Sister Chromatid Cohesion Research Articles

Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms

Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesin's cohesion and loop extrusion activities can be partially separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wild-type cohesin. This suggests that cohesion establishment and cohesin's interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.

ISW1a modulates cohesin distribution in centromeric and pericentromeric regions.

Cohesin is a highly conserved, multiprotein complex whose canonical function is to hold sister chromatids together to ensure accurate chromosome segregation. Cohesin association with chromatin relies on the Scc2-Scc4 cohesin loading complex that enables cohesin ring opening and topological entrapment of sister DNAs. To better understand how sister chromatid cohesion is regulated, we performed a proteomic screen in budding yeast that identified the Isw1 chromatin remodeler as a cohesin binding partner. In addition, we found that Isw1 also interacts with Scc2-Scc4. Lack of Isw1 protein, the Ioc3 subunit of ISW1a or Isw1 chromatin remodeling activity resulted in increased accumulation of cohesin at centromeres and pericentromeres, suggesting that ISW1a may promote efficient translocation of cohesin from the centromeric site of loading to neighboring regions. Consistent with the role of ISW1a in the chromatin organization of centromeric regions, Isw1 was found to be recruited to centromeres. In its absence we observed changes in the nucleosomal landscape at centromeres and pericentromeres. Finally, we discovered that upon loss of RSC functionality, ISW1a activity leads to reduced cohesin binding and cohesion defect. Taken together, our results support the notion of a key role of chromatin remodelers in the regulation of cohesin distribution on chromosomes.

Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions

RNA binding motif protein X‐linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene’s importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

The histone methyltransferase NSD3 contributes to sister chromatid cohesion and to cohesin loading at mitotic exit.

Sister chromatid cohesion is a multi-step process implemented throughout the cell cycle to ensure the correct transmission of chromosomes to daughter cells. Although cohesion establishment and mitotic cohesion dissolution have been extensively explored, the regulation of cohesin loading is still poorly understood. Here, we report that the methyltransferase NSD3 is essential for mitotic sister chromatid cohesion before mitosis entry. NSD3 interacts with the cohesin loader complex kollerin (composed of NIPBL and MAU2) and promotes the chromatin recruitment of MAU2 and cohesin at mitotic exit. We also show that NSD3 associates with chromatin in early anaphase, prior to the recruitment of MAU2 and RAD21, and dissociates from chromatin when prophase begins. Among the two NSD3 isoforms present in somatic cells, the long isoform is responsible for regulating kollerin and cohesin chromatin-loading, and its methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring proper kollerin recruitment and thus cohesin loading.

Replisome-cohesin interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors

The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate successful establishment of sister chromatid cohesion during DNA replication.

Preprint Highlight: Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms

Cohesins organize chromatin within the nucleus by promoting loop extrusion via cis contacts on chromatin and sister chromatid cohesion via trans contacts between chromatids. Whether cohesins employ a distinct mechanism for these two functions is not known. The authors use a carefully designed conditional expression of cohesin, mutated at its hinge domain, and assess its function with a variety of in vitro and in vivo experiments including live TIRF imaging, loop extrusion assay, Hi-C, and calibrated ChIP sequencing. A mutation in the hinge domain separates the two functions of cohesin, as the mutant supports loop extrusion but not sister chromatid cohesion. A unique mechanism of cohesins in promoting loop extrusion has implications for their distinct role in genome organization and for transcriptional regulation and cell fate determination. This study will be of broad interest to genome biologists with respect to these phenomena. This preprint has been assigned the following badges: New Hypothesis, New Materials. Read the preprint on bioRxiv ( Nagasaka et al., 2022 ): https://doi.org/10.1101/2022.09.23.509019 .

