC2MicroC and regression methods can markedly improve Hi-C towards Micro-C, and our DDPM-based HiC2MicroC outperforms regression in various terms. First, HiC2MicroC successfully recovers most of the Micro-C loops even those not detected in Hi-C maps. Second, a majority of the HiC2MicroC-recovered loops anchor CTCF binding sites in a convergent orientation. Third, HiC2MicroC loops share genomic and epigenetic properties with Micro-C loops, including linking promoters and enhancers, and their anchors are enriched for structural proteins (CTCF and cohesin) and histone modifications. Lastly, we find our recovered loops are also consistent with the loops identified from promoter capture Micro-C (PCMicro-C) and Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET). Overall, HiC2MicroC is an effective tool for further studying Hi-C data with Micro-C as a template. HiC2MicroC is publicly available at https://github.com/zwang-bioinformatics/HiC2MicroC/. Author summary: The Micro-C assay, an improved variant of Hi-C, can capture genome-wide DNA interactions at nucleosome resolution, which makes it possible to detect more chromatin loops that relate to regulatory elements. However, there are not enough experimental Micro-C data publicly available compared with widely performed Hi-C experiments. Here we use deep learning methods to predict Micro-C from Hi-C. Our evaluation indicates that our method can successfully recover the loops that are detected in Micro-C but not identified in Hi-C. Our method provides an effective way to further take advantage of Hi-C. [ABSTRACT FROM AUTHOR]

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45–MCM2-7–GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination. [ABSTRACT FROM AUTHOR]

Accurate duplication and separation of long linear genomic DNA molecules is associated with a number of purely mechanical problems. SMC complexes are key components of the cellular machinery that ensures decatenation of sister chromosomes and compaction of genomic DNA during division. Cohesin, one of the essential eukaryotic SMC complexes, has a typical ring structure with intersubunit pore through which DNA molecules can be threaded. Capacity of cohesin for such topological entrapment of DNA is crucial for the phenomenon of post-replicative association of sister chromatids better known as cohesion. Recently, it became apparent that cohesin and other SMC complexes are, in fact, motor proteins with a very peculiar movement pattern leading to formation of DNA loops. This specific process has been called loop extrusion. Extrusion underlies multiple functions of cohesin beyond cohesion, but molecular mechanism of the process remains a mystery. In this review, we summarized the data on molecular architecture of cohesin, effect of ATP hydrolysis cycle on this architecture, and known modes of cohesin–DNA interactions. Many of the seemingly disparate facts presented here will probably be incorporated in a unified mechanistic model of loop extrusion in the not-so-distant future. [ABSTRACT FROM AUTHOR]

The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bacterial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the current understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its involvement in sister chromatid cohesion. [ABSTRACT FROM AUTHOR]

Cohesin is a highly conserved ring-shaped complex involved in topologically embracing chromatids, gene expression regulation, genome compartmentalization, and genome stability maintenance. Genomic analyses have detected mutations in the cohesin complex in a wide array of human tumors. These findings have led to increased interest in cohesin as a potential target in cancer therapy. Synthetic lethality has been suggested as an approach to exploit genetic differences in cancer cells to influence their selective killing. In this study, we show that mutations in ESCO1, NIPBL, PDS5B, RAD21, SMC1A, SMC3, STAG2, and WAPL genes are synthetically lethal with stimulation of WNT signaling obtained following LY2090314 treatment, a GSK3 inhibitor, in several cancer cell lines. Moreover, treatment led to the stabilization of β-catenin and affected the expression of c-MYC, probably due to the occupancy decrease in cohesin at the c-MYC promoter. Finally, LY2090314 caused gene expression dysregulation mainly involving pathways related to transcription regulation, cell proliferation, and chromatin remodeling. For the first time, our work provides the underlying molecular basis for synthetic lethality due to cohesin mutations and suggests that targeting the WNT may be a promising therapeutic approach for tumors carrying mutated cohesin. [ABSTRACT FROM AUTHOR]

