Publications by authors named "Katsuhiko Shirahige"

207 Publications

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer.

Proc Natl Acad Sci U S A 2021 Aug;118(35)

Project for Cellular Senescence, Cancer Institute, Japanese Foundation for Cancer Research, 135-8550 Tokyo, Japan;

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.
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http://dx.doi.org/10.1073/pnas.2025647118DOI Listing
August 2021

Codependency and mutual exclusivity for gene community detection from sparse single-cell transcriptome data.

Nucleic Acids Res 2021 Oct;49(18):e104

Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Single-cell RNA-seq (scRNA-seq) can be used to characterize cellular heterogeneity in thousands of cells. The reconstruction of a gene network based on coexpression patterns is a fundamental task in scRNA-seq analyses, and the mutual exclusivity of gene expression can be critical for understanding such heterogeneity. Here, we propose an approach for detecting communities from a genetic network constructed on the basis of coexpression properties. The community-based comparison of multiple coexpression networks enables the identification of functionally related gene clusters that cannot be fully captured through differential gene expression-based analysis. We also developed a novel metric referred to as the exclusively expressed index (EEI) that identifies mutually exclusive gene pairs from sparse scRNA-seq data. EEI quantifies and ranks the exclusive expression levels of all gene pairs from binary expression patterns while maintaining robustness against a low sequencing depth. We applied our methods to glioblastoma scRNA-seq data and found that gene communities were partially conserved after serum stimulation despite a considerable number of differentially expressed genes. We also demonstrate that the identification of mutually exclusive gene sets with EEI can improve the sensitivity of capturing cellular heterogeneity. Our methods complement existing approaches and provide new biological insights, even for a large, sparse dataset, in the single-cell analysis field.
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http://dx.doi.org/10.1093/nar/gkab601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8501962PMC
October 2021

Suv4-20h2 protects against influenza virus infection by suppression of chromatin loop formation.

iScience 2021 Jun 29;24(6):102660. Epub 2021 May 29.

Laboratory of Regulation for Intractable Infectious Diseases, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085, Japan.

The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates disease pathogenesis. Here, we show that upon influenza virus infection, the H4K20me3 methyltransferase Suv4-20h2 binds the viral protein NP, which results in the inactivation of Suv4-20h2 and the dissociation of cohesin from Suv4-20h2. Inactivation of Suv4-20h2 by viral infection or genetic deletion allows the formation of an active chromatin loop at the HoxC8-HoxC6 loci coincident with cohesin loading. HoxC8 and HoxC6 proteins in turn enhance viral replication by inhibiting the Wnt-β-catenin mediated interferon response. Importantly, loss of Suv4-20h2 augments the pathology of influenza infection . Thus, Suv4-20h2 acts as a safeguard against influenza virus infection by suppressing cohesin-mediated loop formation.
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http://dx.doi.org/10.1016/j.isci.2021.102660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209278PMC
June 2021

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

Mol Cell 2021 07 30;81(13):2778-2792.e4. Epub 2021 Apr 30.

Center for Biological Research Margarita Salas (CIB-CSIC), Spanish National Research Council, Madrid, Spain. Electronic address:

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.
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http://dx.doi.org/10.1016/j.molcel.2021.04.006DOI Listing
July 2021

Erratum to "Functional control of Eco1 through the MCM complex in sister chromatid cohesion" [Gene 784C (2021) 145584].

Gene 2021 Jun 16;787:145634. Epub 2021 Apr 16.

Laboratory of Genome Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.gene.2021.145634DOI Listing
June 2021

Small extracellular vesicles derived from interferon-γ pre-conditioned mesenchymal stromal cells effectively treat liver fibrosis.

NPJ Regen Med 2021 Mar 30;6(1):19. Epub 2021 Mar 30.

Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.