Preprint Highlight: Age-dependent loss of cohesion protection in human oocytes

Age-dependent loss of cohesion is a determinant of aneuploidy in human oocytes. Mechanisms for how this loss is triggered with age are not known. Using oocytes from human participants of various ages, the authors correlate loss of Sgo2, a protector of cohesins, with age-related meiosis II cohesion defects including increased inter-kinetochore distances and single chromatids. High-resolution microscopy reveals age-dependent loss of Sgo2 specifically from pericentromeric bridges, but not from centromeric regions. This study highlights the role of pericentromeric cohesins in upholding sister-chromatid cohesion, even when centromeric cohesins are protected. This work is relevant to researchers interested in chromosome biology, meiosis, aneuploidy mechanisms, and fertility. This preprint has been assigned the following badges: New Hypothesis. Read the preprint on bioRxiv ( Mihalas et al., 2023 ): https://doi.org/10.1101/2023.01.13.523952 .

Structural basis of centromeric cohesion protection

In the early stages of mitosis, cohesin is released from chromosome arms but not from centromeres. The protection of centromeric cohesin by SGO1 maintains the sister chromatid cohesion that resists the pulling forces of microtubules until all chromosomes are attached in a bipolar manner to the mitotic spindle. Here we present the X-ray crystal structure of a segment of human SGO1 bound to a conserved surface of the cohesin complex. SGO1 binds to a composite interface formed by the SA2 and SCC1RAD21 subunits of cohesin. SGO1 shares this binding interface with CTCF, indicating that these distinct chromosomal regulators control cohesin through a universal principle. This interaction is essential for the localization of SGO1 to centromeres and protects centromeric cohesin against WAPL-mediated cohesin release. SGO1–cohesin binding is maintained until the formation of microtubule–kinetochore attachments and is required for faithful chromosome segregation and the maintenance of a stable karyotype.

Chromosome organization by fine-tuning an ATPase.

Cohesin is an ATPase that drives chromosome organization through the generation of intramolecular loops and sister chromatid cohesion. Cohesin's ATPase is stimulated by Scc2 binding but attenuated by acetylation of its Smc3 subunit. In this issue of Genes & Development, Boardman and colleagues (pp. 277-290) take a genetic approach to generate a mechanistic model for the opposing regulation of cohesin's ATPase by Scc2 and Smc3 acetylation. Their findings provide in vivo insight into how this important genome organizer functions in vivo.

Function and inhibition of Haspin kinase: targeting multiple cancer therapies by antimitosis.

Haploid germ cell-specific nuclear protein kinase (Haspin) is a serine/threonine kinase as an atypical kinase, which is structurally distinct from conventional protein kinases. Functionally, Haspin is involved in important cell cycle progression, particularly in critical mitosis regulating centromeric sister chromatid cohesion during prophase and prometaphase, and subsequently ensuring proper chromosome alignment during metaphase and the normal chromosome segregation during anaphase. However, increasing evidence has demonstrated that Haspin is significantly upregulated in a variety of cancer cells in addition to normal proliferating somatic cells. Its knockdown or small molecule inhibition could prevent cancer cell growth and induce apoptosis by disrupting the regular mitotic progression. Given the specificity of its expressed tissues or cells and the uniqueness of its current known substrate, Haspin can be a promising target against cancer. Consequently, selective synthetic and natural inhibitors of Haspin have been widely developed to determine their inhibitory power for various cancer cells in vivo and in vitro. Here our perspective includes a comprehensive review of the roles and structure of Haspin, its relatively potent and selective inhibitors and Haspin's preliminary studies in a variety of cancers.

A model for Scc2p stimulation of cohesin's ATPase and its inhibition by acetylation of Smc3p.

