Biology subjects

Sakata, T.

Publications and source records attributed to Sakata, T..

13 recordsLinked to original sources

bioRxiv2026

DNA catenation is essential for Sister Chromatid Cohesion

From S phase until anaphase, sister chromatids remain physically linked to ensure accurate chromosome segregation. This linkage, called sister chromatid cohesion, must withstand the pulling forces generated by the mitotic spindle and is thought to require entrapment of sister DNAs inside the ring-shaped cohesin complex. DNA catenations that arise naturally during replication could also tether sister chromatids, but whether they contribute to cohesion has remained unresolved. Here, using budding yeast, we show that cohesin-mediated sister DNA entrapment alone cannot withstand spindle-generated pulling forces and that robust sister chromatid cohesion requires DNA catenation. Selective removal of DNA catenations causes catastrophic cohesion loss, delays chromosome biorientation and increases chromosome mis-segregation, even when cohesin rings remain intact. Importantly, DNA catenation similarly underpins force-resistant cohesion in metazoan chromosomes. Our findings fundamentally redefine the physical basis of sister chromatid cohesion by establishing DNA catenation as an evolutionarily conserved component of the force-resistant linkage between sister chromatids that enables accurate chromosome segregation.

molecular biology↗

bioRxiv2026

Absence of nascent peptides triggers nonfunctional ribosome decay

The nonfunctional rRNA decay (NRD) pathway eliminates defective ribosomes to maintain protein synthesis integrity. Although the Mms1-Rtt101 E3 ligase is known to trigger 25S NRD to clear defective 60S subunits, the specific molecular marks and targets it recognizes remain unknown. Here, we demonstrate that the late-stage maturation factor Reh1 functions as a specific molecular sensor for these defects. While Reh1 is normally displaced from the polypeptide exit tunnel (PET) during successful translation initiation, it is persistently retained on nonfunctional 80S ribosomes incapable of peptide bond formation. This prolonged retention, driven by the absence of a nascent peptide, provides a physical mark that recruits the Mms1 complex via Reh1s N-terminal domain, triggering site-specific ubiquitination of ribosomal protein Rpl19 (uL6). Our findings reveal that the cell repurposes a maturation factor as a sentinel to monitor translation competence, achieving robust detection of diverse ribosomal and maturation defects through a single molecular pathway.

molecular biology↗

bioRxiv2026

Region-specific generation and retention of acetylated cohesin shape the genome-wide pattern of sister chromatid cohesion

Sister chromatid cohesion depends on ESCO2-mediated acetylation of the cohesin subunit SMC3, yet how this modification is established and maintained across the genome remains poorly understood. Here, we examined the chromatin-binding dynamics of ESCO2 and its enzymatic activity using quantitative ChIP-seq of human cells synchronously progressing through S phase. ESCO2 bound broadly along chromosomes without forming discrete peaks and preferentially localized to transcriptionally inactive regions, but was not confined to H3K9me3- or H3K27me3-enriched heterochromatin. Consistent with this binding pattern, ESCO2-dependent SMC3 acetylation showed a similarly broad distribution in these regions. In contrast, ESCO1, a paralog of ESCO2, primarily mediated SMC3 acetylation at cohesin peak sites. Notably, most of the broadly distributed, ESCO2-dependent SMC3 acetylation generated during S phase was progressively lost from chromatin after replication, whereas the fraction that persisted into late G2 accumulated in domains enriched for the repressive H3K27me3 modification. Our work identifies selective post-replicative retention of acetylated cohesin as a previously unappreciated step in shaping the genome-wide architecture of sister chromatid cohesion.

molecular biology↗

bioRxiv2025

Cohesin constrains histone modification-driven chromatin dynamics

Gene expressions are regulated by an interplay between epigenetics and spatial genome organization, the deregulation of which has been implicated in the development of cancers, including myeloid neoplasms. However, it is unclear how they coordinately contribute to normal and malignant hematopoiesis. Here, we show that simultaneous dysregulations of histone modifications and chromatin structures caused by mutations of the epigenetic modulator Asxl1 and cohesin subunit Stag2 cooperatively induce ectopic interactions between polycomb-regulated promoters and active enhancers, leading to the aberrant upregulation of hematopoietic stem cell related genes and development of myelodysplastic syndromes (MDS). De-repression of polycomb-regulated genes induces their translocation to the transcriptionally active loci, where active promoters and enhancers are assembled, in the absence of Stag2-mediated chromatin organization. Our findings revealed that cohesin counteracts histone modification-driven chromatin conformations, manifesting the coordinate roles of histone modifiers and cohesin in regulating genome architectures and gene expressions to prevent malignant transformation.