Mesenchymal stromal cells (MSCs) are used for ameliorating liver fibrosis and aiding liver regeneration after cirrhosis; Here, we analyzed the therapeutic potential of small extracellular vesicles (sEVs) derived from interferon-γ (IFN-γ) pre-conditioned MSCs (γ-sEVs). γ-sEVs effectively induced anti-inflammatory macrophages with high motility and phagocytic abilities in vitro, while not preventing hepatic stellate cell (HSC; the major source of collagen fiber) activation in vitro. The proteome analysis of MSC-derived sEVs revealed anti-inflammatory macrophage inducible proteins (e.g., annexin-A1, lactotransferrin, and aminopeptidase N) upon IFN-γ stimulation. Furthermore, by enabling CXCR1+ macrophage accumulation in the damaged area, γ-sEVs ameliorated inflammation and fibrosis in the cirrhosis mouse model more effectively than sEVs. Single cell RNA-Seq analysis revealed diverse effects, such as induction of anti-inflammatory macrophages and regulatory T cells, in the cirrhotic liver after γ-sEV administration. Overall, IFN-γ pre-conditioning altered sEVs resulted in efficient tissue repair indicating a new therapeutic strategy.
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http://dx.doi.org/10.1038/s41536-021-00132-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010072PMC
March 2021

Functional control of Eco1 through the MCM complex in sister chromatid cohesion.

Gene 2021 Jun 20;784:145584. Epub 2021 Mar 20.

Laboratory of Genome Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address:

Sister chromatid cohesion (SCC) is essential for the maintenance of genome integrity. The establishment of SCC is coupled to DNA replication, and this is achieved in budding yeast Saccharomyces cerevisiae by a mechanism that is dependent on the interaction between Eco1 acetyltransferase and PCNA in the DNA replication complex. In vertebrates, the Eco1 homolog ESCO2 has been reported to interact with MCM complex in the DNA replication complex to establish DNA replication-dependent cohesion. Here we show that budding yeast Eco1 is also physically interacted with the MCM complex. We found that Eco1 was specifically bound to Mcm2 subunit in the MCM complex and they interacted via their N-terminal regions, using yeast two-hybrid system. The underlying mechanism of the interaction was different between yeast and vertebrates. Intensive molecular dissection of Eco1 identified residues important for interaction with Mcm2 and/or PCNA. Mutant forms of Eco1 (Eco1 and Eco1), where sets of the identified residues were substituted with alanine, resulted in impaired SCC, decreased level of acetylation of Smc3, and a reduction of Eco1 protein amount in yeast cells. We, hence, suggest that Eco1 is stabilized by its interactions with MCM complex and PCNA, which allows it to promote DNA replication-coupled SCC establishment.
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http://dx.doi.org/10.1016/j.gene.2021.145584DOI Listing
June 2021

Single-cell transcriptional analysis reveals developmental stage-dependent changes in retinal progenitors in the murine early optic vesicle.

Biochem Biophys Res Commun 2021 03 3;543:80-86. Epub 2021 Feb 3.

Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan. Electronic address:

The optic vesicle in the developing embryonic eye contains a multitude of neuroepithelial progenitors that subsequently differentiate into functionally distinct domains of the optic cup, such as the neural retina, pigment epithelium, and optic stalk. To investigate cell-type diversity across early optic vesicles before regionalization of the optic cup, we performed single-cell RNA-sequencing (scRNA-seq) using 7989 cells from the presumptive eye area in mouse embryos at the 12-26-somite stages at five developmental time points. We demonstrated the presence of seven optic vesicle populations. Moreover, the four populations of retinal progenitor cells could be classified according to their stage-dependent time point, and these cells exhibited altered expression of several structural and metabolic key genes, such as Col9a1 and Ckb, just before regionalization of the optic cup. From these data, we provide the first report on stage-dependent transcriptional profiles during initial retinal specification at single-cell resolution and highlight the unexpected developmental heterogeneity of the murine optic vesicle structure.
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http://dx.doi.org/10.1016/j.bbrc.2021.01.043DOI Listing
March 2021

Comparative proteomic analysis to identify the novel target gene of angiotensin II in adrenocortical H295R cells.

Endocr J 2021 Apr 26;68(4):441-450. Epub 2020 Dec 26.

Department of Molecular Endocrinology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.

