Language：English   Publishing type：Research paper (scientific journal)  

The "histone code," defined by the combinatorial patterns of post-translational modifications (PTMs) on histones, plays a pivotal role in chromatin structure and gene expression. Tools for the regioselective introduction of histone PTMs in living cells are critical for dissecting the functions of these epigenetic marks. Here, we report the design and development of three regioselective catalysts that acetylate distinct lysine residues (K43, K108, and K120) on histone H2B. Using a combination of molecular dynamics simulations of catalyst-nucleosome complexes and systematic experimental optimization of catalyst structures, we identified key design principles for achieving regioselectivity. Specifically, excluding highly reactive off-target lysine residues from the catalyst effective region (CER) while maintaining proximity to a target lysine residue proved crucial. Biochemical and cellular analyses of the catalytic histone acetylation revealed that each lysine acetylation elicited unique effects on the binding affinity and activity of nucleosome-interacting molecules, as well as on transcriptional programs and cellular phenotypes. These findings establish a framework for designing regioselective histone acetylation catalysts and advance our understanding of the regulatory mechanisms underlying histone PTMs.

DOI： 10.1021/jacs.5c01699

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Authorship：Corresponding author   Language：English  

DOI： 10.1101/2025.04.09.648027

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Authorship：Corresponding author   Language：English  

DOI： 10.1101/2025.02.06.636921

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/pnasnexus/pgae527

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.13149

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Language：English   Publishing type：Research paper (scientific journal)  

G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9Cre/+ mice with G9afl/fl mice to generate G9afl/fl; Sox9Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development.

DOI： 10.1038/s41598-024-71570-5

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-024-51153-8

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DOI： 10.1101/2024.07.25.605169

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Language：English   Publishing type：Research paper (scientific journal)  

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

DOI： 10.1096/fj.202400449RR

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Publisher：Research Square Platform LLC  

Abstract <p>Transcription factor-bound chromatin structures regulate cell lineages in multicellular organisms. Single-cell epigenomics has the potential to reveal lineage determination on chromatin structure, but the methodology is still in development. Here, we develop single-cell combinatorial-indexing multi-target Chromatin Integration Labeling followed by sequencing (sci-mtChIL-seq) as a single-cell multi-epigenomics approach, which enables simultaneous single-cell analysis of both RNA polymerase II binding to chromatin and epigenomic factors such as transcription factors/histones. We apply sci-mtChIL-seq to analyze the binding dynamics of skeletal-muscle-specific transcription factor MyoD during mouse embryonic myogenesis. Based on RNA polymerase II-bound gene profiles, single-cells are efficiently classified into myogenic-clusters and ordered pseudotemporally. MyoD exhibits genome-wide binding in the muscle-progenitor-cell population, but in myocytes, this transitions toward enrichment in muscle-specific genes on active chromatin. Thus, sci-mtChIL-seq can be a powerful tool to analyze epigenomic dynamics in cell fate determination.</p>

DOI： 10.21203/rs.3.rs-4503255/v1

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Other Link： https://www.researchsquare.com/article/rs-4503255/v1.html

Publisher：American Chemical Society (ACS)  

The combination of post-translational modifications (PTMs) of histones, a major component of chromatin, defines “histone code” having distinct impact on chromatin structures and gene expression. Therefore, methods to introduce histone PTMs at desired positions are useful tools for investigating the functions of epigenetic marks, especially to understand consequences of specific modifications in histones. Chemical catalysts, such as lysine (Lys)-acetylating catalysts, have been emerged to regioselectively promote histone PTMs without relying on histone-modifying enzymes. However, it has been poorly under-stood what factors determine Lys-residue selectivity of the histone acetylating catalysts. Here we show that the intrinsic reac-tivity, distance from the catalytically active site, and conformational flexibility of Lys residues, are the critical factors for determining acetylation yield and regioselectivity. Combining molecular dynamics simulations of catalyst-nucleosome com-plexes and experimental optimization of the catalyst structure, we developed three catalysts that selectively acetylated K43, K108, and K120 of H2B, respectively. Biochemical analyses of the regioselectively acetylated nucleosomes revealed that each Lys acetylation showed distinct impacts on inter-nucleosomal interactions and the affinity to a nucleosome-binding molecule. Our data provide a guideline for developing regioselective histone acetylation catalysts and may further accelerate studies of epigenetics regulated by histone PTMs.

DOI： 10.26434/chemrxiv-2024-d1l1b

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.

DOI： 10.1083/jcb.202310084

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.13109

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Language：English   Publishing type：Research paper (scientific journal)  

Mitochondria play a vital role in non-shivering thermogenesis in both brown and subcutaneous white adipose tissues (BAT and scWAT, respectively). However, specific regulatory mechanisms driving mitochondrial function in these tissues have been unclear. Here we demonstrate that prolonged activation of β-adrenergic signaling induces epigenetic modifications in scWAT, specifically targeting the enhancers for the mitochondria master regulator genes Pgc1a/b. This is mediated at least partially through JMJD1A, a histone demethylase that in response to β-adrenergic signals, facilitates H3K9 demethylation of the Pgc1a/b enhancers, promoting mitochondrial biogenesis and the formation of beige adipocytes. Disruption of demethylation activity of JMJD1A in mice impairs activation of Pgc1a/b driven mitochondrial biogenesis and limits scWAT beiging, contributing to reduced energy expenditure, obesity, insulin resistance, and metabolic disorders. Notably, JMJD1A demethylase activity is not required for Pgc1a/b dependent thermogenic capacity of BAT especially during acute cold stress, emphasizing the importance of scWAT thermogenesis in overall energy metabolism.

