福世 真樹

Familial adenomatous polyposis (FAP) is an inherited disorder characterized by numerous colorectal adenomatous polyps with predisposition to the development of colorectal cancer (CRC). Here, we conducted genome-wide DNA methylation analysis of FAP neoplasms, including seven cancer samples and 16 adenoma samples, using an Infinium 450K BeadArray. As controls for sporadic colorectal neoplasms and mucosae, we used Infinium 450k data from 297 CRC samples, 45 colorectal adenoma samples, and 37 normal mucosa samples with reference to The Cancer Genome Atlas and other databases. Unsupervised two-way hierarchical clustering analysis of FAP and sporadic CRC/adenoma revealed that CRC was classified into four DNA methylation epigenotypes (MEs): high-ME (HME), intermediate-ME (IME), low-ME (LME), and normal-like ME (NME). Five FAP neoplasms (two cancer and three adenoma) were clustered with IME, whereas 18 FAP neoplasms (five cancer and 13 adenoma) were clustered into NME. IME FAP neoplasms significantly correlated with KRAS mutations, similar to sporadic CRC. Within IME cases, however, aberrant DNA methylation was significantly less frequent in FAP neoplasms than sporadic neoplasms, and these unmethylated genes included WNT family genes and several types of oncogenes. In summary, FAP neoplasms were classified into at least two molecular subtypes, i.e., NME in the majority of cases showing mostly no aberrant methylation and IME in some cases accompanied by KRAS mutations but less frequent aberrant DNA methylation than sporadic neoplasms, suggesting that FAP may follow a tumorigenesis pathway different from that of sporadic CRC.

Epigenome regulates gene expression to determine cell fate, and accumulation of epigenomic aberrations leads to diseases, including cancer. NCD38 inhibits lysine-specific demethylase-1 (LSD1), a histone demethylase targeting H3K4me1 and H3K4me2, but not H3K4me3. In this study, we conjugated NCD38 with a potent small molecule called pyrrole (Py) imidazole (Im) polyamide, to analyze whether targets of the inhibitor could be regulated in a sequence-specific manner. We synthesized two conjugates using β-Ala (β) as a linker, i.e., NCD38-β-β-Py-Py-Py-Py (NCD38-β2P4) recognizing WWWWWW sequence, and NCD38-β-β-Py-Im-Py-Py (NCD38-β2PIPP) recognizing WWCGWW sequence. When RKO cells were treated with NCD38, H3K4me2 levels increased in 103 regions with significant activation of nearby genes (P = 0.03), whereas H3K4me3 levels were not obviously increased. H3K27ac levels were also increased in 458 regions with significant activation of nearby genes (P = 3 × 10-10), and these activated regions frequently included GC-rich sequences, but less frequently included AT-rich sequences (P < 1 × 10-15) or WWCGWW sequences (P = 2 × 10-13). When treated with NCD38-β2P4, 234 regions showed increased H3K27ac levels with significant activation of nearby genes (P = 2 × 10-11), including significantly fewer GC-rich sequences (P < 1 × 10-15) and significantly more AT-rich sequences (P < 1 × 10-15) compared with NCD38 treatment. When treated with NCD38-β2PIPP, 82 regions showed increased H3K27ac levels, including significantly fewer GC-rich sequences (P = 1 × 10-11) and fewer AT-rich sequences (P = 0.005), but significantly more WWCGWW sequences (P = 0.0001) compared with NCD38 treatment. These indicated that target regions of epigenomic inhibitors could be modified in a sequence-specific manner and that conjugation of Py-Im polyamides may be useful for this purpose.

Oropharyngeal squamous cell carcinoma (OPSCC) incidence has increased dramatically due to human papillomavirus (HPV); however, associated epigenetic alterations are not well studied. We performed genome-wide DNA methylation analysis using an Infinium 450k BeadArray for clinical OPSCC and noncancerous samples and cancer cell lines with/without 5-aza-2 '-deoxycytidine and/or trichostatin A treatment. Frequent promoter hypermethylation and methylation-associated silencing were detected in 144 genes, which included those involved in cell-cell signaling and neuron differentiation. The methylation of nine genes (GHSR, ITGA4, RXRG, UTF1, CDH8, FAN19A4, CTNNA2, NEFH, and CASR) was quantitatively validated in 70 pharyngeal SCC cases by pyrosequencing. Hypermethylation significantly correlated with HPV-L1 positivity, but not with age or smoking status. p16INK4A was generally activated in HPV-L1(+) tumors, and p16-positive cases significantly associated with better prognosis. RXRG hypermethylation strongly correlated with positivity of HPV-L1 and p16 (P = 3 x 10(-5) and P = 5 x 10(-4), respectively). RXRG-methylation(+) significantly associated with better prognosis when analyzing all tumor cases (P = 0.04), and when analyzing the p16-negative poorer-outcome group (P = 0.03). Thus, aberrant DNA methylation might be involved in HPV-associated OPSCC; in addition, DNA methylation could serve as a marker to classify subgroups based on outcome. (C) 2017 Elsevier B.V. All rights reserved.

