関 元昭

Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7 upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencing of gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPR interference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.

INTRODUCTION: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remain unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. METHODS: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight non-cancerous colonic epithelial cells using Methylation EPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. RESULTS: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. CONCLUSIONS: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a distinct prognosis, the molecular basis has not been fully elucidated. Here we conducted DNA methylation analysis of 30 lung adenocarcinoma cases annotated with the predominant histological subtypes and three normal lung cases using the Infinium BeadChip. Unsupervised hierarchical clustering analysis revealed three subgroups with different methylation levels: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME). Micropapillary pattern (MPP)-predominant cases and those with MPP components were significantly enriched in HME (p = 0.02 and p = 0.03, respectively). HME cases showed a significantly poor prognosis for recurrence-free survival (p < 0.001) and overall survival (p = 0.006). We identified 365 HME marker genes specifically hypermethylated in HME cases with enrichment of "cell morphogenesis" related genes; 305 IME marker genes hypermethylated in HME and IME, but not in LME, with enrichment "embryonic organ morphogenesis"-related genes; 257 Common marker genes hypermethylated commonly in all cancer cases, with enrichment of "regionalization"-related genes. We extracted surrogate markers for each epigenotype and designed pyrosequencing primers for five HME markers (TCERG1L, CXCL12, FAM181B, HOXA11, GAD2), three IME markers (TBX18, ZNF154, NWD2) and three Common markers (SCT, GJD2, BARHL2). DNA methylation profiling using Infinium data was validated by pyrosequencing, and HME cases defined by pyrosequencing results also showed the worse recurrence-free survival. In conclusion, lung adenocarcinomas are stratified into subtypes with distinct DNA methylation levels, and the high-methylation subtype correlated with MPP-predominant cases and those with MPP components and showed a poor prognosis.

Abstract Aberrant DNA methylation accumulates in non-malignant gastric mucosa after exposure to environmental pathogens such as H. pylori (HP). To understand how environmental factors and DNA methylation interplay to influence primary gastric cancer (GC) risk, we performed an integrated analysis of clinical factors and DNA methylation data of gastric tissues from a longitudinally monitored cohort in Japan, with validation in a separate cohort from Singapore. The Japanese check-up cohort included 4,234 healthy subjects who underwent gastric mucosal biopsy. The median observation period was 4.2 years, and 77 subjects developed GC. GC incidence correlated with age, drinking, smoking, GC family history, and HP status in the multivariable Cox model. Next, we conducted comprehensive DNA methylation analysis using Infinium MethylationEPIC arrays on gastric tissues (n=164), including (1) mucosal biopsies from subjects who later developed GC (“future GC patients”), (2) mucosal biopsies from HP(+) subjects who did not later develop GC (“future non-GC subjects”), (3) mucosal biopsies from future non-GC HP(−) subjects (“control mucosae”), and (4) GCs and surrounding mucosae. Infinium data of gastric mucosae (n=137) collected in the GCEP cohort (Singapore) were also analyzed. DNA methylation of promoter region observed in GCs, accumulated not only in mucosae adjacent to GCs but also in the biopsy mucosae of future GC patients. Mucosae of future non-GC subjects were more methylated than control mucosae but less methylated than mucosae of future GC patients. Similar findings were observed in the GCEP cohort. DNA methylation levels were associated with clinical factors and histopathological alterations - however, in multivariable analyses, DNA methylation remained an independent GC risk factor. Methylation levels were predictive of not only higher GC risk but also a shorter period to GC incidence. We then focused on the associations between environmental factors and methylation. Heavy drinking and smoking were associated with the accumulation of DNA methylation only in HP(+) subjects. The increases in methylation over time in subjects who quit smoking were significantly attenuated compared to continuous smokers. These results suggest that pro-carcinogenic epigenetic alterations initiated by HP exposure are amplified by unfavorable but modifiable lifestyle choices. Furthermore, target genes methylated by each environmental factor may overlap in part; however, they were not necessarily methylated similarly, suggesting that the best markers for stratifying GC risk may differ for each subgroup classified by exposure to environmental factors. Indeed, candidate markers for stratifying GC risk overlapped among subgroups, whereas markers unique to each subgroup were also identified, highlighting the potential of integrating environmental, lifestyle, and epigenetic information to inform GC precision prevention. Citation Format: Genki Usui, Keisuke Matsusaka, Kie K. Huang, Feng Zhu, Tomohiro Shinozaki, Masaki Fukuyo, Bahityar Rahmutulla, Norikazu Yogi, Tomoka Okada, Motoaki Seki, Eiji Sakai, Kazutoshi Fujibayashi, Stephen K. Tsao, Christopher Khor, Tiing L. Ang, Hiroyuki Abe, Hisahiro Matsubara, Masashi Fukayama, Toshiaki Gunji, Nobuyuki Matsuhashi, Teppei Morikawa, Tetsuo Ushiku, Khay G. Yeoh, Patrick Tan, Atsushi Kaneda. Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 761.

