担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Identification of immunoglobulin genes in hybridomas is essential for producing antibodies for research and clinical applications. A couple of methods such as RACE and degenerative PCR have been developed for determination of the Igh and Igl/Igk coding sequences (CDSs) but it has been difficult to process a number of hybridomas both with accuracy and rapidness. Here, we propose a new strategy for antibody sequence determination by mRNA-seq of hybridomas. We demonstrated that hybridomas highly expressed the Igh and Igl/Igk genes and that de novo transcriptome assembly using mRNA-seq data enabled identification of the CDS of both Igh and Igl/Igk accurately. Furthermore, we estimated that only 30,000 sequenced reads are required to identify immunoglobulin sequences from four different hybridoma clones. Thus, our approach would facilitate determining variable CDSs drastically.

DOI： 10.1371/journal.pone.0165473

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.

DOI： 10.1038/srep24318

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The binding of transcription factors (TFs) triggers activation of specific chromatin regions through the recruitment and activation of RNA polymerase. Unique nucleosome positioning (NP) occurs during gene expression and has been suggested to be involved in various other chromatin functions. However, the diversity of NP that can occur for each function has not been clarified. Here we used MNase-Seq data to evaluate NP around 258 cis-regulatory elements in the mouse genome. Principal component analysis of the 258 elements revealed that NP consisted of five major patterns. Furthermore, the five NP patterns had predictive power for the level of gene expression. We also demonstrated that selective NP patterns appeared around TF binding sites. These results suggest that the NP patterns are correlated to specific functions on chromatin.

DOI： 10.1038/srep19620

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled. Results: We screened mouse, rat and human genomes by in silico hybridization using canonical histone sequences. In the mouse genome, we identified 14 uncharacterized H3 genes, among which 13 are similar to H3.3 and do not have human or rat counterparts, and one is similar to human testis-specific H3 variant, H3T/H3.4, and had a rat paralog. Although some of these genes were previously annotated as pseudogenes, their tissue-specific expression was confirmed by sequencing the 3′-UTR regions of the transcripts. Certain new variants were also detected at the protein level by mass spectrometry. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome-wide distributions of most variants were similar to that of H3.3. Moreover, forced expression of H3 variants in chromatin resulted in alternate gene expression patterns after cell differentiation. Conclusions: We comprehensively identified and characterized novel mouse H3 variant genes that encoded highly conserved amino acid sequences compared to known histone H3. We speculated that the diversity of H3 variants acquired after species separation played a role in regulating tissue-specific gene expression in individual species. Their biological relevance and evolutionary aspect involving pseudogene diversification will be addressed by further functional analysis.

DOI： 10.1186/s13072-015-0027-3

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Aggregation plots are frequently used to evaluate signal distributions at user-interested points in ChIP-Seq data analysis. agplus, a new and simple command-line tool, enables rapid and flexible generation of text tables tailored for aggregation plots from which users can easily design multiple groups based on user-definitions such as regulatory regions or transcription initiation sites.

DOI： 10.1093/bioinformatics/btv322

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown.

DOI： 10.1093/nar/gks1010

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Cell Science  

Muscle stem cells (MuSCs) play an indispensable role in postnatal muscle growth and hypertrophy in adults. MuSCs also retain a highly regenerative capacity and are therefore considered a promising stem cell source for regenerative therapy for muscle diseases. In this study, we identify tumor-suppressor protein Tob1 as a Pax7 target protein that negatively controls the population expansion of MuSCs. Tob1 protein is undetectable in the quiescent state but is upregulated during activation in MuSCs. Tob1 ablation in mice accelerates MuSC population expansion and boosts muscle regeneration. Moreover, inactivation of Tob1 in MuSCs ameliorates the efficiency of MuSC transplantation in a murine muscular dystrophy model. Collectively, selective targeting of Tob1 might be a therapeutic option for the treatment of muscular diseases, including muscular dystrophy and age-related sarcopenia.