Epstein-Barr virus (EBV)-positive gastric cancer (GC) shows a higher DNA methylation epigenotype. EBV infection can causally induce genome-wide aberrant DNA methylation, as previously demonstrated by in vitro infection experiments in the low-methylation GC cell line MKN7. However, whether EBV exerts DNA methylation remodelling properties in non-neoplastic epithelial cells remains unclear. Here we performed post-infection time-series DNA methylation analyses using the immortalized normal gastric epithelial cell line GES1. Genome-wide analysis using Illumina's Infinium 450 k BeadArray demonstrated global de novo DNA methylation from post-infection day 17, which was completed by 28 days in a manner similar to that observed in MKN7 cells. De novo methylation of all types of GC-specific methylation marker genes was observed, indicating that EBV infection is sufficient for gastric epithelial cells to acquire an EBV-positive GC epigenotype. Pyrosequencing demonstrated that methylation of the viral genome preceded that of the host cellular genome, suggesting the existence of well-ordered mechanisms that induce methylation. Spatiotemporal representation with differential models revealed dynamic alterations of DNA methylation in promoter regions, occurring from lower-CpG peripheral regions and extending to higher-CpG core regions. In summary, EBV infection exerted powerful pressure to induce global de novo DNA methylation in non-neoplastic cells within a month in a spatiotemporally well-ordered manner. Copyright (C) 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epstein-Barr virus (EBV) infection is associated with tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancer. We previously showed that EBV(+) gastric cancer presents an extremely high-methylation epigenotype and this aberrant DNA methylation causes silencing of multiple tumour suppressor genes. However, the mechanisms that drive EBV infectionmediated tumorigenesis, including other epigenomic alteration, remain unclear. We analysed epigenetic alterations induced by EBV infection especially at enhancer regions, to elucidate their contribution to tumorigenesis. We performed ChIP sequencing on H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K9me3 in gastric epithelial cells infected or not with EBV. We showed that repressive marks were redistributed after EBV infection, resulting in aberrant enhancer activation and repression. Enhancer dysfunction led to the activation of pathways related to cancer hallmarks (e.g., resisting cell death, disrupting cellular energetics, inducing invasion, evading growth suppressors, sustaining proliferative signalling, angiogenesis, and tumour-promoting inflammation) and inactivation of tumour suppressive pathways. Deregulation of cancer-related genes in EBV-infected gastric epithelial cells was also observed in clinical EBV(+) gastric cancer specimens. Our analysis showed that epigenetic alteration associated with EBV-infection may contribute to tumorigenesis through enhancer activation and repression.

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIPsequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation- sensitive genes significantly correlated with loss of H3K27me3 premarks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation- resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

Sporadic colorectal cancer (CRC) is classified into several molecular subtypes. We previously established two groups of DNA methylation markers through genome-wide DNA methylation analysis to classify CRC into distinct subgroups: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME, respectively). HME CRC, also called CpG island methylator phenotype (CIMP)-high CRC, shows methylation of both Group 1 markers (CIMP markers) and Group 2 markers, while IME/CIMP-low CRC shows methylation of Group 2, but not of Group 1 markers, and LME CRC shows no methylation of either Group 1 or Group 2 markers. While BRAF- and KRAS-mutation(+) CRC strongly correlated with HME and IME, respectively, clinicopathological features of NRAS-mutation(+) CRC, including association with DNA methylation, remain unclear. To characterize NRAS-mutation(+) CRC, the methylation levels of 19 methylation marker genes (6 Group 1 and 13 Group 2) were analyzed in 61 NRAS-mutation(+) and 144 NRAS-mutation(-) CRC cases by pyrosequencing, and their correlation with clinicopathological features was investigated. Different from KRAS-mutation(+) CRC, NRAS-mutation(+) CRC significantly correlated with LME. NRAS-mutation(+) CRC showed significantly better prognosis than KRAS-mutation(+) CRC (P = 3 x 10(-4)). NRAS-mutation(+) CRC preferentially occurred in elder patients (P = 0.02) and at the distal colon (P = 0.006), showed significantly less lymph vessel invasion (P = 0.002), and correlated with LME (P = 8 x 10(-5)). DNA methylation significantly accumulated at the proximal colon. NRAS-mutation(+) CRC may constitute a different subgroup from KRAS-mutation(+) CRC, showing significant correlation with LME, older age, distal colon, and relatively better prognosis.