The evolutionarily conserved cohesin complex mediates sister chromatid cohesion and facilitates mitotic chromosome condensation, DNA repair, and transcription regulation. These biological functions require cohesin's two ATPases, formed by the Smc1p and Smc3p subunits. Cohesin's ATPase activity is stimulated by the Scc2p auxiliary factor. This stimulation is inhibited by Eco1p acetylation of Smc3p at an interface with Scc2p. It was unclear how cohesin's ATPase activity is stimulated by Scc2p or how acetylation inhibits Scc2p, given that the acetylation site is distal to cohesin's ATPase active sites. Here, we identify mutations in budding yeast that suppressed the in vivo defects caused by Smc3p acetyl-mimic and acetyl-defective mutations. We provide compelling evidence that Scc2p activation of cohesin ATPase depends on an interface between Scc2p and a region of Smc1p proximal to cohesin's Smc3p ATPase active site. Furthermore, substitutions at this interface increase or decrease ATPase activity to overcome ATPase modulation by acetyl-mimic and acetyl-null mutations. Using these observations and an existing cryo-EM structure, we propose a model for regulating cohesin ATPase activity. We suggest that Scc2p binding to Smc1p causes the adjacent Smc1p residues and ATP to shift, stimulating Smc3p's ATPase. This stimulatory shift is inhibited through acetylation of the distal Scc2p-Smc3p interface.

Symmetric control of sister chromatid cohesion establishment.

Besides entrapping sister chromatids, cohesin drives other high-order chromosomal structural dynamics like looping, compartmentalization and condensation. ESCO2 acetylates a subset of cohesin so that cohesion must be established and only be established between nascent sister chromatids. How this process is precisely achieved remains unknown. Here, we report that GSK3 family kinases provide higher hierarchical control through an ESCO2 regulator, CRL4MMS22L. GSK3s phosphorylate Thr105 in MMS22L, resulting in homo-dimerization of CRL4MMS22L and ESCO2 during S phase as evidenced by single-molecule spectroscopy and several biochemical approaches. A single phospho-mimicking mutation on MMS22L (T105D) is sufficient to mediate their dimerization and rescue the cohesion defects caused by GSK3 or MMS22L depletion, whereas non-phosphorylable T105A exerts dominant-negative effects even in wildtype cells. Through cell fractionation and time-course measurements, we show that GSK3s facilitate the timely chromatin association of MMS22L and ESCO2 and subsequently SMC3 acetylation. The necessity of ESCO2 dimerization implicates symmetric control of cohesion establishment in eukaryotes.

Molecular mechanisms protecting centromeres from self-sabotage and implications for cancer therapy.

Centromeres play a crucial role in DNA segregation by mediating the cohesion and separation of sister chromatids during cell division. Centromere dysfunction, breakage or compromised centromeric integrity can generate aneuploidies and chromosomal instability, which are cellular features associated with cancer initiation and progression. Maintaining centromere integrity is thus essential for genome stability. However, the centromere itself is prone to DNA breaks, likely due to its intrinsically fragile nature. Centromeres are complex genomic loci that are composed of highly repetitive DNA sequences and secondary structures and require the recruitment and homeostasis of a centromere-associated protein network. The molecular mechanisms engaged to preserve centromere inherent structure and respond to centromeric damage are not fully understood and remain a subject of ongoing research. In this article, we provide a review of the currently known factors that contribute to centromeric dysfunction and the molecular mechanisms that mitigate the impact of centromere damage on genome stability. Finally, we discuss the potential therapeutic strategies that could arise from a deeper understanding of the mechanisms preserving centromere integrity.

Cohesin organization, dynamics, and subdomain functions revealed by genetic suppressor screening.

Cohesin is a heteropentameric protein complex that contributes to various aspects of chromosome structure and function, such as sister chromatid cohesion, genome compaction, and DNA damage response. Previous studies have provided abundant information on architecture and regional structures of the cohesin complex, but the configuration and structural dynamics of the whole cohesin complex are still largely unknown, partly due to flexibility of its coiled coils. We studied cohesin organization and dynamics using in vivo functional mutation compensation. Specifically, we developed and applied genetic suppressor screening methods to identify second mutations in cohesin complex genes that rescue lethality caused by various site-specific abnormalities in the cohesin complex. Functional analysis of these missense suppressor mutations revealed novel features of cohesin. Here, we summarize recent genetic suppressor screening results and insights into: 1) cohesin's structural organization when holding chromosomal DNAs; 2) interaction between cohesin head-kleisin and hinge; 3) ATP-driven cohesin conformational changes for genome packaging.

Function and Evolution of the Loop Extrusion Machinery in Animals.