DOI： 10.1016/j.isci.2024.109398

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9—the kinase that releases paused polymerase II—is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.

DOI： 10.1038/s41556-024-01389-9

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Other Link： https://www.nature.com/articles/s41556-024-01389-9

Language：English   Publishing type：Research paper (scientific journal)  

The SWI/SNF chromatin remodeling complex consists of more than ten component proteins that form a large protein complex of >1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem cells (ESCs). Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ESCs, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested that there was an ectopic genomic distribution of the SWI/SNF complex upon disruption of Smarce1, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ESC differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

DOI： 10.1242/jcs.260467

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Publisher：Cold Spring Harbor Laboratory  

Abstract

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into theRosa26locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X-chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

DOI： 10.1101/2024.03.13.584715

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DOI： 10.21203/rs.3.rs-3991504/v1

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Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVES: The tongue comprises multiple tissues of different embryonic origins, including pharyngeal arch, somite, and cranial neural crest (CNC). However, its developmental regulatory mechanisms, especially those involving epigenetic modifiers, remain poorly understood. This study examined the roles of the epigenetic modifier G9a in murine tongue development. METHODS: We deleted G9a using Sox 9 (SRY-related HMG-box gene 9)-Cre recombinase, which acts in tongue progenitor cells, including CNC-derived cells, to generate G9a conditional knockout (cKO) mice. Histochemical and immunohistochemical analyses were conducted on sections prepared from tongue tissues of control and cKO mice. RESULTS: Cre-dependent LacZ reporter mice, generated by crossing Rosa-LacZ mice with sox9-Cre mice, revealed Cre recombinase activity in the mucosal epithelium and tongue connective tissue of the embryonic tongue. Tongue volume was significantly reduced on embryonic day 17.5 (E17.5) and postnatal day 0 (P0) in cKO mice. Histological sections showed that the lingual mucosal epithelium was thinner in cKO mice. Reduced G9a levels were accompanied by decreased levels of a G9a substrate, dimethylated lysine 9 in histone H3, in the embryonic tongue. BrdU injection at E16.5 revealed reduced numbers of BrdU-positive cells in the mucosal epithelium and underlying connective tissue at E17.5 in cKO mice, indicating suppression of cell proliferation in both tissues. Investigation of keratin 5 and 8 protein localization showed significantly suppressed expression in the lingual mucosal epithelium in cKO mice. CONCLUSIONS: G9a is required for proper proliferation and differentiation of sox9-expressing tongue progenitor cells and is thereby involved in tongue development.

DOI： 10.1016/j.job.2023.12.007

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/DGD.12908

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/J.CEB.2023.102319

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DOI： 10.1016/J.BONE.2023.117000

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/J.CELREP.2023.113576

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DOI： 10.1101/2023.12.03.569804

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Language：English  

DOI： 10.1038/s41596-023-00940-6

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Publisher：Cold Spring Harbor Laboratory  

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and inter-cellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving comprehensive acquisition of spatial protein information from cells. However, resolution is still compromised when visualizing activated signals. Here we developed Precise Emission Canceling Antibody (PECAb) that have cleavable fluorescent labeling. PECAbs allow high-resolution sequential imaging using hundreds of antibodies and allow reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

DOI： 10.1101/2023.10.17.561810

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Publisher：Cold Spring Harbor Laboratory  

The nuclear lamina (NL) lines the inner nuclear membrane (INM) of the nuclear envelope (NE) to maintain nuclear structure in metazoan cells. The major NL components, the nuclear lamins contribute to the protection against NE rupture induced by mechanical stress. Lamin C (LC) but not lamin A (LA) is rapidly diffused from the nucleoplasm to sites of NE rupture induced by laser microirradiation in immortalized mouse embryonic fibroblasts (MEFs). However, the molecular mechanism for differentiating LA from LC with respect to the localization/accumulation at the rupture sites still remains unknown. In this study, we reveal by immunofluorescence, live-cell imaging and fluorescence correlation spectroscopy (FCS) that the CaaX motif, the second cleavage site and the LA-characteristic segment (LACS) regulate the localization of LA to the rupture sites. Our data suggest that Hutchinson-Gilford Progeria syndrome (HGPS) might exhibit the delay in localizing LA to the rupture sites through this mechanism.

DOI： 10.1101/2023.09.02.555826

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DOI： 10.1101/2023.09.02.555826

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

Cell motility is related to the higher-order structure of chromatin. Stimuli that induce cell migration change chromatin organization; such stimuli include elevated histone H3 lysine 9 trimethylation (H3K9me3). We previously showed that depletion of histone H3 lysine 9 methyltransferase, SUV39H1, suppresses directional cell migration. However, the molecular mechanism underlying this association between chromatin and cell migration remains elusive. The Golgi apparatus is a cell organelle essential for cell motility. In this study, we show that loss of H3K9 methyltransferase SUV39H1 but not SETDB1 or SETDB2 causes dispersion of the Golgi apparatus throughout the cytoplasm. The Golgi dispersion triggered by SUV39H1 depletion is independent of transcription, centrosomes, and microtubule organization, but is suppressed by depletion of any of the following three proteins: LINC complex components SUN2, nesprin-2, or microtubule plus-end-directed kinesin-like protein KIF20A. In addition, SUN2 is closely localized to H3K9me3, and SUV39H1 affects the mobility of SUN2 in the nuclear envelope. Further, inhibition of cell motility caused by SUV39H1 depletion is restored by suppression of SUN2, nesprin-2, or KIF20A. In summary, these results show the functional association between chromatin organization and cell motility via the Golgi organization regulated by the LINC complex.