DOI： 10.1242/jcs.261886

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DOI： 10.1101/2024.05.28.596135

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記述言語：その他   掲載種別：研究論文（学術雑誌）   出版者・発行元：Communications Biology  

The nucleosome is a fundamental unit of chromatin in which about 150 base pairs of DNA are wrapped around a histone octamer. The overlapping di-nucleosome has been proposed as a product of chromatin remodeling around the transcription start site, and previously found as a chromatin unit, in which about 250 base pairs of DNA continuously bind to the histone core composed of a hexamer and an octamer. In the present study, our genome-wide analysis of human cells suggests another higher nucleosome stacking structure, the overlapping tri-nucleosome, which wraps about 300-350 base-pairs of DNA in the region downstream of certain transcription start sites of actively transcribed genes. We determine the cryo-electron microscopy (cryo-EM) structure of the overlapping tri-nucleosome, in which three subnucleosome moieties, hexasome, hexasome, and octasome, are associated by short connecting DNA segments. Small angle X-ray scattering and coarse-grained molecular dynamics simulation analyses reveal that the cryo-EM structure of the overlapping tri-nucleosome may reflect its structure in solution. Our findings suggest that nucleosome stacking structures composed of hexasome and octasome moieties may be formed by nucleosome remodeling factors around transcription start sites for gene regulation.

DOI： 10.1038/s42003-023-05694-1

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記述言語：英語   出版者・発行元：Nature Protocols  

Correction to: Nature Protocolshttps://doi.org/10.1038/s41596-020-0375-8, published online 17 August 2020. In the version of this article initially published, a merged image in the fourth column (Primary–, Anti-mouse IgG–, and ChIL probe +) in Fig. 2c did not correspond with the single-channel images above it. This was due to inadvertently placing another image from the same experiment. The original and corrected Fig. 2c appear as Fig. 1, below. The error has been corrected in the HTML and PDF versions of the article. Original and revised Fig. 2c.

DOI： 10.1038/s41596-023-00940-6

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：STAR Protocols  

Photo-isolation chemistry (PIC) enables isolation of transcriptome information from locally defined areas by photo-irradiation. Here, we present an optimized PIC protocol for formalin-fixed frozen and paraffin mouse sections and fresh-frozen mouse sections. We describe tissue section preparation and permeabilization, followed by in situ reverse transcription using photo-caged primers. We then detail immunostaining and UV-mediated uncaging to the target areas, followed by linear amplification of uncaged cDNAs, library preparation, and quantification. This protocol can be applied to various animal tissue types. For complete details on the use and execution of this protocol, please refer to Honda et al. (2021).

DOI： 10.1016/j.xpro.2022.101346

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers in Cell and Developmental Biology  

The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-β2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-β2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.

DOI： 10.3389/fcell.2022.780038

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記述言語：英語   出版者・発行元：Cell Stem Cell  

Adaptation to mechanical load, leading to enhanced force and power output, is a characteristic feature of skeletal muscle. Formation of new myonuclei required for efficient muscle hypertrophy relies on prior activation and proliferation of muscle stem cells (MuSCs). However, the mechanisms controlling MuSC expansion under conditions of increased load are not fully understood. Here we demonstrate that interstitial mesenchymal progenitors respond to mechanical load and stimulate MuSC proliferation in a surgical mouse model of increased muscle load. Mechanistically, transcriptional activation of Yes-associated protein 1 (Yap1)/transcriptional coactivator with PDZ-binding motif (Taz) in mesenchymal progenitors results in local production of thrombospondin-1 (Thbs1), which, in turn, drives MuSC proliferation through CD47 signaling. Under homeostatic conditions, however, CD47 signaling is insufficient to promote MuSC proliferation and instead depends on prior downregulation of the Calcitonin receptor. Our results suggest that relayed signaling between mesenchymal progenitors and MuSCs through a Yap1/Taz-Thbs1-CD47 pathway is critical to establish the supply of MuSCs during muscle hypertrophy.

DOI： 10.1016/j.stem.2021.11.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

An effective combination of multi-omic datasets can enhance our understanding of complex biological phenomena. To build a context-dependent network with multiple omic layers, i.e., a trans-omic network, we perform phosphoproteomics, transcriptomics, proteomics, and metabolomics of murine liver for 4 h after insulin administration and integrate the resulting time series. Structural characteristics and dynamic nature of the network are analyzed to elucidate the impact of insulin. Early and prominent changes in protein phosphorylation and persistent and asynchronous changes in mRNA and protein levels through non-transcriptional mechanisms indicate enhanced crosstalk between phosphorylation-mediated signaling and protein expression regulation. Metabolic response shows different temporal regulation with transient increases at early time points across categories and enhanced response in the amino acid and nucleotide categories at later time points as a result of process convergence. This extensive and dynamic view of the trans-omic network elucidates prominent regulatory mechanisms that drive insulin responses through intricate interlayer coordination.