Activation of the p53 tumor suppressor triggers a transcriptional program to control cellular response to stress. However, the molecular mechanisms by which p53 controls gene transcription are not completely understood. Here, we uncover the critical role of spatio-temporal genome architecture in this process. We demonstrate that p53 drives direct and indirect changes in genome compartments, topologically associating domains, and DNA loops prior to one hour of its activation, which escort the p53 transcriptional program. Focusing on p53-bound enhancers, we report 340 genes directly regulated by p53 over a median distance of 116 kb, with 74% of these genes not previously identified. Finally, we showcase that p53 controls transcription of distal genes through newly formed and pre-existing enhancer-promoter loops in a cohesin dependent manner. Collectively, our findings demonstrate a previously unappreciated architectural role of p53 as regulator at distinct topological layers and provide a reliable set of new p53 direct target genes that may help designs of cancer therapies. Here the authors uncover p53's role as the master regulator of spatio-temporal genome organization. p53 controls the expression of 340 distal genes through newly formed and pre-existing loops between p53-bound enhancers and promoters. [ABSTRACT FROM AUTHOR]

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase–anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail. [ABSTRACT FROM AUTHOR]

Background Material‐Methods Results‐Discussion Conclusions Telomeres are unique structures situated at the ends of chromosomes. Preserving the structure and function of telomeres is essential for maintaining genomic stability and promoting genetic diversity during male meiosis in mammals.This review compiled recent literature on the function and regulation of telomeres during male meiosis in both mice and humans, and also highlighted the critical roles of telomeres in reproductive biology and medicine.Various structures, consisting of the LINC complex (SUN‐KASH), SPDYA‐CDK2, TTM trimer (TERB1‐TERB2‐MAJIN), and shelterin, are critical in controlling telomeric activities, such as nuclear envelope attachment and bouquet formation. Other than telomere‐related proteins, cohesins and genes responsible for regulating telomere function are also highlighted, though the exact mechanism remains unclear. The gene‐mutant mouse models with meiotic defects directly reveal the essential roles of telomeres in male meiosis. Recently reported mutant genes associated with telomere activity in clinical practice have also been illustrated in detail.Proper regulation of telomere activities is essential for male meiosis progression in mice and humans. [ABSTRACT FROM AUTHOR]

Compartment formation in interphase chromosomes is a result of spatial segregation between euchromatin and heterochromatin on a few megabase pairs (Mbp) scale. On the sub-Mbp scales, topologically associating domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are present even without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer–promoter/promoter–promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines [ABSTRACT FROM AUTHOR]

Summary: EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long‐range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q− myeloid leukaemia cells, the EVI1 promoter interacts via long‐range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long‐range interactions appear to not depend on CTCF/cohesin, though EVI1+3q− cells utilise an EVI1 promoter‐proximal site to enhance its expression that is also involved in CTCF‐mediated looping in 3q+ cells. Long‐range interactions in 3q− cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q− cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference‐induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q− leukaemia and consolidate the view that EVI1 in 3q− leukaemia is largely promoter‐driven, potentially involving long‐distance promoter clustering. [ABSTRACT FROM AUTHOR]

The connection between the patterns observed in 3C-type experiments and the modeling of polymers remains unresolved. This paper presents a simulation pipeline that generates thermodynamic ensembles of 3D structures for topologically associated domain (TAD) regions by loop extrusion model (LEM). The simulations consist of two main components: a stochastic simulation phase, employing a Monte Carlo approach to simulate the binding positions of cohesins, and a dynamical simulation phase, utilizing these cohesins' positions to create 3D structures. In this approach, the system's total energy is the combined result of the Monte Carlo energy and the molecular simulation energy, which are iteratively updated. The structural maintenance of chromosomes (SMC) protein complexes are represented as loop extruders, while the CCCTC-binding factor (CTCF) locations on DNA sequence are modeled as energy minima on the Monte Carlo energy landscape. Finally, the spatial distances between DNA segments from ChIA-PET experiments are compared with the computer simulations, and we observe significant Pearson correlations between predictions and the real data. LoopSage model offers a fresh perspective on chromatin loop dynamics, allowing us to observe phase transition between sparse and condensed states in chromatin. • We improved the already-existing LEM (loop extrusion model) algorithm for the prediction of 3D structure of chromatin. • We present a simulation pipeline that generates thermodynamic ensembles of 3D structures for topologically associated domain (TAD) regions. • The CCCTC-binding factor (CTCF) locations on DNA sequence are modeled as energy minima on the Monte Carlo energy landscape. • The spatial distances between DNA segments from ChIA-PET experiments show high correlation with the averaged simulated distances. • LoopSage allows us to observe phase transition between sparse and condensed states in chromatin. [ABSTRACT FROM AUTHOR]