DOI： 10.1371/journal.pone.0283490

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Language：English   Publishing type：Research paper (scientific journal)  

Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.

DOI： 10.1093/nar/gkad387

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Language：English   Publishing type：Research paper (scientific journal)  

Higher-order genomic structures play a critical role in regulating gene expression by influencing the spatial proximity of promoters and enhancers. Live-cell imaging studies have demonstrated that three-dimensional genome structures undergo dynamic changes over time. Transcription is also dynamic, with genes frequently switching between active and inactive states. Recent observations suggest that the formation of condensates, composed of transcription-related factors, RNA, and RNA-binding proteins, around genes can regulate transcription. Advancements in technology have facilitated the visualization of the intricate spatiotemporal relationship between higher-order genomic structures, condensate formation, and transcriptional activity in living cells.

DOI： 10.1016/j.sbi.2023.102615

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

ABSTRACT

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.

DOI： 10.1242/bio.059783

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Other Link： https://journals.logists.com/bio/bio/article-pdf/doi/10.1242/bio.059783/2857331/bio059783.pdf

Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, including Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

DOI： 10.1093/nar/gkad342

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Language：English   Publisher：(一社)日本生理学会  

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DOI： 10.1021/acs.analchem.2c04133

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DOI： 10.1093/stmcls/sxac082

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DOI： 10.3390/molecules28062524

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1101/2023.03.02.530854

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1101/2023.01.18.524584

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.cub.2022.11.015

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DOI： 10.1016/j.bbrc.2022.11.102

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

DOI： 10.1083/jcb.202201024

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1063/5.0133277

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DOI： 10.1101/2022.10.09.511457

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DOI： 10.1063/5.0107395

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Protein kinase A promotes beige adipogenesis downstream from β-adrenergic receptor signaling by phosphorylating proteins, including histone H3 lysine 9 (H3K9) demethylase JMJD1A. To ensure homeostasis, this process needs to be reversible however, this step is not well understood. We show that myosin phosphatase target subunit 1- protein phosphatase 1β (MYPT1-PP1β) phosphatase activity is inhibited via PKA-dependent phosphorylation, which increases phosphorylated JMJD1A and beige adipogenesis. Mechanistically, MYPT1-PP1β depletion results in JMJD1A-mediated H3K9 demethylation and activation of the Ucp1 enhancer/promoter regions. Interestingly, MYPT1-PP1β also dephosphorylates myosin light chain which regulates actomyosin tension-mediated activation of YAP/TAZ which directly stimulates Ucp1 gene expression. Pre-adipocyte specific Mypt1 deficiency increases cold tolerance with higher Ucp1 levels in subcutaneous white adipose tissues compared to control mice, confirming this regulatory mechanism in vivo. Thus, we have uncovered regulatory cross-talk involved in beige adipogenesis that coordinates epigenetic regulation with direct activation of the mechano-sensitive YAP/TAZ transcriptional co-activators.

DOI： 10.1038/s41467-022-33363-0

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

<jats:p>Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that is an aggregation-induced emission luminogen (AIEgen). AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.</jats:p>

DOI： 10.26434/chemrxiv-2022-p8jq4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

DOI： 10.1242/dev.200243

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DOI： 10.1083/jcb.202104134

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ceb.2022.01.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

Summary

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

DOI： 10.1101/2022.01.06.472721

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Publishing type：Research paper (scientific journal)  

DOI： 10.1266/GGS.22-00012

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/S43587-021-00147-Y

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Other Link： https://www.nature.com/articles/s43587-021-00147-y

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Although intracellular biomarkers can be imaged with fluorescent dye(s)-labeled antibodies, the use of such probes for precise imaging of intracellular biomarkers in living cells remains challenging due to background noise...

DOI： 10.1039/d2sc02355e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells.

DOI： 10.1083/jcb.202103078

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

DOI： 10.1093/nar/gkab1068

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>The morphology of the Golgi complex is influenced by the cellular context, which strictly correlates with nuclear functions; however, the mechanism underlying this association remains elusive. The inner nuclear membrane SUN proteins, SUN1 and SUN2, have diverse functions together with the outer nuclear membrane nesprin proteins, which comprise the LINC complex. We found that depletion of SUN1 leads to Golgi complex dispersion with maintenance of ministacks and retained function for vesicle transport through the Golgi complex. In addition, SUN2 associates with microtubule plus-end-directed motor KIF20A, possibly via nesprin-2. KIF20A plays a role in the Golgi dispersion in conjunction with the SUN2-nesprin-2 LINC complex in SUN1-depleted cells, suggesting that SUN1 suppresses the function of the SUN2-nesprin-2 LINC complex under a steady-state condition. Further, SUN1-knockout mice, which show impaired cerebellar development and cerebellar ataxia, presented altered Golgi morphology in Purkinje cells. These findings revealed a regulation of the Golgi organization by the LINC complex.