All restriction enzymes examined are phosphodiesterases generating 3'-OH and 5'-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R. PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R. PabI homologs from Campylobacter coli (R. CcoLI) and Helicobacter py-lori (R. HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37. C. The AP lyase activity is more coupled with glycosylase activity in R. CcoLI than in R. PabI. R. CcoLI/R. PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37 degrees C. The R. PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biologi-cal significance. These results support generalization of the concept of restriction-modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

Aberrant DNA methylation causes major epigenetic changes and has been implicated in cancer following the inactivation of tumor suppressor genes by hypermethylation of promoter CpG islands. Although methylated DNA regions can be randomly demethylated by 5-azacytidine and 5-aza-2'-deoxycytidine, site-specific inhibition of DNA methylation, for example, in the promoter region of a specific gene, has yet to be technically achieved. Hairpin pyrrole (Py)-imidazole (Im) polyamides are small molecules that can be designed to recognize and bind to particular DNA sequences. In this study, we synthesized the hairpin polyamide MLH1_-16 (Py-Im-β-Im-Im-Py-γ-Im-Py-β-Im-Py-Py) to target a CpG site 16 bp upstream of the transcription start site of the human MLH1 gene. MLH1 is known to be frequently silenced by promoter hypermethylation, causing microsatellite instability and a hypermutation phenotype in cancer. We show that MLH1_-16 binds to the target site and that CpG methylation around the binding site is selectively inhibited in vitro. MLH1_non, which does not have a recognition site in the MLH1 promoter, neither binds to the sequence nor inhibits DNA methylation in the region. When MLH1_-16 was used to treat RKO human colorectal cancer cells in a remethylating system involving the MLH1 promoter under hypoxic conditions (1% O2), methylation of the MLH1 promoter was inhibited in the region surrounding the compound binding site. Silencing of the MLH1 expression was also inhibited. Promoter methylation and silencing of MLH1 were not inhibited when MLH1_non was added. These results indicate that Py-Im polyamides can act as sequence-specific antagonists of CpG methylation in living cells.

Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5' regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2, were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A, and also by seven human miRNAs targeting TET2, e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2. Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.

Decoding of closely related genomes is now revealing the process of population evolution. In bacteria, population divergence appears associated with a unique set of sequence-specific epigenetic DNA methylation systems, often within restriction-modification (RM) systems. They might define a unique gene expression pattern and limit genetic flux between lineages in population divergence. We addressed the contribution of methylation systems to population diversification in panmictic bacterial species, Helicobacter pylori, which shows an interconnected population structure through frequent mutual recombination. We analyzed complete genome sequences of 28 strains collected in Fukui, Japan. Their nucleotide sequences are closely related although fine-scale analyses revealed two subgroups likely reflecting human subpopulations. Their sequences are tightly connected by homologous recombination. Our extensive analysis of RM systems revealed an extreme variability in DNA methyltransferases, especially in their target recognition domains. Their diversity was, however, not immediately related to the genome sequence diversity, except for very closely related strains. An interesting exception is a hybrid strain, which likely has conserved the methylation gene repertoire from one parent but diversified in sequence by massive acquisition of fragmentary DNA sequences from the other parent. Our results demonstrate how a bacterial population can be extremely divergent in epigenetics and yet homogenized in sequence.