Clonal heterogeneity underlies diverse biological processes, including cancer progression, cell differentiation, and microbial evolution. Cell tagging strategies with DNA barcodes have recently enabled analysis of clone size dynamics and clone-restricted transcriptomic landscapes of heterogeneous populations. However, isolating a target clone that displays a specific phenotype from a complex population remains challenging. Here, we present a new multi-kingdom genetic barcoding system, CloneSelect, in which a target cell clone can be triggered to express a reporter gene for isolation through barcode-specific CRISPR base editing. In CloneSelect, cells are first barcoded and propagated so their subpopulation can be subjected to a given experiment. A clone that shows a phenotype or genotype of interest at a given time can then be isolated from the initial or subsequent cell pools stored throughout the experimental timecourse. This novel CRISPR-barcode genetics platform provides many new ways of analyzing and manipulating mammalian, yeast, and bacterial systems. Teaser A multi-kingdom CRISPR-activatable barcoding system enables the precise isolation of target barcode-labeled clones from a complex cell population.

Background: We have recently revealed that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mu variant shows a pronounced resistance to antibodies elicited by natural SARS-CoV-2 infection and vaccination. Methods: However, it remains unclear which mutations determine the resistance of SARS-CoV-2 Mu to antiviral sera. In addition, it is unclear how SARS-CoV-2 Mu infection induces antiviral immunity. Results: In this study, we reveal that the 2 mutations in the SARS-CoV-2 Mu spike protein, YY144-145TSN and E484K, are responsible for the resistance to coronavirus disease 2019 convalescent sera during early 2020 and vaccine sera. Conclusions: It is notable that the convalescent sera of SARS-CoV-2 Mu-infected individuals are broadly antiviral against Mu as well as other SARS-CoV-2 variants of concern and interest.

Infection with certain viruses is an important cause of cancer. The Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium recently analyzed the whole-genome sequencing (WGS) data from 2656 cases across 21 cancer types, and indicated that Epstein-Barr virus (EBV) is detected in many different cancer cases at a higher frequency than previously reported. However, whether EBV-positive cancer cases detected by WGS-based screening correspond to those detected by conventional histopathological techniques is still unclear. In this study, to elucidate the involvement of EBV in various cancers, we reanalyzed the WGS data of the PCAWG cohort combined with the analysis of clinical samples of gastric and pancreatic cancer in our cohort. Based on EBV copy number in each case, we classified tumors into three subgroups: EBV-High, EBV-Low, and EBV-Negative. The EBV-High subgroup was found to be EBV-positive in the cancer cells themselves, whereas the EBV-Low subgroup was EBV-positive in the surrounding lymphocytes. Further, the EBV-Low subgroup showed a significantly worse prognosis for both gastric cancer and across cancer types. In summary, we classified tumors based on EBV copy number and found a unique cancer subgroup, EBV-positive in the surrounding lymphocytes, which was associated with a poor prognosis.