Angiotensin II (Ang II) is a well-known peptide that maintains the balance of electrolytes in the higher vertebrates. Ang II stimulation in the adrenal gland induces the synthesis of mineralocorticoids, mainly aldosterone, through the up-regulation of aldosterone synthase (CYP11B2) gene expression. Additionally, it has been reported that Ang II activates multiple signaling pathways such as mitogen-activated protein kinase (MAPK) and Ca signaling. Although Ang II has various effects on the cellular signaling in the adrenal cells, its biological significance, except for the aldosterone synthesis, is still unclear. In this study, we attempted to search the novel target gene(s) of Ang II in the human adrenal H295R cells using a proteomic approach combined with stable isotopic labeling using amino acid in cell culture (SILAC). Interestingly, we found that Ang II stimulation elevated the expression of phosphofructokinase type platelet (PFKP) in both protein and mRNA levels. Moreover, transactivation of PFKP by Ang II was dependent on extracellular-signal-regulated kinase (ERK) 1/2 activation. Finally, we observed that Ang II treatment facilitated glucose uptake in the H295R cells. Taken together, we here identified PFKP as a novel target gene of Ang II, indicating that Ang II not only stimulates steroidogenesis but also affects glucose metabolism.
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http://dx.doi.org/10.1507/endocrj.EJ20-0144DOI Listing
April 2021

Transcription-dependent cohesin repositioning rewires chromatin loops in cellular senescence.

Nat Commun 2020 11 27;11(1):6049. Epub 2020 Nov 27.

Cancer Research UK Cambridge Institute, University of Cambridge, Robinson Way, Cambridge, UK.

Senescence is a state of stable proliferative arrest, generally accompanied by the senescence-associated secretory phenotype, which modulates tissue homeostasis. Enhancer-promoter interactions, facilitated by chromatin loops, play a key role in gene regulation but their relevance in senescence remains elusive. Here, we use Hi-C to show that oncogenic RAS-induced senescence in human diploid fibroblasts is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks often at the 3' end of a subset of active genes. RAS-induced de novo cohesin peaks are transcription-dependent and enriched for senescence-associated genes, exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Similar IL1B induction with de novo cohesin appearance and new loop formation are observed in terminally differentiated macrophages, but not TNFα-treated cells. These results suggest that RAS-induced senescence represents a cell fate determination-like process characterised by a unique gene expression profile and 3D genome folding signature, mediated in part through cohesin redistribution on chromatin.
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http://dx.doi.org/10.1038/s41467-020-19878-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695716PMC
November 2020

Parsing multiomics landscape of activated synovial fibroblasts highlights drug targets linked to genetic risk of rheumatoid arthritis.

Ann Rheum Dis 2020 Nov 2. Epub 2020 Nov 2.

Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

Objectives: Synovial fibroblasts (SFs) are one of the major components of the inflamed synovium in 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.

Methods: SFs from RA and osteoarthritis (OA) patients (n=30 each) were stimulated with eight different cytokines (interferon (IFN)-α, IFN-γ, tumour necrosis factor-α, interleukin (IL)-1β, IL-6/sIL-6R, IL-17, transforming growth factor-β1, IL-18) or a combination of all 8 (8-mix). Peripheral blood mononuclear cells were fractioned into five immune cell subsets (CD4 T cells, CD8 T cells, B cells, natural killer (NK) cells, monocytes). Integrative analyses including mRNA expression, histone modifications (H3K27ac, H3K4me1, H3K4me3), three-dimensional (3D) genome architecture and genetic variations of single nucleotide polymorphisms (SNPs) were performed.

Results: Unstimulated RASFs differed markedly from OASFs in the transcriptome and epigenome. Meanwhile, most of the responses to stimulations were shared between the diseases. Activated SFs expressed pathogenic genes, including whose induction by IFN-γ was significantly affected by an RA risk SNP (rs6074022). On chromatin remodelling in activated SFs, RA risk loci were enriched in clusters of enhancers (super-enhancers; SEs) induced by synergistic proinflammatory cytokines. An RA risk SNP (rs28411362), located in an SE under synergistically acting cytokines, formed 3D contact with the promoter of 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.