Patients with gastric cancer typically face gastrectomies even when few or no nodal metastases are reported. Current procedures poorly predict lymphatic metastases; thus, evaluation of target molecules expressed on cancer cell membranes is necessary for in vivo detection. However, marker development is limited by the intratumoral heterogeneity of gastric cancer cells. In this study, multiple gene expression arrays of 42 systemic normal tissue samples and 56 gastric cancer samples were used to investigate two adhesion molecules, cadherin 17 (CDH17) and claudin 18 (CLDN18), which are intestinal and gastric markers, respectively. Expression of CDH17 and CLDN18 was partially redundant, but overlapped in 50 of 56 cases (89.3%). Tissue microarrays constructed using primary lesions and nodal metastases of 106 advanced gastric cancers revealed CDH17 and CLDN18 expression in 98 positive cases of 106 (92%). Hierarchical clustering classified gastric cancers into three subgroups, CDH17(++)/CLDN18(+/-), CDH17(++)/CLDN18(++) or CDH17(+)/CLDN18(+), and CDH17(-)/CLDN18(++/+/-). Whole tissue sections displayed strong, homogeneous staining for CDH17 and CLDN18. Together, these results indicate that CDH17 and CLDN18 are useful target molecules; moreover, their coupling can aid in the comprehensive detection and localization of gastric cancer metastases in vivo to overcome challenges associated with intratumoral heterogeneity.

Genomes of a given bacterial species can show great variation in gene content and thus systematic analysis of the entire gene repertoire, termed the pan-genome, is important for understanding bacterial intra-species diversity, population genetics, and evolution. Here, we analyzed the pan-genome from 30 completely sequenced strains of the human gastric pathogen Helicobacter pylori belonging to various phylogeographic groups, focusing on 991 accessory (not fully conserved) orthologous groups (OGs). We developed a method to evaluate the mobility of genes within a genome, using the gene order in the syntenically conserved regions as a reference, and classified the 991 accessory OGs into five classes: Core, Stable, Intermediate, Mobile, and Unique. Phylogenetic networks based on the gene content of Core and Stable classes are highly congruent with that created from the concatenated alignment of fully conserved core genes, in contrast to those of Intermediate and Mobile classes, which show quite different topologies. By clustering the accessory OGs on the basis of phylogenetic pattern similarity and chromosomal proximity, we identified 60 co-occurring gene clusters (CGCs). In addition to known genomic islands, including cag pathogenicity island, bacteriophages, and integrating conjugative elements, we identified some novel ones. One island encodes TerY-phosphorylation triad, which includes the eukaryote-type protein kinase/phosphatase gene pair, and components of type VII secretion system. Another one contains a reverse-transcriptase homolog, which may be involved in the defense against phage infection through altruistic suicide. Many of the CGCs contained restriction-modification (RM) genes. Different RM systems sometimes occupied the same (orthologous) locus in the strains. We anticipate that our method will facilitate pangenome studies in general and help identify novel genomic islands in various bacterial species.

Objective Pulmonary adenocarcinoma with a micropapillary component (MPC) has aggressive malignant behavior even if resectable. The aim of this study was to determine clinicopathologic features of patients who underwent surgery for pulmonary adenocarcinoma harboring MPCs, with particular focus on coexistent free tumor clusters (FTCs). Methods We retrospectively reviewed 444 patients with pulmonary adenocarcinoma who underwent surgery from March 2007 to July 2013. An MPC was defined as a &gt 5% micropapillary pattern. We also defined FTCs to be a group of more than 3 small clusters containing &lt 20 nonintegrated micropapillary tumor cells that were spreading within air spaces, &gt 3 mm apart from the main tumor. The clinicopathologic characteristics of patients with and without FTCs were retrospectively investigated in MPC-positive patients. Results MPCs were identified in 67 patients (15.1%), 31 of whom (46.3%) were positive for FTCs. The distance between the furthest edge of FTCs and main tumors did not exceed the diameter of the main tumor in each case (average, 7.3 mm). Locoregional recurrences were frequently observed in FTC-positive patients. FTC-positive patients experienced a significantly lower 5-year recurrence-free survival rate compared with FTC-negative/MPC-positive patients (20.4% vs 52.2%, P &lt .001). Recurrence-free survival of FTC-negative and -positive patients was equivalent to that of patients with p-T2 and p-T3 MPC-negative adenocarcinoma, respectively. Conclusions Coexistence of FTCs resulted in a further negative impact on postoperative prognosis among MPC-positive adenocarcinomas and should be considered for upstaging the p-T factor and during evaluation of surgical margins.