<title>Abstract</title> Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein–protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for &amp;gt;11 000 bait-prey pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.

<title>Abstract</title>Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

<title>Abstract</title>Morphological profiling is a combination of established optical microscopes and cutting-edge machine vision technologies, which stacks up successful applications in high-throughput phenotyping. One major question is how much information can be extracted from an image to identify genetic differences between cells. While fluorescent microscopy images of specific organelles have been broadly used for single-cell profiling, the potential ability of bright-field (BF) microscopy images of label-free cells remains to be tested. Here, we examine whether single-gene perturbation can be discriminated based on BF images of label-free cells using a machine learning approach. We acquired hundreds of BF images of single-gene mutant cells, quantified single-cell profiles consisting of texture features of cellular regions, and constructed a machine learning model to discriminate mutant cells from wild-type cells. Interestingly, the mutants were successfully discriminated from the wild type (area under the receiver operating characteristic curve = 0.773). The features that contributed to the discrimination were identified, and they included those related to the morphology of structures that appeared within cellular regions. Furthermore, functionally close gene pairs showed similar feature profiles of the mutant cells. Our study reveals that single-gene mutant cells can be discriminated from wild-type cells based on BF images, suggesting the potential as a useful tool for mutant cell profiling.

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.

Epstein-Barr virus (EBV) is associated with several human malignancies including 8-10% of gastric cancers (GCs). Genome-wide analysis of 3D chromatin topologies across GC lines, primary tissue and normal gastric samples revealed chromatin domains specific to EBV-positive GC, exhibiting heterochromatin-to-euchromatin transitions and long-range human-viral interactions with non-integrated EBV episomes. EBV infection in vitro suffices to remodel chromatin topology and function at EBV-interacting host genomic loci, converting H3K9me3+ heterochromatin to H3K4me1+/H3K27ac+ bivalency and unleashing latent enhancers to engage and activate nearby GC-related genes (for example TGFBR2 and MZT1). Higher-order epigenotypes of EBV-positive GC thus signify a novel oncogenic paradigm whereby non-integrative viral genomes can directly alter host epigenetic landscapes ('enhancer infestation'), facilitating proto-oncogene activation and tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

Although high-throughput biological technologies have been producing a vast amount of multi-omits data regarding cancer genomics and several disease susceptible genes have been reported, many of these genes are likely to be irrelevant for the cancer process because only one feature of the tumor pathway could be focused on. By identifying 'CpG core', which was extracted from CpG sites in genomic DNA by our newly developed method, we performed integrated analysis using gene expression and DNA methylation profiles of 116 colorectal cancer samples. First, based on gene expression values, colorectal cancer samples were divided into three clusters (Cluster-1, -2, and -3) by k-means clustering. The 5-year overall survival rates of colorectal cancer patients were 74.8%, 29.2%, and 29.4% in Cluster-1, -2, and -3, respectively, and the prognosis of Cluster-2 was significantly poorer than that of the other two clusters owing to liver metastasis (P &lt; 0.001). Second, each cluster was divided into two subgroups based on methylation status, and the 5-year overall survival rate of Cluster-1H (36.8%) was significantly shorter than that of Cluster-1L (96.1%) due to the accumulation of aberrant DNA methylation (P = 0.014). Third, network-based analysis using expression and methylation profiles demonstrated that nucleoporin family genes were downregulated in Cluster-2 and that the PTX3 gene was highly methylated in Cluster-1H. These combined data indicate that integrated analysis can identify disease characteristics that would be missed using single comprehensive analysis, and that multiple pathways would play pivotal roles in the liver metastasis of colorectal cancer. (C) 2016 Elsevier Inc. All rights reserved.