DOI： 10.1038/s41598-021-84750-4

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Other Link： http://www.nature.com/articles/s41598-021-84750-4

Language：English   Publishing type：Research paper (scientific journal)  

The analysis of gene expression regulation, or the epigenome analysis, at the single-cell level is at the forefront of genomics research. To elucidate the mechanisms that regulate gene expression, chromatin immunoprecipitation has been conventionally used for determining the binding sites of DNA-binding proteins, such as histones and transcription factors. Now several new approaches have been emerged to reveal epigenome states at the single-cell level. Instead of using immunoprecipitation of fragmented chromatin, in situ reactions using cells or nuclei, combining with transposase tagging and other methods, have enabled single-cell analysis. Furthermore, single-cell multiomics techniques to simultaneously profiling transcriptome and open chromatin or histone modification have been developed. These single-cell analyses have the potential to identify different cell types in a cell population and reveal the dynamic changes of gene regulation, although those technologies have not yet reached a level for general application.

DOI： 10.1016/j.sbi.2021.06.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41467-021-23417-0

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Other Link： http://www.nature.com/articles/s41467-021-23417-0

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41467-021-21589-3

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Other Link： http://www.nature.com/articles/s41467-021-21589-3

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Spatiotemporal changes in general transcription levels play a vital role in the dynamic regulation of various critical activities. Phosphorylation levels at Ser2 in heptad repeats within the C-terminal domain of RNA polymerase II, representing the elongation form, is an indicator of transcription. However, rapid transcriptional changes during tissue development and cellular phenomena are difficult to capture in living organisms. We introduced a genetically encoded system termed modification-specific intracellular antibody (mintbody) into <italic>Arabidopsis thaliana</italic>. We developed a protein processing- and 2A peptide-mediated two-component system for real-time quantitative measurement of endogenous modification level. This system enables quantitative tracking of the spatiotemporal dynamics of transcription. Using this method, we observed that the transcription level varies among tissues in the root and changes dynamically during the mitotic phase. The approach is effective for achieving live visualization of the transcription level in a single cell and facilitates an improved understanding of spatiotemporal transcription dynamics.

DOI： 10.1038/s42003-021-02106-0

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Other Link： http://www.nature.com/articles/s42003-021-02106-0

Publisher：Cold Spring Harbor Laboratory  

Abstract

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, including various combinations of transcriptome, genome and epigenome. However, comprehensive profiling of the histone post-translational modifications that influence gene expression at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of genetically engineered proteins. By fusing Dam to single-chain variable fragment antibodies, engineered chromatin reader domains, or endogenous chromatin-binding proteins, we render the DamID technology and all its implementations compatible with the genome-wide identification of histone post-translational modifications. Importantly, this enables the joint analysis of chromatin marks and transcriptome in a variety of biological systems at the single-cell level. In this study, we use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. We further demonstrate the applicability of this method to in vivo systems by mapping H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specifically in the notochord. Overall, EpiDamID is a new addition to a vast existing toolbox for obtaining systematic insights into the role of chromatin states during dynamic cellular processes.

DOI： 10.1101/2021.10.26.465688

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

DOI： 10.1093/jmicro/dfab030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/gtc.12893

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/gtc.12893

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/gtc.12868

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{eLife} Sciences Publications, Ltd  

In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

DOI： 10.7554/eLife.66290

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1097/WNR.0000000000001636

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Publisher：Cold Spring Harbor Laboratory  

<title>Abstract</title>In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription “factories” in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters.

<sec><title>Summary</title>The authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

</sec>

DOI： 10.1101/2021.04.27.441582

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acsomega.0c06281

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Life Science Alliance, LLC  

SET8 is solely responsible for histone H4 lysine-20 (H4K20) monomethylation, which preferentially occurs in nucleosomal H4. However, the underlying mechanism by which SET8 specifically promotes the H4K20 monomethylation in the nucleosome has not been elucidated. Here, we report the cryo-EM structures of the human SET8–nucleosome complexes with histone H3 and the centromeric H3 variant, CENP-A. Surprisingly, we found that the overall cryo-EM structures of the SET8–nucleosome complexes are substantially different from the previous crystal structure models. In the complexes with H3 and CENP-A nucleosomes, SET8 specifically binds the nucleosomal acidic patch via an arginine anchor, composed of the Arg188 and Arg192 residues. Mutational analyses revealed that the interaction between the SET8 arginine anchor and the nucleosomal acidic patch plays an essential role in the H4K20 monomethylation activity. These results provide the groundwork for understanding the mechanism by which SET8 specifically accomplishes the H4K20 monomethylation in the nucleosome.

DOI： 10.26508/lsa.202000919

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Language：English   Publishing type：Research paper (scientific journal)  

Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.

DOI： 10.1016/j.molcel.2021.02.034

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Publisher：Research Square Platform LLC  

<title>Abstract</title>
Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes <italic>Cebpa</italic> and <italic>Pparg</italic> early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.

DOI： 10.21203/rs.3.rs-250361/v1

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Other Link： https://www.researchsquare.com/article/rs-250361/v1.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/gtc.12840

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EMBO  

DOI： 10.15252/embr.202051989

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.15252/embr.202051989

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Optical Society  

DOI： 10.1364/OL.416006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome’s acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.