Cohesin holds together the sister chromatids from DNA replication onwards. How cohesion is established has long remained a black box. Through recent studies, a model is emerging in which a replisome–cohesin encounter results in the establishment of cohesive linkages at sites of replication termination. Cohesin holds together the sister chromatids from DNA replication onwards. How cohesion is established has long remained a black box. Through recent studies, a model is emerging in which a replisome–cohesin encounter results in the establishment of cohesive linkages at sites of replication termination. [ABSTRACT FROM AUTHOR]

Meiotic cohesion loss underlies elevated rates of infertility and chromosome abnormalities in children of older women. A new study shows that cohesins are turned over throughout meiotic prophase, suggesting that cohesion loss is likely not solely due to early establishment of cohesion. [ABSTRACT FROM AUTHOR]

Turning centromere DNA into a mechanical spring is central to the fidelity of chromosome segregation. A recent study shows how centromere DNA loops and partitioning cohesin and condensin convert centromeres and pericentromeres into bipartite bottlebrushes. [ABSTRACT FROM AUTHOR]

Oncogene-induced replication stress is a crucial driver of genomic instability and one of the key events contributing to the onset and evolution of cancer. Despite its critical role in cancer, the mechanisms that generate oncogene-induced replication stress remain not fully understood. Here, we report that an oncogenic c-Myc-dependent increase in cohesins on DNA contributes to the induction of replication stress. Accumulation of cohesins on chromatin is not sufficient to cause replication stress, but also requires cohesins to accumulate at specific sites in a CTCF-dependent manner. We propose that the increased accumulation of cohesins at CTCF site interferes with the progression of replication forks, contributing to oncogene-induced replication stress. This is different from, and independent of, previously suggested mechanisms of oncogene-induced replication stress. This, together with the reported protective role of cohesins in preventing replication stress-induced DNA damage, supports a double-edge involvement of cohesins in causing and tolerating oncogene-induced replication stress. Here the authors report that oncogenic c-Myc induces replication stress via increasing the amount of cohesins bound to chromatin at CTCF sites. [ABSTRACT FROM AUTHOR]

Background: SMC1A is a subunit of the cohesin complex that participates in many DNA- and chromosome-related biological processes. Previous studies have established that SMC1A is involved in cancer development and in particular, is overexpressed in chromosomally unstable human colorectal cancer (CRC). This study aimed to investigate whether SMC1A could serve as a therapeutic target for CRC. Methods: At first, we studied the effects of either SMC1A overexpression or knockdown in vitro. Next, the outcome of SMC1A knocking down (alone or in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor) was analyzed in vivo. Results: We found that SMC1A knockdown affects cell proliferation and reduces the ability to grow in anchorage-independent manner. Next, we demonstrated that the silencing of SMC1A and the combo treatment were effective in increasing overall survival in a xenograft mouse model. Functional analyses indicated that both treatments lead to atypical mitotic figures and gene expression dysregulation. Differentially expressed genes were implicated in several pathways including gene transcription regulation, cellular proliferation, and other transformation-associated processes. Conclusions: These results indicate that SMC1A silencing, in combination with bevacizumab, can represent a promising therapeutic strategy for human CRC. [ABSTRACT FROM AUTHOR]

During meiosis, cohesin and meiosis-specific proteins organize chromatin into an axis-loop architecture, coordinating homologous synapsis, recombination, and ordered chromosome segregation. However, how the meiotic chromosome axis is assembled and differentiated with meiotic progression remains elusive. Here, we explore the dynamic recruitment of two long arms of the bivalent proteins, LAB-1 and LAB-2, in Caenorhabditis elegans. LAB proteins directly interact with the axis core HORMA complexes and weak interactions contribute to their recruitment. LAB proteins phase separate in vitro, and this capacity is promoted by HORMA complexes. During early prophase, synapsis oppositely regulates the axis enrichment of LAB proteins. After the pachytene exit, LAB proteins switch from a reciprocal localization pattern to a colocalization pattern, and the normal dynamic pattern of LAB proteins is altered in meiotic mutants. We propose that LAB recruitment senses axis differentiation, and phase separation of meiotic structures helps subdomain establishment and accurate segregation of the chromosomes. [ABSTRACT FROM AUTHOR]