DOI： 10.1016/j.celrep.2021.109569

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<title>Abstract</title>In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 10⁴ unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions.

DOI： 10.1038/s41467-021-24691-8

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner and that there were genes whose expression was changed independently of the enzyme treatment time, amount and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods.

DOI： 10.1111/gtc.12870

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non-coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3-specific intracellular antibody or H3K27me3-mintbody. By combining live-cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP-seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.

DOI： 10.15252/embr.202051989

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3, and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription.

DOI： 10.1093/jb/mvab001

記述言語：英語   掲載種別：研究論文（学術雑誌）  

MCF7 cells acquire estrogen-independent proliferation after long-term estrogen deprivation (LTED), which recapitulates endocrine therapy resistance. LTED cells can become primed for apoptosis, but the underlying mechanism is largely unknown. We previously reported that Eleanor non-coding RNAs (ncRNAs) upregulate the ESR1 gene in LTED cells. Here, we show that Eleanors delineate the topologically associating domain (TAD) of the ESR1 locus in the active nuclear compartment of LTED cells. The TAD interacts with another transcriptionally active TAD, which is 42.9 Mb away from ESR1 and contains a gene encoding the apoptotic transcription factor FOXO3. Inhibition of a promoter-associated Eleanor suppresses all genes inside the Eleanor TAD and the long-range interaction between the two TADs, but keeps FOXO3 active to facilitate apoptosis in LTED cells. These data indicate a role of ncRNAs in chromatin domain regulation, which may underlie the apoptosis-prone nature of therapy-resistant breast cancer cells and could be good therapeutic targets.

DOI： 10.1038/s41467-019-11378-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c).

DOI： 10.7554/eLife.46667

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCβ) via dephosphorylation and activation of Brg1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of Brg1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.

DOI： 10.1128/MCB.00063-19

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In overloaded and regenerating muscle, the generation of new myonuclei depends on muscle satellite cells (MuSCs). Because MuSC behaviors in these two environments have not been considered separately, MuSC behaviors in overloaded muscle remain unexamined. Here, we show that most MuSCs in overloaded muscle, unlike MuSCs in regenerating muscle, proliferate in the absence of MyoD expression. Mechanistically, MuSCs in overloaded muscle sustain the expression of Heyl, a Notch effector gene, to suppress MyoD expression, which allows effective MuSC proliferation on myofibers and beneath the basal lamina. Although Heyl-knockout mice show no impairment in an injury model, in a hypertrophy model, their muscles harbor fewer new MuSCderived myonuclei due to increased MyoD expression and diminished proliferation, which ultimately causes blunted hypertrophy. Our results show that sustained HeyL expression is critical for MuSC proliferation specifically in overloaded muscle, and thus indicate that the MuSCproliferation mechanism differs in overloaded and regenerating muscle.

DOI： 10.7554/eLife.48284

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tn5 transposase is a bacterial enzyme that integrates a DNA fragment into genomic DNA, and is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes. However, in chromatin, the DNA targeting by Tn5 transposase has remained unclear. In the present study, we reconstituted well-positioned 601 dinucleosomes, in which two nucleosomes are connected with a linker DNA, and studied the DNA integration sites in the dinucleosomes by Tn5 transposase in vitro. We found that Tn5 transposase preferentially targets near the entry–exit DNA regions within the nucleosome. Tn5 transposase minimally cleaved the dinucleosome without a linker DNA, indicating that the linker DNA between two nucleosomes is important for the Tn5 transposase activity. In the presence of a 30 base-pair linker DNA, Tn5 transposase targets the middle of the linker DNA, in addition to the entry–exit sites of the nucleosome. Intriguingly, this Tn5-targeting characteristic is conserved in a dinucleosome substrate with a different DNA sequence from the 601 sequence. Therefore, the Tn5-targeting preference in the nucleosomal templates reported here provides important information for the interpretation of Tn5 transposase-based genomics methods, such as ATAC-seq.

DOI： 10.1098/rsob.190116

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon Dmrt3 and Dmrta2 show a medialhigh/laterallow expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore double-mutant progenitors in the medial region exhibit upregulated Pax6 andmore lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex.

DOI： 10.1242/dev.174243

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts May be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA.