DOI： 10.1073/pnas.2019554118

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Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.2019554118

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.celrep.2021.108912

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.stemcr.2021.03.007

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/tpj.15420

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s13258-021-01046-7

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer US  

DOI： 10.1007/978-1-0716-1538-6_17

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer US  

DOI： 10.1007/978-1-0716-1593-5_3

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Language：English   Publisher：Cold Spring Harbor Laboratory  

<title>Abstract</title>Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate <italic>in vivo</italic> cell-type dynamics of tissues.

DOI： 10.1101/2020.12.18.423434

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

DOI： 10.1038/s41467-020-15428-0

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Language：English   Publishing type：Research paper (scientific journal)  

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated (CA) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions.

DOI： 10.1038/s41467-020-19621-z

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41467-020-19878-4

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications.

DOI： 10.1038/s41596-020-0375-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science ({AAAS})  

DOI： 10.1126/sciadv.abc0330

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/jcs.243303

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Language：English   Publishing type：Research paper (scientific journal)  

The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.

DOI： 10.1016/j.bone.2020.115332

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Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO Human Artificial Chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase, we removed heterochromatin mark H3K9me3 specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. Yet, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A/-C and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.

DOI： 10.1242/jcs.242610

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Language：English   Publishing type：Research paper (scientific journal)  

Post-translational modifications on histones can be stable epigenetic marks and transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during the G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. We also screened chemical compounds in marine organism extracts that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

DOI： 10.1242/jcs.243444

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Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

DOI： 10.1126/sciadv.aaz6699

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{eLife} Sciences Publications, Ltd  

DOI： 10.7554/eLife.53885

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Language：English   Publishing type：Research paper (scientific journal)  

Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.

DOI： 10.15252/embj.2019103949

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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.

DOI： 10.1186/s13072-019-0319-0

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Language：English   Publishing type：Research paper (scientific journal)  

Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.

DOI： 10.1093/nar/gkz1040

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Language：English   Publishing type：Research paper (scientific journal)  

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

DOI： 10.1093/jb/mvz059

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s42003-019-0663-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

DOI： 10.1038/s41467-019-08314-x

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DOI： 10.1038/s41467-019-10846-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

DOI： 10.1038/s42003-018-0251-z

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DOI： 10.1038/s41598-019-46573-2

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Mitochondrial dysfunction causes various diseases. Mutations in the SLC25A46 gene have been identified in mitochondrial diseases that are sometimes classified as Charcot-Marie-Tooth disease type 2, optic atrophy, and Leigh syndrome. A homolog of SLC25A46 was identified in Drosophila and designated as dSLC25A46 (CG5755). We previously established mitochondrial disease model targeting of dSLC25A46, which causes locomotive dysfunction and morphological defects at neuromuscular junctions, such as reduced synaptic branch lengths and decreased numbers of boutons. The diverse symptoms of mitochondrial diseases carrying mutations in SLC25A46 may be associated with the dysregulation of some epigenetic regulators. To investigate the involvement of epigenetic regulators in mitochondrial diseases, we examined candidate epigenetic regulators that interact with human SLC25A46 by searching Gene Expression Omnibus (GEO). We discovered that HDAC1 binds to several SLC25A46 genomic regions in human cultured CD4 (+) cells, and attempted to prove this in an in vivo Drosophila model. By demonstrating that Rpd3, Drosophila HDAC1, regulates the histone H4K8 acetylation state in dSLC25A46 genomic regions, we confirmed that Rpd3 is a novel epigenetic regulator modifying the phenotypes observed with the mitochondrial disease model targeting of dSLC25A46. The functional reduction of Rpd3 rescued the deficient locomotive ability and aberrant morphology of motoneurons at presynaptic terminals induced by the dSLC25A46 knockdown. The present results suggest that the inhibition of HDAC1 suppresses the pathogenic processes that lead to the degeneration of motoneurons in mitochondrial diseases.

DOI： 10.1016/j.yexcr.2019.111673

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While nuclear lamina abnormalities are hallmarks of human diseases, their interplay with epigenetic regulators and precise epigenetic landscape remain poorly understood. Here, we show that loss of the lysine acetyltransferase MOF or its associated NSL-complex members KANSL2 or KANSL3 leads to a stochastic accumulation of nuclear abnormalities with genomic instability patterns including chromothripsis. SILAC-based MOF and KANSL2 acetylomes identified lamin A/C as an acetylation target of MOF. HDAC inhibition or acetylation-mimicking lamin A derivatives rescue nuclear abnormalities observed in MOF-deficient cells. Mechanistically, loss of lamin A/C acetylation resulted in its increased solubility, defective phosphorylation dynamics and impaired nuclear mechanostability. We found that nuclear abnormalities include EZH2-dependent histone H3 Lys 27 trimethylation and loss of nascent transcription. We term this altered epigenetic landscape "heterochromatin enrichment in nuclear abnormalities" (HENA). Collectively, the NSL-complex-dependent lamin A/C acetylation provides a mechanism that maintains nuclear architecture and genome integrity.

DOI： 10.1038/s41556-019-0397-z

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Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.