Conclusions: Our findings established the dynamic landscape of activated SFs and yielded potential therapeutic targets associated with genetic risk of RA.
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http://dx.doi.org/10.1136/annrheumdis-2020-218189DOI Listing
November 2020

Publisher Correction: Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

Nat Immunol 2020 Dec;21(12):1611-1612

Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Suita, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41590-020-00809-wDOI Listing
December 2020

Age-Dependent Ribosomal DNA Variations in Mice.

Mol Cell Biol 2020 10 26;40(22). Epub 2020 Oct 26.

Institute for Quantitative Biosciences, University of Tokyo, Tokyo, Japan

The rRNA gene, which consists of tandem repetitive arrays (ribosomal DNA [rDNA] repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in aging mammalian cells. Using quantitative single-cell analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to levels in 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice, and two of them were found in the conserved region in budding yeast. We established yeast strains with the old-mouse-specific mutations and found that they shortened the life span of the cells. Our findings suggest that rDNA is also fragile in mammalian cells and that alterations within this region have a profound effect on cellular function.
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http://dx.doi.org/10.1128/MCB.00368-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588874PMC
October 2020

Bioinformatical dissection of fission yeast DNA replication origins.

Open Biol 2020 07 22;10(7):200052. Epub 2020 Jul 22.

Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Replication origins in eukaryotes form a base for assembly of the pre-replication complex (pre-RC), thereby serving as an initiation site of DNA replication. Characteristics of replication origin vary among species. In fission yeast , DNA of high AT content is a distinct feature of replication origins; however, it remains to be understood what the general molecular architecture of fission yeast origin is. Here, we performed ChIP-seq mapping of Orc4 and Mcm2, two representative components of the pre-RC, and described the characteristics of their binding sites. The analysis revealed that fission yeast efficient origins are associated with two similar but independent features: a ≥15 bp-long motif with stretches of As and an AT-rich region of a few hundred bp. The A-rich motif was correlated with chromosomal binding of Orc, a DNA-binding component in the pre-RC, whereas the AT-rich region was associated with efficient binding of the DNA replicative helicase Mcm. These two features, in combination with the third feature, a transcription-poor region of approximately 1 kb, enabled to distinguish efficient replication origins from the rest of chromosome arms with high accuracy. This study, hence, provides a model that describes how multiple functional elements specify DNA replication origins in fission yeast genome.
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http://dx.doi.org/10.1098/rsob.200052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574548PMC
July 2020

Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

Nat Immunol 2020 08 22;21(8):950-961. Epub 2020 Jun 22.

Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Suita, Japan.

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.
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http://dx.doi.org/10.1038/s41590-020-0700-yDOI Listing
August 2020

Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells.

Commun Biol 2020 05 4;3(1):208. Epub 2020 May 4.

RIKEN Cluster for Pioneering Research, Tsukuba, Ibaraki, Japan.

Paternal environmental factors can epigenetically influence gene expressions in offspring. We demonstrate that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes.
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http://dx.doi.org/10.1038/s42003-020-0935-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198565PMC
May 2020

A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint.

EMBO Rep 2020 06 19;21(6):e50257. Epub 2020 Apr 19.

CRBM, University of Montpellier, CNRS, Montpellier, France.

The Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1-3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways.
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http://dx.doi.org/10.15252/embr.202050257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271318PMC
June 2020

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

Cancer Discov 2020 06 5;10(6):836-853. Epub 2020 Apr 5.

Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including . However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in -cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269820PMC
June 2020

Tex19.1 inhibits the N-end rule pathway and maintains acetylated SMC3 cohesin and sister chromatid cohesion in oocytes.

J Cell Biol 2020 05;219(5)

Medical Research Council Human Genetics Unit, Medical Research Council Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, UK.