Structural maintenance of chromosomes (SMC) complexes are essential proteins found in genomes of all cellular organisms. Essential functions of these proteins, such as mitotic chromosome formation and sister chromatid cohesion, were discovered a long time ago. Recent advances in chromatin biology showed that SMC proteins are involved in many other genomic processes, acting as active motors extruding DNA, which leads to the formation of chromatin loops. Some loops formed by SMC proteins are highly cell type and developmental stage specific, such as SMC-mediated DNA loops required for VDJ recombination in B-cell progenitors, or dosage compensation in Caenorhabditis elegans and X-chromosome inactivation in mice. In this review, we focus on the extrusion-based mechanisms that are common for multiple cell types and species. We will first describe an anatomy of SMC complexes and their accessory proteins. Next, we provide biochemical details of the extrusion process. We follow this by the sections describing the role of SMC complexes in gene regulation, DNA repair, and chromatin topology.

Cohesin regulates alternative splicing.

Cohesin, a trimeric complex that establishes sister chromatid cohesion, has additional roles in chromatin organization and transcription. We report that among those roles is the regulation of alternative splicing through direct interactions and in situ colocalization with splicing factors. Degradation of cohesin results in marked changes in splicing, independent of its effects on transcription. Introduction of a single cohesin point mutation in embryonic stem cells alters splicing patterns, demonstrating causality. In primary human acute myeloid leukemia, mutations in cohesin are highly correlated with distinct patterns of alternative splicing. Cohesin also directly interacts with BRD4, another splicing regulator, to generate a pattern of splicing that is distinct from either factor alone, documenting their functional interaction. These findings identify a role for cohesin in regulating alternative splicing in both normal and leukemic cells and provide insights into the role of cohesin mutations in human disease.

Application of neural network-based image analysis to detect sister chromatid cohesion defects

Sister chromatid cohesion (SCC) is mediated by the cohesin complex and its regulatory proteins. To evaluate the involvement of a protein in cohesin regulation, preparations of metaphase chromosome spreads and classifications of chromosome shapes after depletion of the target protein are commonly employed. Although this is a convenient and approved method, the evaluation and classification of each chromosome shape has to be performed manually by researchers. Therefore, this method is time consuming, and the results might be affected by the subjectivity of researchers. In this study, we developed neural network-based image recognition models to judge the positional relationship of sister chromatids, and thereby detect SCC defects. Transfer learning models based on SqueeezeNet or ResNet-18 were trained with more than 600 chromosome images labeled with the type of chromosome, which were classified according to the positional relationship between sister chromatids. The SqueezeNet-based trained model achieved a concordance rate of 73.1% with the sample answers given by a researcher. Importantly, the model successfully detected the SCC defect in the CTF18 deficient cell line, which was used as an SCC-defective model. These results indicate that neural-network-based image recognition models are valuable tools for examining SCC defects in different genetic backgrounds.

The cohesin modifier ESCO2 is stable during DNA replication

Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein following replication. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we also did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using both flow cytometry and live-cell imaging. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells.

Non-Rabl chromosome organization in endoreduplicated nuclei of barley embryo and endosperm tissues.

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner.

A replication fork determinant for the establishment of sister chromatid cohesion

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Cohesion establishment requires acetylation of conserved cohesin lysine residues by Eco1 acetyltransferase. Here, we explore how cohesin acetylation is linked to DNA replication. Biochemical reconstitution of replication-coupled cohesin acetylation reveals that transient DNA structures, which form during DNA replication, control the acetylation reaction. As polymerases complete lagging strand replication, strand displacement synthesis produces DNA flaps that are trimmed to result in nicked double-stranded DNA. Both flaps and nicks stimulate cohesin acetylation, while subsequent nick ligation to complete Okazaki fragment maturation terminates the acetylation reaction. A flapped or nicked DNA substrate constitutes a transient molecular clue that directs cohesin acetylation to a window behind the replication fork, next to where cohesin likely entraps both sister chromatids. Our results provide an explanation for how DNA replication is linked to sister chromatid cohesion establishment.