DOI： 10.1242/dev.179127

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Gliomas are classified by combining histopathologic and molecular features, including isocitrate dehydrogenase (IDH) status. Although IDH-wild-type diffuse astrocytic glioma (DAG) shows a more aggressive phenotype than IDH-mutant type, lack of knowledge regarding relevant molecular drivers for this type of tumor has hindered the development of therapeutic agents. Here, we examined human IDH-wild-type DAGs and a glioma mouse model with a mosaic analysis with double markers (MADM) system, which concurrently lacks p53 and NF1 and spontaneously develops tumors highly comparable with human IDH-wild-type DAG without characteristic molecular features of glioblastoma (DAG-nonMF). During tumor formation, enhancer of zeste homolog (EZH2) and the other polycomb repressive complex 2 (PRC2) components were upregulated even at an early stage of tumorigenesis, together with an increased number of genes with H3K27me3 or H3K27me3 and H3K4me3 bivalent modifications. Among the epigenetically dysregulated genes, frizzled-8 (Fzd8), which is known to be a cancer- and stem cell reprogramming-related gene, was gradually silenced during tumorigenesis. Genetic and pharmacologic inhibition of EZH2 in MADM mice showed reactivation of aberrant H3K27me3 target genes, including Fzd8, together with significant reduction of tumor size. Our study clarifies a pathogenic molecular pathway of IDH-wild-type DAG-nonMF that depends on EZH2 activity and provides a strong rationale for targeting EZH2 as a promising therapeutic approach for this type of glioma. SIGNIFICANCE: EZH2 is involved in the generation of IDH-wild-type diffuse astrocytic gliomas and is a potential therapeutic target for this type of glioma. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/4814/F1.large.jpg.

DOI： 10.1158/0008-5472.can-19-1272

DOI： 10.1158/0008-5472.CAN-19-1272

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

DOI： 10.3390/molecules24142620

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Dysregulation of enhancer of zeste homologue 2 (EZH2), a methyltransferase component of polycomb repressive complex 2, is found in many types of cancers especially those that are highly progressive and aggressive. Specific catalytic inhibitors of EZH2 have high anti-tumour activity, particularly in lymphomas with EZH2 activating mutations. However, the clinical benefits of EZH2 catalytic inhibitors in tumours overexpressing EZH2 are still limited. Here, we identified NPD13668, a novel modulator of EZH2-mediated gene silencing, from 329,049 small chemical compounds using a cell-based high-throughput screening assay. NPD13668 reactivated the expression of silenced H3K27me3 target genes together with depletion of the H3K27me3 modification. In addition, NPD13668 repressed the cell growth of prostate cancer cell lines (PC3 and LNCaP) and ovarian cancer cell lines (SKOV3 and NIH-OVCAR3). NPD13668 partially inhibited the methyltransferase activity of EZH2 in vitro. Genome-wide expression analysis revealed that after NPD13668 treatment, about half of the upregulated genes overlapped with genes upregulated after treatment with GSK126, well-known EZH2 catalytic inhibitor, indicating that NPD13668 is a potential modulator of EZH2 methyltransferase activity. Our data demonstrated that targeting the pharmacological inhibition of EZH2 activity by NPD13668 might be a novel cancer treatment.

DOI： 10.1093/jb/mvz007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

The nucleosome containing the histone H2A.Z.1 variant is unstable, as compared to the canonical nucleosome in vitro, and the incorporation of H2A.Z.1 into chromatin is less stable than that of the canonical H2A in vivo. In the present study, we designed a human H2A.Z.1(S42R) mutant, in which the Ser42 residue is replaced by Arg. In the crystal structure of the nucleosome containing H2A.Z.1(S42R), the Arg residue inserted at the H2A.Z.1-Ser42 position forms additional hydrogen bonds and electrostatic interactions with the DNA backbone phosphates. The Arg42 residue is located in the L1-loop region of H2A.Z.1, but the backbone geometry of the L1-loop is not affected by the H2A.Z.1(S42R) substitution. The nucleosome containing H2A.Z.1(S42R) exhibited enhanced thermal stability, as compared to that containing wild-type H2A.Z.1 in vitro. Fluorescence recovery after photobleaching experiments revealed that H2A.Z.1(S42R) was more stably incorporated in chromatin than wild-type H2A.Z.1 in living cells. Therefore, the H2A.Z.1(S42R) mutant stabilizes the nucleosome in vitro and in vivo, and may be useful as a tool to study the functional significance of the unstable nature of the H2A.Z.1 nucleosome.

DOI： 10.1016/j.bbrc.2019.06.012

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Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.

DOI： 10.1038/s41598-019-44036-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

DOI： 10.1083/jcb.201811090

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The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

DOI： 10.1038/s41598-019-40546-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Nature  

DOI： 10.1038/s41556-018-0248-3

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Minimal sets of transcription factors can directly reprogram somatic cells into neurons. However, epigenetic remodeling during neuronal reprogramming has not been well reconciled with transcriptional regulation. Here we show that NeuroD1 achieves direct neuronal conversion from mouse microglia both in vitro and in vivo. Exogenous NeuroD1 initially occupies closed chromatin regions associated with bivalent trimethylation of histone H3 at lysine 4 (H3K4me3) and H3K27me3 marks in microglia to induce neuronal gene expression. These regions are resolved to a monovalent H3K4me3 mark at later stages of reprogramming to establish the neuronal identity. Furthermore, the transcriptional repressors Scrt1 and Meis2 are induced as NeuroD1 target genes, resulting in a decrease in the expression of microglial genes. In parallel, the microglial epigenetic signature in promoter and enhancer regions is erased. These findings reveal NeuroD1 pioneering activity accompanied by global epigenetic remodeling for two sequential events: onset of neuronal property acquisition and loss of the microglial identity during reprogramming.