cancer biology↗

bioRxiv2025

Cohesin acts as a transcriptional gatekeeper by restraining pause-release to promote processive elongation

Cohesin organizes 3D chromatin architecture, including promoter-enhancer loops, yet its loss has surprisingly modest effects on steady-state gene expression. We address this paradox by demonstrating that cohesin acts at different stages of transcription. First, it promotes Pol II promoter recruitment by facilitating enhancer-promoter communication that maintains active promoter chromatin states. Second, it delays pause release by transiently associating with the transcriptional machinery during the pause-release transition. Kinetic modelling suggests that reduced Pol II recruitment and enhanced pause release have compensatory effects, contributing to minimal changes in steady-state gene expression across genes upon cohesin loss. In contrast, cohesin depletion impairs robust transcriptional induction in response to external stimuli. Moreover, cohesin ensures sufficient pausing duration as a quality-control-like step to promote elongation complex assembly and transcription processivity. Here, we show that cohesin regulates multiple steps of transcription, offering mechanistic insight into cohesin-related diseases, including cancers and cohesinopathies.

molecular biology↗

bioRxiv2024

Controlling mechanism of the Scc2-cohesin interaction to restrict peri-centromeric DNA loop expansion and facilitate mitotic chromosome segregation

Cohesin exhibits DNA loop extrusion activity when bound to an ATPase activator Scc2 (NIPBL in humans), thereby organizing higher-order chromosome folding. In budding yeast, the majority of the chromosome-bound cohesins lack association with Scc2. It remains unknown how the interaction between Scc2 and cohesin is regulated on the chromosome and what physiological consequences malfunction in this regulatory mechanism causes. Here, we show that simultaneous deletion of Wpl1 and Eco1, two of the known cohesin regulators, resulted in Scc2 co-localization with cohesin around the centromeres of metaphase chromosomes. In these cells, the pericentromeric DNA loops were enlarged to connect the centromere to the cohesin/Scc2 co-bound sites at distances of up to a few hundred kb, indicating highly active loop extrusion by the Scc2-associated cohesin. Furthermore, we demonstrated that {Delta}wpl1 {Delta}eco1 cells exhibited a delay in the progression of mitotic chromosome segregation, a phenotype dependent on the presence of Scc2 in metaphase. These findings suggest that Wpl1 and Eco1 cooperatively regulate Scc2-cohesin interaction on chromosomes, restrict the size of the pericentromeric DNA loops, and facilitate mitotic chromosome segregation.

molecular biology↗

bioRxiv2024

Cohesin regulates promoter-proximal pausing of RNA Polymerase II by limiting recruitment of super elongation complex

Cohesin is a ring-shaped complex, responsible for establishing sister chromatid cohesion and forming topologically associating domains (TADs) and chromatin loops. Loss-of-function mutations in cohesin subunits and its regulatory factors can cause Cornelia de Lange syndrome (CdLS). Because dysregulated gene expression was observed in CdLS, it has long been thought that cohesin plays a regulatory role in transcription. Here, we investigated the effect of acute cohesin depletion on transcription and observed that a small number of genes exhibited differential expression. Analysis of RNA polymerase II (Pol II) distribution revealed that the depletion reduced Pol II promoter binding and pausing simultaneously at the majority of genes. This implies that at most genes, the two decreases counterbalance each other, resulting in unchanged gene expression. Additionally, we find that cohesin loss increased promoter binding of super elongation complex (SEC), which mediates the release of Pol II from paused state. Moreover, the reduction in pausing caused by cohesin depletion was no longer observed when SEC was inhibited. These observations suggest that cohesin regulates Pol II pausing by restricting SEC recruitment to promoters. Together, our study demonstrates the involvement of cohesin in transcriptional regulation, particularly in Pol II pause and release.

molecular biology↗

bioRxiv2023

Extracellular Matrix Proteins Changes in the Left Ventricular Unloading and Overloading after Myocardial Infarction