Age-dependent oocyte aneuploidy, a major cause of Down syndrome, is associated with declining sister chromatid cohesion in postnatal oocytes. Here we show that cohesion in postnatal mouse oocytes is regulated by Tex19.1. We show Tex19.1-/- oocytes have defects maintaining chiasmata, missegregate their chromosomes during meiosis, and transmit aneuploidies to the next generation. Furthermore, we show that mouse Tex19.1 inhibits N-end rule protein degradation mediated by its interacting partner UBR2, and that Ubr2 itself has a previously undescribed role in negatively regulating the acetylated SMC3 subpopulation of cohesin in mitotic somatic cells. Lastly, we show that acetylated SMC3 is associated with meiotic chromosome axes in mouse oocytes, and that this population of cohesin is specifically depleted in the absence of Tex19.1. These findings indicate that Tex19.1 regulates UBR protein activity to maintain acetylated SMC3 and sister chromatid cohesion in postnatal oocytes and prevent aneuploidy from arising in the female germline.
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http://dx.doi.org/10.1083/jcb.201702123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199850PMC
May 2020

ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet.

Mol Cell 2020 05 19;78(3):445-458.e6. Epub 2020 Mar 19.

RIKEN Cluster for Pioneering Research, Tsukuba, Ibaraki 305-0074, Japan; Department of Functional Genomics, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. Electronic address:

Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7 mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7 mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.
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http://dx.doi.org/10.1016/j.molcel.2020.02.028DOI Listing
May 2020

The Microbiome of the Meibum and Ocular Surface in Healthy Subjects.

Invest Ophthalmol Vis Sci 2020 02;61(2):18

,.

Purpose: The purpose of this study was to investigate the microbiome in the meibum, conjunctival sac, and eyelid skin in young and elderly healthy subjects, and analyze the effect that age, sex, and region have on microbiome composition.

Methods: This study involved 36 healthy subjects (young-age subjects: 9 men/9 women, age range: 20-35 years; elderly age subjects: 9 men/9 women, age range: 60-70 years). In all subjects, lower-eyelid meibum, lower conjunctival sac, and lower-eyelid skin specimens were collected from one eye, and then stored at -20°C. Taxonomic composition of the microbiome was obtained via 16S rRNA gene sequencing, and then analyzed.

Results: The meibum microbiome showed a high α-diversity (within-community diversity), particularly in the young subjects. However, in approximately 30% of the elderly subjects, a low-diversity microbiome dominated by Corynebacterium sp. or Neisseriaceae was observed. In the young subjects, the microbiome of the meibum resembled that of the conjunctival-sac, yet in the elderly subjects, the microbiome of the conjunctival-sac became more similar to that of the eyelid skin. The eyelid-skin microbiome was relatively simple, and was typically dominated by Propionibacterium acnes in the young subjects, or by Corynebacterium sp. or Neisseriaceae in the elderly subjects. In both age groups, no significant difference was seen between the men and women in regard to the meibum, conjunctival-sac, and eyelid-skin microbiome.

Conclusions: Our findings confirmed that the meibum of healthy adult-age subjects harbors highly diverse microbiota, and revealed that the meibum microbiome, especially the decrease of its diversity, alters with aging and may affect the homeostasis of the ocular surface.
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http://dx.doi.org/10.1167/iovs.61.2.18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326502PMC
February 2020

The Phosphatase PP1 Promotes Mitotic Slippage through Mad3 Dephosphorylation.

Curr Biol 2020 01 9;30(2):335-343.e5. Epub 2020 Jan 9.

CRBM, University of Montpellier, CNRS, 1919 Route de Mende, 34293 Montpellier, France. Electronic address:

Accurate chromosome segregation requires bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the spindle assembly checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays anaphase onset until all chromosomes are bipolarly attached [1]. SAC signaling fires at kinetochores and involves a soluble mitotic checkpoint complex (MCC) that inhibits the anaphase-promoting complex (APC) [2, 3]. The mitotic delay imposed by SAC, however, is not everlasting. If kinetochores fail to establish bipolar connections, cells can escape from the SAC-induced mitotic arrest through a process called mitotic slippage [4]. Mitotic slippage occurs in the presence of SAC signaling at kinetochores [5, 6], but whether and how MCC stability and APC inhibition are actively controlled during slippage is unknown. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached [7, 8]. PP1 turns off SAC signaling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores [9-11]. Here, we show that, in budding yeast, PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilizes the MCC without affecting the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.
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http://dx.doi.org/10.1016/j.cub.2019.11.054DOI Listing
January 2020

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

Epigenetics Chromatin 2019 12 19;12(1):77. Epub 2019 Dec 19.