DOI： 10.1016/j.neuron.2018.12.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

DOI： 10.1083/jcb.201811170

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

DOI： 10.1038/s41467-018-03868-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

DOI： 10.1038/s41467-018-04283-9

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DOI： 10.1038/s41467-018-03845-1

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Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may p

DOI： 10.1093/nar/gky661

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ange.201807534

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Endocytosis is an important route for the intracellular delivery of biomacromolecules, wherein their inefficient endosomal escape into the cytosol remains a major barrier. Based on the understanding that endosomal membranes are negatively charged, we focused on the potential of cationic lytic peptides for developing endosomolysis agents to release such entrapped molecules. As such, a venom peptide, Mastoparan X, was employed and redesigned to serve as a delivery tool. Appending a tri-glutamate unit to the N-terminus attenuates the cytotoxicity of Mastoparan X by about 40 fold, while introduction of a NiII -dipicolylamine complex enhances cellular uptake of the peptide by about 17 fold. Using the optimized peptide, various fluorescently labeled macromolecules were successfully delivered to the cytosol, enabling live-cell imaging of acetylated histones.

DOI： 10.1002/anie.201807534

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Press  

DOI： 10.1016/j.molcel.2018.05.018

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AIM: The aim of this study was to reveal the epigenome landscape of human normal hepatocytes. MATERIALS & METHODS: Cells purified from partial hepatectomy specimens of Japanese patients were subjected to whole-genome bisulfite sequencing using postbisulfite adaptor tagging, chromatin immunoprecipitation sequencing, RNA sequencing and whole-genome sequencing. RESULTS: CHG and CHH methylations were inversely associated with gene expression. Histone modification profiles of personal differentially methylated regions (pDMRs) differed considerably among samples. pDMRs were observed around the transcription start sites of genes whose expression is reportedly regulated by CpG methylation. pDMRs were frequently observed in the vicinity of single-nucleotide variations and insertions/deletions. CONCLUSION: Genetic variations may induce epigenetic variations, generating individual differences in the phenotypes of normal hepatocytes through variations in expression.

DOI： 10.2217/epi-2017-0111

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Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases.

DOI： 10.1016/j.celrep.2018.03.141

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Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome.

DOI： 10.1016/j.stemcr.2018.02.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-VCH Verlag  

DOI： 10.15252/embj.201797677

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bpj.2017.11.1410

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.1717509115

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

DOI： 10.1101/gr.224717.117

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The inactive X chromosome (Xi) harbors characteristic epigenetic features, including the enrichment of histone H3 lysine 27 trimethylation (H3K27me3) and H4 lysine 20 monomethylation (H4K20me1) as well as a lack of histone acetylation. Recently, these modifications have been visualized not only in fixed specimen, but also in living cells via probes derived from modification-specific antibodies. The probes include fluorescently labeled antigen binding fragments (Fabs), which can be loaded into cells, as well as genetically encoded single-chain variable fragments tagged with the green fluorescent protein. We refer to the latter as modification specific intracellular antibodies, or "mintbodies" for short. By using Fabs or mintbodies to target Xi-specific modifications, the dynamics of Xi in living cells can be visualized.

DOI： 10.1007/978-1-4939-8766-5_8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

DOI： 10.1111/andr.12438

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The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

DOI： 10.1080/15592294.2018.1469891

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DOI： 10.1073/pnas.1714579115

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DOI： 10.1038/s41598-017-12608-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{eLife} Sciences Organisation, Ltd.  

DOI： 10.7554/eLife.31641

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DOI： 10.7554/eLife.25317

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DOI： 10.1016/j.yexcr.2017.05.016

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DOI： 10.1016/j.celrep.2017.06.056

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DOI： 10.1038/srep45894

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DOI： 10.1016/j.juro.2017.02.277

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DOI： 10.1111/gtc.12479

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DOI： 10.1038/srep41912

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DOI： 10.1016/j.celrep.2016.12.065

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DOI： 10.1038/cddiscovery.2016.77

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DOI： 10.1371/journal.pone.0167554

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DOI： 10.1038/ncomms13465

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DOI： 10.1371/journal.pone.0165473

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DOI： 10.1016/j.jmb.2016.08.010

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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DOI： 10.1016/j.celrep.2016.07.079

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DOI： 10.1182/blood-2016-01-694810

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DOI： 10.1016/j.yexcr.2016.06.013

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DOI： 10.1093/nar/gkw202

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DOI： 10.1016/j.devcel.2016.05.006

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DOI： 10.1093/nar/gkw008

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Language：English   Publishing type：Part of collection (book)   Publisher：CRC Press  

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DOI： 10.1111/gtc.12349

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DOI： 10.1038/srep24318

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：{eLife} Sciences Publications, Ltd  

DOI： 10.7554/eLife.09540

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

DOI： 10.1007/978-4-431-55873-6_19

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DOI： 10.1091/mbc.E15-08-0605

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DOI： 10.1016/j.cell.2016.11.007

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DOI： 10.1038/ng.3662

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DOI： 10.1186/s13072-016-0051-y