BackgroundThe cardiac extracellular matrix (ECM) is a dynamic scaffold that transmits and responds to forces that act on the heart during the cardiac cycle. While acute left ventricle (LV) unloading by percutaneous LV assist device (pLVAD) is now a therapeutic option, its impact on ECM is unknown. We hypothesize that the composition of LV ECM proteins changes in response to reduced wall stress with mechanical LV unloading. MethodsTen Yorkshire pigs underwent myocardial infarction, and one week later were subjected to either 2 hours of LV unloading with pLVAD or 2 hours of LV overloading by mechanically-induced acute aortic regurgitation. Non-ischemic remote myocardium was processed for ECM specific proteomics. Volcano plots were constructed to determine differentially expressed proteins between the groups. Gene ontology (GO) analysis was performed to identify associated molecular processes and biological functions. Immunofluorescence microscopy was performed to validate differential protein expression. ResultsOf the 986 proteins analyzed, 39 were significantly differentially expressed between overloaded and unloaded myocardium. GO analysis by molecular function revealed that differences tending towards RNA binding and lipid binding proteins. Analysis by biological process showed that cell differentiation, vesicle transport, and programmed cell death were the processes most affected. Staining for three of the identified differentially expressed proteins had results congruent with proteomic analysis. ConclusionsThere were significant differences in the protein composition of the cardiac ECM between acutely unloaded and overloaded myocardium, suggesting that it actively responds to altered LV load.

molecular biology↗

bioRxiv2023

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils

The Structural Maintenance of Chromosome (SMC) protein complexes cohesin, condensin and the Smc5/6 complex (Smc5/6) are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6s recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled chromosomal DNA at cohesin-dependent chromosome loop boundaries. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes, and efficiently initiates loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

molecular biology↗

bioRxiv2022

High salt induces cognitive impairment via the angiotensin II-AT1 and prostaglandin E2-EP1 systems

High salt (HS) intake is a known risk factor for hypertension and dementia. Clinical studies have shown that antihypertensive drugs can decrease the incidence of dementia. Accordingly, a strong relationship can be suggested between hypertension and cognitive impairment. It is well-known that angiotensin II (Ang II)-AT1 and prostaglandin E2 (PGE2)-EP1 systems are involved in hypertension and neurotoxicity. However, the involvement of these systems in HS-mediated hypertension and emotional and cognitive impairments remains unclear. Herein, we demonstrated that hypertension and impaired social behavior and object recognition memory following HS intake could be associated with tau hyperphosphorylation, decreased phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and postsynaptic density protein 95 (PSD95) expression in the prefrontal cortex and hippocampus of mice. These changes were blocked by pharmacological treatment with losartan, an Ang II receptor blocker (ARB), or EP1 gene knockout. Our findings suggest that Ang II-AT1 and PGE2-EP1 systems could be novel therapeutic targets for hypertension-induced dementia.

neuroscience↗

bioRxiv2022

Context-dependent 3D genome regulation by cohesin and related factors

Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, acetyltransferase, and the insulation factor CTCF. Although various models of regulation have been proposed (e.g., loop extrusion), how cohesin and related factors collectively or individually regulate the hierarchical chromatin structure and gene expression remains unclear. In this study, we have depleted cohesin and related factors and then conducted a comprehensive evaluation of the resulting 3D genome, transcriptome and epigenome data. We observed substantial variation in depletion effects among factors at topologically associating domain (TAD) boundaries and on interTAD interactions, which were partly related to epigenomic status. Gene expression changes were highly correlated with direct cohesin binding and gain of TAD boundaries than with the loss of boundaries. Our results suggested that cohesin positively regulates gene expression, whereas other mechanisms (e.g., cohesin turnover and acetylation) add to the diversity of this pattern of dysregulation. Moreover, cohesin was broadly enriched in active compartment A, but not in compartment B, which were retained even after CTCF depletion. Our rich dataset and the subsequent data-driven analysis support the context-specific regulation of chromatin folding by cohesin and related factors.