The human STAG2 protein is an essential component of the cohesin complex involved in cellular processes of gene expression, DNA repair, and genomic integrity. Somatic mutations in the STAG2 sequence have been associated with various types of cancer, while congenital variants have been linked to developmental disorders such as Mullegama–Klein–Martinez syndrome, X-linked holoprosencephaly-13, and Cornelia de Lange syndrome. In the cohesin complex, the direct interaction of STAG2 with DNA and with NIPBL, RAD21, and CTCF proteins has been described. The function of STAG2 within the complex is still unknown, but it is related to its DNA binding capacity and is modulated by its binding to the other three proteins. Every missense variant described for STAG2 is located in regions involved in one of these interactions. In the present work, we model the structure of 12 missense variants described for STAG2, as well as two other variants of NIPBl and two of RAD21 located at STAG2 interaction zone, and then analyze their behavior through molecular dynamic simulations, comparing them with the same simulation of the wild-type protein. This will allow the effects of variants to be rationalized at the atomic level and provide clues as to how STAG2 functions in the cohesin complex. [ABSTRACT FROM AUTHOR]

The mitotic cohesin complex necessary for sister chromatid cohesion and chromatin loop formation shows local and global association to chromosomes in response to DNA double‐strand breaks (DSBs). Here, by genome‐wide binding analysis of the meiotic cohesin with Rec8, we found that the Rec8‐localization profile along chromosomes is altered from middle to late meiotic prophase I with cleavage‐independent dissociation. Each Rec8‐binding site on the chromosome axis follows a unique alternation pattern with dissociation and probably association. Centromeres showed altered Rec8 binding in late prophase I relative to mid‐prophase I, implying chromosome remodeling of the regions. Rec8 dissociation ratio per chromosome is correlated well with meiotic DSB density. Indeed, the spo11 mutant deficient in meiotic DSB formation did not change the distribution of Rec8 along chromosomes in late meiotic prophase I. These suggest the presence of a meiosis‐specific regulatory pathway for the global binding of Rec8‐cohesin in response to DSBs. [ABSTRACT FROM AUTHOR]

Background: Cornelia de Lange syndrome (CdLS) is mainly characterized by specific facial features, growth retardation, and bone deformities. Seven genes reportedly cause CdLS. Recent research has reported that loss‐of‐function variants affecting MAU2, which encodes a regulator of the cohesin complex, can cause CdLS. Thus far, only one MAU2‐CdLS case has been reported worldwide. Methods: We detected a novel variant in MAU2 gene, NM_015329, c.526C>T (p.Arg176Trp) in a Chinese patient with CdLS, constructed a plasmid for in vitro transcriptional and protein level analysis, and analyzed the interaction between the MAU2/NIPBL complex using molecular dynamics (MD). Results: The results showed that the level of the exogenous MAU2 mutant protein was significantly reduced compared with that of the exogenous wild‐type protein. However, MD analysis predicted an increased binding free energy between the MAU2 and NIPBL proteins that may impact the structural stability of the complex. Conclusion: We investigated a MAU2‐CdLS case in a Chinese family, which strengthens the association between MAU2 variants and CdLS phenotypes. We therefore propose that MAU2 be included in the CdLS gene screening list. [ABSTRACT FROM AUTHOR]

Stromal antigen 2 (STAG2), a subunit of the cohesin complex, is recurrently mutated in various tumors. However, the role of STAG2 in DNA repair and its therapeutic implications are largely unknown. Here it is reported that knockout of STAG2 results in increased double‐stranded breaks (DSBs) and chromosomal aberrations by reducing homologous recombination (HR) repair, and confers hypersensitivity to inhibitors of ataxia telangiectasia mutated (ATMi), Poly ADP Ribose Polymerase (PARPi), or the combination of both. Of note, the impaired HR by STAG2‐deficiency is mainly attributed to the restored expression of KMT5A, which in turn methylates H4K20 (H4K20me0) to H4K20me1 and thereby decreases the recruitment of BRCA1‐BARD1 to chromatin. Importantly, STAG2 expression correlates with poor prognosis of cancer patients. STAG2 is identified as an important regulator of HR and a potential therapeutic strategy for STAG2‐mutant tumors is elucidated. [ABSTRACT FROM AUTHOR]