DOI： 10.1098/rsob.170255

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In metazoan cells, only a limited number of mini chromosome maintenance (MCM) complexes are fired during S phase, while the majority remain dormant. Several methods have been used to map replication origins, but such methods cannot identify dormant origins. Herein, we determined MCM7-binding sites in human cells using ChIP-Seq, classified them into firing and dormant origins using origin data and analysed their association with various chromatin signatures. Firing origins, but not dormant origins, were well correlated with open chromatin regions and were enriched upstream of transcription start sites (TSSs) of transcribed genes. Aggregation plots of MCM7 signals revealed minimal difference in the efficacy of MCM loading between firing and dormant origins. We also analysed common fragile sites (CFSs) and found a low density of origins at these sites. Nevertheless, firing origins were enriched upstream of the TSSs. Based on the results, we propose a model in which excessive MCMs are actively loaded in a genome-wide manner, irrespective of chromatin status, but only a fraction are passively fired in chromatin areas with an accessible open structure, such as regions upstream of TSSs of transcribed genes. This plasticity in the specification of replication origins may minimize collisions between replication and transcription.

DOI： 10.1093/nar/gky476

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method.

DOI： 10.1371/journal.pone.0191532

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state.

DOI： 10.1093/nar/gkx475

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4^{CDT 2}-mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns.

DOI： 10.1111/gtc.12481

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate "overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process.

DOI： 10.1126/science.aak9867

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

DOI： 10.1021/acs.biochem.6b01098

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that MyoD expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of MyoD mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N⁶-methyladenosine (m⁶A) modifications of RNA. Knockdown of Mettl3 revealed that MyoD RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m⁶A modification sites were profiled by m⁶A sequencing and identified within the 5⁰ untranslated region (UTR) of MyoD mRNA. Deletion of the 5⁰ UTR revealed that it has a role in MyoD mRNA processing. These data showed that Mettl3 is required for MyoD mRNA expression in proliferative myoblasts.

DOI： 10.1098/rsob.170119

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

DOI： 10.1016/j.celrep.2016.12.065

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3.Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3.Y nucleosome was stable in vitro and in vivo. Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3.Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3.Y nucleosome are also conserved in the H3.Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3.Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes.

DOI： 10.1093/nar/gkw202

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51.

DOI： 10.1038/srep24228

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function. J. Cell. Biochem. 117: 780-792, 2016.

DOI： 10.1002/jcb.25368

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.

DOI： 10.7554/eLife.09540

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Human histone H3.5 is a non-allelic H3 variant evolutionally derived from H3.3. The H3.5 mRNA is highly expressed in human testis. However, the function of H3.5 has remained poorly understood. Results: We found that the H3.5 nucleosome is less stable than the H3.3 nucleosome. The crystal structure of the H3.5 nucleosome showed that the H3.5-specific Leu103 residue, which corresponds to the H3.3 Phe104 residue, reduces the hydrophobic interaction with histone H4. Mutational analyses revealed that the H3.5-specific Leu103 residue is responsible for the instability of the H3.5 nucleosome, both in vitro and in living cells. The H3.5 protein was present in human seminiferous tubules, but little to none was found in mature sperm. A chromatin immunoprecipitation coupled with sequencing analysis revealed that H3.5 accumulated around transcription start sites (TSSs) in testicular cells. Conclusions: We performed comprehensive studies of H3.5, and found the instability of the H3.5 nucleosome and the accumulation of H3.5 protein around TSSs in human testis. The unstable H3.5 nucleosome may function in the chromatin dynamics around the TSSs, during spermatogenesis.

DOI： 10.1186/s13072-016-0051-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.

DOI： 10.1007/s10577-015-9486-4

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methyl-ation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

DOI： 10.1098/rsob.150128

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Estrogen receptor-α (ER)-positive breast cancer cells undergo hormone-independent proliferation after deprivation of oestrogen, leading to endocrine therapy resistance. Up-regulation of the ER gene (ESR1) is critical for this process, but the underlying mechanisms remain unclear. Here we show that the combination of transcriptome and fluorescence in situ hybridization analyses revealed that oestrogen deprivation induced a cluster of noncoding RNAs that defined a large chromatin domain containing the ESR1 locus. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating noncoding RNAs). Eleanors were present in ER-positive breast cancer tissues and localized at the transcriptionally active ESR1 locus to form RNA foci. Depletion of one Eleanor, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. Eleanor-mediated gene activation represents a new type of locus control mechanism and plays an essential role in the adaptation of breast cancer cells.