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DOI： 10.1247/csf.16013

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Drosophila Jumonji/Jarid2 (dJmj) has been identified as a component of Polycomb repressive complex 2. However, it is suggested that dJmj has both PRC-dependent and -independent roles. Subcellular localization of dJmj during spermatogenesis is unknown. We therefore performed immunocytochemical analyses with specific antibodies to dJmj and tri-methylation at lysine 27 on histone H3 (H3K27me3). Interestingly, dJmj exclusively localizes at nucleolus in the late growth stage. Examination of the dJmj localization in various Polycomb group (PcG) mutant lines at the late growth stage allowed identification of some PcG genes, including Polycomb (Pc), to be responsible for dJmj recruitment to nucleolus. In addition, we found that size of nucleolus was decreased in some of these mutant lines. In a mutant of testis-specific TAF homolog (tTAF) that is responsible for nucleolus localization of Pc, dJmj signals were detected not only at nucleolus but also on the condensed chromatin in the late growth stage. Duolink In situ Proximity ligation assay clarified that Pc interacts with dJmj at nucleolus in the late growth stage. Furthermore, the level of H3K27me3 decreased in nuclei at this stage. Taken together, we conclude that tTAF is responsible for recruitments of dJmj to nucleolus in the late growth stage that appears to be mediated by Pc. Compartmentalization of dJmj in nucleolus together with some of PcG may be necessary to de-repress the expression of genes required to cellular growth and proliferation in the following meiotic divisions.

DOI： 10.1080/21565562.2016.1232023

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DOI： 10.1242/jcs.180786

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DOI： 10.1508/cytologia.80.383

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DOI： 10.1007/s10577-015-9486-4

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DOI： 10.1093/nar/gkv841

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DOI： 10.1016/j.molcel.2015.10.025

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DOI： 10.1111/gtc.12281

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DOI： 10.1186/s13072-015-0036-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

DOI： 10.1186/s13072-015-0027-3

Scopus

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DOI： 10.1007/s00418-015-1344-0

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DOI： 10.1038/srep12720

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DOI： 10.1016/j.stem.2015.03.002

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1089/cell.2014.0086

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DOI： 10.1007/978-1-4939-1804-1_16

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0131204

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Language：Japanese   Publisher：Japan Society for Comparative Endocrinology  

DOI： 10.5983/nl2008jsce.41.85

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JLC/20012692031?from=CiNii

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CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep09628

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gku1346

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DOI： 10.4049/jimmunol.1302456

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DOI： 10.1016/j.cell.2015.03.027

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DOI： 10.1242/bio.201410850

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DOI： 10.1007/s00418-015-1318-2

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DOI： 10.1016/j.yexcr.2015.03.010

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DOI： 10.7554/eLife.11215

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DOI： 10.1038/ncomms8052

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DOI： 10.1038/ncomms8753

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DOI： 10.1371/journal.pgen.1005053

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：7530  

DOI： 10.1038/nature13714

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer New York  

DOI： 10.1007/978-1-4939-1594-1_10

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Language：Japanese   Publisher：(公社)日本生化学会  

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DOI： 10.1016/j.stemcr.2014.06.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：9  

DOI： 10.1371/journal.pone.0106271

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S128_3

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DOI： 10.1016/j.stemcr.2014.05.008

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S128_1

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DOI： 10.1371/journal.pone.0096005

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DOI： 10.1091/mbc.E13-09-0558

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DOI： 10.1093/nar/gku640

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DOI： 10.1016/j.ab.2014.07.023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：7  

DOI： 10.1038/nsmb.2845

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DOI： 10.1038/ncomms4634

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DOI： 10.1371/journal.pone.0091330

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DOI： 10.1016/j.molcel.2014.06.024

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DOI： 10.1016/j.ymeth.2014.08.002

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DOI： 10.1186/bcr3681

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DOI： 10.1016/j.devcel.2014.05.001

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DOI： 10.1007/s00418-014-1284-0

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DOI： 10.1186/1756-8935-7-17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Union of Crystallography (IUCr)  

The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

DOI： 10.1107/S090744491302252X

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DOI： 10.1101/gad.217281.113

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DOI： 10.1038/srep02436

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DOI： 10.1111/cas.12166

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DOI： 10.1038/jhg.2013.66

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pt 6  

DOI： 10.1242/jcs.112623

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DOI： 10.1038/srep03510

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DOI： 10.1095/biolreprod.112.103572

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DOI： 10.1093/nar/gks1464

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DOI： 10.1007/s00428-013-1428-y

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DOI： 10.1016/j.jmb.2012.10.018

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DOI： 10.1016/j.gep.2013.01.002

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DOI： 10.1016/j.devcel.2013.02.009

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DOI： 10.1038/nsmb.2532

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DOI： 10.1095/biolreprod.113.108597

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DOI： 10.1172/JCI70739

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DOI： 10.1016/j.molcel.2013.08.007

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DOI： 10.1007/s10577-013-9375-7

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DOI： 10.1038/hr.2012.125

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DOI： 10.1038/emboj.2012.197

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DOI： 10.1016/j.stem.2011.12.017

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DOI： 10.1016/j.molcel.2012.06.010

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DOI： 10.1007/s10577-012-9276-1

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DOI： 10.1093/pcp/pcs053

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DOI： 10.1074/jbc.M112.397950

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DOI： 10.1007/s10577-012-9317-9

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DOI： 10.1186/1471-2164-13-424

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DOI： 10.1371/journal.pgen.1002933

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DOI： 10.1007/s10577-012-9318-8