Department of Cardiology, Saitama Medical University International Medical Center, Saitama, 350-1298, Japan.

Background: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.

Results: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.

Conclusions: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.
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http://dx.doi.org/10.1186/s13072-019-0319-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921469PMC
December 2019

Chromosome-associated RNA-protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe.

Nat Commun 2019 12 6;10(1):5598. Epub 2019 Dec 6.

Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, Kobe, 651-2492, Japan.

Pairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA-protein complexes is required for robust pairing. Moreover, the lncRNA-protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA-protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.
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http://dx.doi.org/10.1038/s41467-019-13609-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898681PMC
December 2019

Missense Mutations in NKAP Cause a Disorder of Transcriptional Regulation Characterized by Marfanoid Habitus and Cognitive Impairment.

Am J Hum Genet 2019 11 3;105(5):987-995. Epub 2019 Oct 3.

Zebrafish Core Facility, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

NKAP is a ubiquitously expressed nucleoplasmic protein that is currently known as a transcriptional regulatory molecule via its interaction with HDAC3 and spliceosomal proteins. Here, we report a disorder of transcriptional regulation due to missense mutations in the X chromosome gene, NKAP. These mutations are clustered in the C-terminal region of NKAP where NKAP interacts with HDAC3 and post-catalytic spliceosomal complex proteins. Consistent with a role for the C-terminal region of NKAP in embryogenesis, nkap mutant zebrafish with a C-terminally truncated NKAP demonstrate severe developmental defects. The clinical features of affected individuals are highly conserved and include developmental delay, hypotonia, joint contractures, behavioral abnormalities, Marfanoid habitus, and scoliosis. In affected cases, transcriptome analysis revealed the presence of a unique transcriptome signature, which is characterized by the downregulation of long genes with higher exon numbers. These observations indicate the critical role of NKAP in transcriptional regulation and demonstrate that perturbations of the C-terminal region lead to developmental defects in both humans and zebrafish.
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http://dx.doi.org/10.1016/j.ajhg.2019.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848994PMC
November 2019

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken.

Sci Rep 2019 08 29;9(1):12560. Epub 2019 Aug 29.

Department of Systems BioMedicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9's functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9's regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.
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http://dx.doi.org/10.1038/s41598-019-48979-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715657PMC
August 2019

Aging of spermatogonial stem cells by Jnk-mediated glycolysis activation.

Proc Natl Acad Sci U S A 2019 08 29;116(33):16404-16409. Epub 2019 Jul 29.

Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan;

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant expression activated c- N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Down-regulation of probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the -deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.
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http://dx.doi.org/10.1073/pnas.1904980116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697785PMC
August 2019

The novel lncRNA CALIC upregulates AXL to promote colon cancer metastasis.

EMBO Rep 2019 08 29;20(8):e47052. Epub 2019 Jul 29.

Laboratory of Molecular and Genetic Information, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Long non-coding RNAs (lncRNAs) are aberrantly expressed in many disease conditions, including cancer. Accumulating evidence indicates that some lncRNAs may play critical roles in cancer progression and metastasis. Here, we identify a set of lncRNAs that are upregulated in metastatic subpopulations isolated from colon cancer HCT116 cells in vivo and show that one of these lncRNAs, which we name CALIC, is required for the metastatic activity of colon cancer cells. We show that CALIC associates with the RNA-binding protein hnRNP-L and imparts specificity to hnRNP-L-mediated gene expression. Furthermore, we demonstrate that the CALIC/hnRNP-L complex upregulates the tyrosine kinase receptor AXL and that knockdown of CALIC or AXL using shRNA in colon cancer cells attenuates their ability to form metastases in mice. These results suggest that the CALIC/hnRNP-L complex enhances the metastatic potential of colon cancer cells.
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http://dx.doi.org/10.15252/embr.201847052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6680119PMC
August 2019
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