ROBO1 is a receptor mediating Slit-induced repulsive action on axon guidance and differentially expressed in human cancers. Although ROBO1 ectodomainhas been detected, the cleavage site had not been determined. In this study we identified the precise cleavage site of ROBO1. We also report multi-step proteolysis of ROBO1 by metalloproteinases and gamma-secretase, producing two carboxy-terminal fragments, ROBO1-CTF1 at 129-kDa and ROBO1-CTF2 at 118-kDa. We have further demonstrated nuclear accumulation of ROBO1, which is abolished by either a metalloproteinase inhibitor TAPI-1 or a gamma-secretase inhibitor L-685,458. ROBO1 may function beyond the receptor through stepwise cleavages and translocation to the nucleus. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Bombyx mori is a female-heterogametic organism (female, ZW; male, ZZ) that appears to have a putative feminizing gene (Fem) on the W chromosome. The paternally transmitted mutant W chromosome, Df(p(Sa) + W-p + (od))Fem, derived from the translocation-carrying W chromosome (p (Sa) + W-p + (od)), is inert as femaleness determinant. Moreover, this Df(p (Sa) + (p) W + (od))Fem chromosome has been thought to have a female-killing factor because no female larvae having the Df(p(Sa) + W-p + (od))Fem chromosome are produced. Initially, to investigate whether the Df(p(Sa) + W-p + (od))Fem chromosome contains any region of the W chromosome or not, we analyzed the presence or absence of 12 W-specific RAPD markers. The Df(p(Sa) + W-p + (od))Fem chromosome contained 3 of 12 W-specific RAPD markers. These results strongly indicate that the Df(p(Sa) + W-p + (od))Fem chromosome contains the region of the W chromosome. Moreover, by using phenotypic and molecular markers, we confirmed that the Df(p(Sa) + W-p + (od))Fem chromosome is connected with a partially deleted Z chromosome and that this fused chromosome behaves as a Z chromosome during male meiosis. Furthermore, we demonstrated that the ZZW-type triploid female having the Df(p(Sa) supercript stop + (p) W + (od))Fem chromosome is viable. Therefore, we concluded that the Df(p(Sa) + W-p + (od))Fem chromosome does not have a female-killing factor but that partial deletion of the Z chromosome causes the death of the ZW-type diploid female having the Df(p(Sa) + W-p + (od))Fem chromosome. Additionally, our results of detailed genetic analyses strongly indicate that the female-killing chromosome composed of the Df(p(Sa) + W-p + (od))Fem chromosome and deleted Z chromosome was generated by translocation between the Z chromosome and the translocation-carrying W chromosome, p(Sa) + W-p + (od).

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Wing discs of holometabolous insects undergo dramatic morphological changes during metamorphosis, a process that is controlled by the actions of hundreds of gene products. Using cDNA microarrays constructed from 5086 ESTs, we monitored the gene expression profiles in wing discs of Bombyx mori at 13 time points during pupal ecdysis (day-4 fifth instar larvae to day-0 pupae). Of the 5086 ESTs on the microarrays, 2998 ESTs had significant signals in more than half of the experiments. Of the 2998 ESTs, genes represented by 683 ESTs showed significant perturbations during pupal ecdysis. Genes previously known to be induced during metamorphosis were identified, including E75, Urbain, Chitinases, and cuticle proteins. The expressions of genes represented by 59 ESTs induced at the beginning of wandering contained genes predicted to be involved in protein degradation, amino acid metabolism, and amino acid transport. The expressions of genes represented by 147 ESTs induced after the ecdysteroid peak had a role in cuticle synthesis, pigmentation, ion transport, protein transport, and transcription regulation. The expressions of genes represented by 85 ESTs repressed after the ecdysteroid peak were predicted to be involved in nucleotide and nucleic acid metabolism and cell cycle. This indicates the involvement of several biological processes in wing disc development during metamorphosis. (C) 2004 Elsevier Ltd. All rights reserved.