A single Aurora kinase found in non-vertebrate deuterostomes is assumed to represent the ancestor of vertebrate Auroras A/B/C. However, the tunicate Oikopleura dioica, a member of the sister group to vertebrates, possesses two Aurora kinases (Aurora1 and Aurora2) that are expressed in proliferative cells and reproductive organs. Previously, we have shown that Aurora kinases relocate from organizing centers to meiotic nuclei and were enriched on centromeric regions as meiosis proceeds to metaphase I. Here, we assessed their respective functions in oogenic meiosis using dsRNA interferences. We found that Aurora1 (Aur1) was involved in meiotic spindle organization and chromosome congression, probably through the regulation of microtubule dynamics, whereas Aurora2 (Aur2) was crucial for chromosome condensation and meiotic spindle assembly. In vitro kinase assays showed that Aur1 and Aur2 had comparable levels of kinase activities. Using yeast two-hybrid library screening, we identified a few novel interaction proteins for Aur1, including c-Jun-amino-terminal kinase-interacting protein 4, cohesin loader Scc2, and mitochondrial carrier homolog 2, suggesting that Aur1 may have an altered interaction network and participate in the regulation of microtubule motors and cohesin complexes in O. dioica. [ABSTRACT FROM AUTHOR]

Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2) are conserved epigenetic regulators that promote transcriptional gene silencing. PRC1 and PRC2 converge on shared targets, catalyzing repressive histone modifications. Additionally, a subset of PRC1/PRC2 targets engage in long-range interactions whose functions in gene silencing are poorly understood. Using a CRISPR screen in mouse embryonic stem cells, we found that the cohesin regulator PDS5A links transcriptional silencing by Polycomb and 3D genome organization. PDS5A deletion impairs cohesin unloading and results in derepression of a subset of endogenous PRC1/PRC2 target genes. Importantly, derepression is not linked to loss of Polycomb chromatin domains. Instead, PDS5A removal causes aberrant cohesin activity leading to ectopic insulation sites, which disrupt the formation of ultra-long Polycomb loops. We show that these loops are important for robust silencing at a subset of PRC1/PRC2 target genes and that maintenance of cohesin-dependent genome architecture is critical for Polycomb regulation. Through a genetic screen, the authors find that the cohesin regulator PDS5A is required for Polycomb target gene silencing. Derepression upon cohesin dysregulation is linked to loss of Polycomb loops without change in repressive chromatin domains. [ABSTRACT FROM AUTHOR]

Mesophilic, anaerobic, and cellulolytic Ruminiclostridium-type bacterial species can secrete an extracellular, multi-enzyme machinery cellulosome, which efficiently degrades cellulose. In this study, we first reported the complete genome of Ruminiclostridium papyrosolvens DSM2782, a single circular 5,027,861-bp chromosome with 37.1% G  +  C content, and compared it with other Ruminiclostridium-type species. Pan-genome analysis showed that Ruminiclostridium-type species share a large number of core genes to conserve basic functions, although they have a high level of intraspecific genetic diversity. Especially, KEGG mapping revealed that Ruminiclostridium-type species mainly use ABC transporters regulated by two-component systems (TCSs) to absorb extracellular sugars but not phosphotransferase systems (PTSs) that are employed by solventogenic clostridia, such as Clostridium acetobutylicum. Furthermore, we performed comparative analyses of the species-specific repertoire of CAZymes for each of the Ruminiclostridium-type species. The high similarity of their cohesins suggests a common ancestor and potential cross-species recognition. Additionally, both differences between the C-terminal cohesins and other cohesins of scaffoldins and between the dockerins linking with cellulases and other catalytic domains indicate a preference for the location of cellulosomal catalytic subunits at scaffoldins. The information gained in this study may be utilized directly or developed further by genetic engineering and optimizing enzyme systems or cell factories for enhanced biotechnological biomass deconstruction and biofuel production. [ABSTRACT FROM AUTHOR]

Transient DNA loops occur throughout the genome due to thermal fluctuations of DNA and the function of SMC complex proteins such as condensin and cohesin. Transient crosslinking within and between chromosomes and loop extrusion by SMCs have profound effects on high-order chromatin organization and exhibit specificity in cell type, cell cycle stage, and cellular environment. SMC complexes anchor one end to DNA with the other extending some distance and retracting to form a loop. How cells regulate loop sizes and how loops distribute along chromatin are emerging questions. To understand loop size regulation, we employed bead–spring polymer chain models of chromatin and the activity of an SMC complex on chromatin. Our study shows that (1) the stiffness of the chromatin polymer chain, (2) the tensile stiffness of chromatin crosslinking complexes such as condensin, and (3) the strength of the internal or external tethering of chromatin chains cooperatively dictate the loop size distribution and compaction volume of induced chromatin domains. When strong DNA tethers are invoked, loop size distributions are tuned by condensin stiffness. When DNA tethers are released, loop size distributions are tuned by chromatin stiffness. In this three-way interaction, the presence and strength of tethering unexpectedly dictates chromatin conformation within a topological domain. [ABSTRACT FROM AUTHOR]