DOI： 10.1038/ncomms7966

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications.

DOI： 10.1093/nar/gku1346

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.

DOI： 10.1093/nar/gkv509

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

DOI： 10.1038/nature13714

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states.

DOI： 10.1016/j.stemcr.2014.05.008

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Raw data from ChIP-seq (chromatin immunoprecipitation combined with massively parallel DNA sequencing) experiments are deposited in public databases as SRAs (Sequence Read Archives) that are publically available to all researchers. However, to graphically visualize ChIP-seq data of interest, the corresponding SRAs must be downloaded and converted into BigWig format, a process that involves complicated command-line processing. This task requires users to possess skill with script languages and sequence data processing, a requirement that prevents a wide range of biologists from exploiting SRAs. To address these challenges, we developed SraTailor, a GUI (Graphical User Interface) software package that automatically converts an SRA into a BigWig-formatted file. Simplicity of use is one of the most notable features of SraTailor: entering an accession number of an SRA and clicking the mouse are the only steps required to obtain BigWig-formatted files and to graphically visualize the extents of reads at given loci. SraTailor is also able to make peak calls, generate files of other formats, process users' own data, and accept various command-line-like options. Therefore, this software makes ChIP-seq data fully exploitable by a wide range of biologists. SraTailor is freely available at http://www.devbio.med.kyushu-u.ac.jp/sra_tailor/, and runs on both Mac and Windows machines.

DOI： 10.1111/gtc.12190

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Microarray analysis using human HeLa cells revealed that a subset of genes was differentially expressed on Thoc5 knockdown. Notably, the depletion of Thoc5 selectively attenuated the expression of mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) indicated that CFIm68 preferentially associated with the 5′ regions of genes; strikingly, the 5′ peak of CFIm68 was significantly and globally reduced on Thoc5 knockdown. We suggest a model in which human Thoc5 controls polyadenylation site choice through the co-transcriptional loading of CFIm68 onto target genes.

DOI： 10.1093/nar/gkt414

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.Results: We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.Conclusions: We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

DOI： 10.1186/1471-2164-12-516

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

開催年月日： 2023年8月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：Waseda University, Tokyo   国名：日本国  

We propose a method based on Hodge decomposition for analyzing high-dimensional and complex molecular dynamics using single-cell omics data. Drawing inspiration from topology and differential geometry, we developed a data-driven vector field reconstruction method that smoothly captures key features of dynamics (e.g., potential, divergence, curl, and Jacobian) with reduced computational costs through appropriate connections and regularization. Our approach has the potential to contribute to biological discoveries and understanding.

開催年月日： 2023年5月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：科学技術振興機構 東京本部   国名：日本国  

開催年月日： 2022年10月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：パシフィコ横浜   国名：日本国  

開催年月日： 2022年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：オンライン   国名：日本国  

開催年月日： 2022年3月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

国名：日本国  

開催年月日： 2020年10月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：久留米大学   国名：日本国  

開催年月日： 2020年1月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：道後温泉   国名：日本国  

開催年月日： 2020年1月

記述言語：日本語  

国名：日本国  

開催年月日： 2019年6月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：神戸国際会議場   国名：日本国  

開催年月日： 2018年10月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：Kyushu University   国名：日本国  

開催年月日： 2018年9月

記述言語：英語  

開催地：Hilton Metropole Florence   国名：イタリア共和国  

開催年月日： 2018年4月

記述言語：英語  

開催地：Max Delbrück Communications Center (Berlin)   国名：ドイツ連邦共和国  

開催年月日： 2017年12月

記述言語：日本語  

開催地：東京大学　武田ホール　（東京都）   国名：日本国  

開催年月日： 2017年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸ポートアイランド（兵庫県）   国名：日本国  

開催年月日： 2017年7月

記述言語：英語  

開催地：IGBMC in Strasbourg   国名：フランス共和国  

開催年月日： 2014年7月

記述言語：日本語  

記述言語：日本語   会議種別：口頭発表（一般）  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

記述言語：日本語  

記述言語：日本語   会議種別：口頭発表（一般）  

記述言語：英語  

国名：カナダ  

記述言語：日本語   会議種別：口頭発表（一般）  

記述言語：日本語   会議種別：口頭発表（一般）  

記述言語：日本語  

記述言語：日本語  

記述言語：英語  

記述言語：英語