genomics↗

bioRxiv2020

Transcription and replication organize cohesin-dependent chromosome loops

Genome function and stability is strictly dependent on regulated folding of chromosomes in space and time. Chromosome loop formation by the protein complex cohesin is a central feature of this multilayer organization1. Accumulating evidence indicates that cohesin creates loops by extruding chromosomal DNA through its ring-like structure in a process that is controlled by the cohesin loading factor Scc2 and the unloader Wpl11-4. Cohesins chromosomal positioning is affected by transcription in both yeast and human cells5,6, and the complex localizes in the vicinity of replication forks7. However, if transcription directly influences chromosome looping remains an unresolved question, and the threedimensional organization of replicating chromosomes is unknown. Here we show that transcription and replication machineries create chromosome loop boundaries. We find that drug-induced depletion of chromosome-bound RNA polymerases triggers a rapid expansion of cohesin-dependent chromosome loops in the budding yeast Saccharomyces cerevisiae. New loop boundaries also form at a few highly expressed genes induced by the cellular stress response caused by the drug. The results also reveal that S-phase chromosomes are shaped by cohesin-dependent loops organized by transcription, with additional loop anchors at replication forks. Together, our results show that replication and transcription control the three-dimensional organization of the genome by blocking the progression of loop-forming cohesin. They also open for the possibility that the resulting positioning of loop-forming cohesin in the vicinity of transcription and replication machineries is part of cohesins functions in transcription control, sister chromatid cohesion and maintenance of fork stability.

molecular biology↗

bioRxiv2019

Synovial fibroblasts contribute to the genetic risk of rheumatoid arthritis through the synergistic action of cytokines

ObjectivesSynovial fibroblasts (SFs) produce a variety of pathogenic molecules in the inflamed synovium of rheumatoid arthritis (RA). We aimed to gain insight into the pathogenic mechanisms of SFs through elucidating the genetic contribution to molecular regulatory networks under inflammatory condition. MethodsSFs from RA and osteoarthritis (OA) patients (n=30 each) were stimulated with 8 different cytokines (IFN-, IFN-{gamma}, TNF-, IL-1{beta}, IL-6/sIL-6R, IL-17, TGF-{beta}1, IL-18) or a combination of all 8 (8-mix). Peripheral blood mononuclear cells (PBMCs) from the same patients were fractioned into five major immune cell subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes). Integrative analyses including mRNA expression, histone modifications (H3K27ac, H3K4me1, H3K4me3), 3D genome architecture and genetic variations of SNPs were performed. ResultsSFs exposed to synergistically acting cytokines produced markedly higher levels of pathogenic molecules, including CD40 whose expression was significantly affected by a RA risk SNP (rs6074022). Upon chromatin remodeling in activated SFs, RA risk loci were enriched in clusters of enhancers (super-enhancers; SEs) induced by synergistic proinflammatory cytokines. A RA risk SNP (rs28411362), located in a SE under synergistically acting cytokines, formed three-dimensional contact with the promoter of MTF1 gene, whose binding motif showed significant enrichment in stimulation specific-SEs. Consistently, inhibition of MTF1 suppressed cytokine and chemokine production from SFs and ameliorated mice model of arthritis. ConclusionsOur findings established the dynamic landscape of activated SFs, and yielded potential therapeutic targets associated with genetic risk of RA. Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIIn rheumatoid arthritis (RA), a variety of dysregulated molecules from immune cells and mesenchymal cells drive disease progression. Synovial fibroblasts (SFs), the most abundant resident mesenchymal cells in the inflamed synovium, produce a variety of pathogenic molecules including IL-6. C_LIO_LIGenome-wide association studies (GWAS) have identified more than 100 RA susceptibility loci. To gain insight into the pathogenic mechanisms of SFs, understanding the genetic contribution to molecular regulatory networks under inflammatory condition is crucial. C_LI What does this study add?O_LIIntegrated analyses of activated SFs demonstrated that SFs exposed to synergistically acting cytokines produced markedly higher levels of pathogenic molecules. Some of which were significantly affected by RA risk loci in a stimulation-specific manner. C_LIO_LIChromatin remodeling induced by synergistic proinflammatory cytokines were associated with RA heritability. Some transcription factors (MTF1, RUNX1) could be crucial for this structural rearrangement and the formation of inflammatory arthritis. C_LI How might this impact on clinical practice or future developments?O_LIOur findings established the dynamic landscape of activated SFs, and yielded potential therapeutic targets associated with genetic risk of RA. C_LI

immunology↗