Objective: Pulmonary adenocarcinoma with a micropapillary component (MPC) has aggressive malignant behavior even if resectable. The aim of this study was to determine clinicopathologic features of patients who underwent surgery for pulmonary adenocarcinoma harboring MPCs, with particular focus on coexistent free tumor clusters (FTCs). Methods: We retrospectively reviewed 444 patients with pulmonary adenocarcinoma who underwent surgery from March 2007 to July 2013. An MPC was defined as a&gt; 5% micropapillary pattern. We also defined FTCs to be a group of more than 3 small clusters containing&lt; 20 nonintegrated micropapillary tumor cells that were spreading within air spaces,&gt; 3mm apart from the main tumor. The clinicopathologic characteristics of patients with and without FTCs were retrospectively investigated in MPC-positive patients. Results: MPCs were identified in 67 patients (15.1%), 31 of whom (46.3%) were positive for FTCs. The distance between the furthest edge of FTCs and main tumors did not exceed the diameter of the main tumor in each case (average, 7.3 mm). Locoregional recurrences were frequently observed in FTC-positive patients. FTC-positive patients experienced a significantly lower 5-year recurrence-free survival rate compared with FTC-negative/MPC-positive patients (20.4% vs 52.2%, P &lt;.001). Recurrence-free survival of FTC-negative and -positive patients was equivalent to that of patients with p-T2 and p-T3 MPC-negative adenocarcinoma, respectively. Conclusions: Coexistence of FTCs resulted in a further negative impact on postoperative prognosis among MPC-positive adenocarcinomas and should be considered for upstaging the p-T factor and during evaluation of surgical margins.

Although most sporadic colorectal cancers (CRC) are thought to develop from protruded adenomas through the adenoma-carcinoma sequence, some CRC develop through flat lesions, so-called laterally spreading tumors (LST). We previously analyzed epigenetic aberrations in LST and found that LST are clearly classified into two molecular subtypes: intermediate-methylation with KRAS mutation and low-methylation with absence of oncogene mutation. Intermediate-methylation LST were mostly granular type LST (LST-G) and low-methylation LST were mostly non-granular LST (LST-NG). In the present study, we conducted a targeted exon sequencing study including 38 candidate CRC driver genes to gain insight into how these genes modulate the development of LST. We identified a mean of 11.5 suspected nonpolymorphic variants per sample, including indels and non-synonymous mutations, although there was no significant difference in the frequency of total mutations between LST-G and LST-NG. Genes associated with RTK/RAS signaling pathway were mutated more frequently in LST-G than LST-NG (P = 0.004), especially KRAS mutation occurring at 70% (30/43) of LST-G but 26% (13/50) of LST-NG (P &lt; 0.0001). Both LST showed high frequency of APC mutation, even at adenoma stage, suggesting its involvement in the initiation stage of LST, as it is involved at early stage of colorectal carcinogenesis via adenoma-carcinoma sequence. TP53 mutation was never observed in adenomas, but was specifically detected in cancer samples. TP53 mutation occurred during development of intramucosal cancer in LST-NG, but during development of cancer with submucosal invasion in LST-G. It is suggested that TP53 mutation occurs in the early stages of cancer development from adenoma in both LST-G and LST-NG, but is involved at an earlier stage in LST-NG.

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high-methylation CRC. The methylation levels of six Group-1 and 14 Group-2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group-1 and Group-2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high-methylation CRC. But TSA showed low-methylation levels of Group-1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high-methylation CRC. High-methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p &lt; 0.0001) and insertion/deletions (p = 0.002). MLH1-methylated SSA/P showed lower methylation level of MLH1 compared with high-methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1-methylated and MLH1-unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF-beta and BMP signaling (but not in TP53 signaling) were significantly involved in high-methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.

AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf. METHODS: Cellular senescence was induced into mouse embryonic fibroblasts (MEFs) by infecting retrovirus to express oncogenic Raf (RafV600E). RNA was collected from RafV600E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV600E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection. RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by shRNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss. CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.

The restriction-modification systems use epigenetic modification to distinguish between self and non-self DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.Pabl) of the Pabl superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5/-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5/-GT# C, #=AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.Pabl reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.Pabl is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Pathogen infection often leads to the expression of virulence and host death when the host-pathogen symbiosis seems more beneficial for the pathogen. Previously proposed explanations have focused on the pathogen's side. In this work, we tested a hypothesis focused on the host strategy. If a member of a host population dies immediately upon infection aborting pathogen reproduction, it can protect the host population from secondary infections. We tested this "Suicidal Defense Against Infection" (SDAI) hypothesis by developing an experimental infection system that involves a huge number of bacteria as hosts and their virus as pathogen, which is linked to modeling and simulation. Our experiments and simulations demonstrate that a population with SDAI strategy is successful in the presence of spatial structure but fails in its absence. The infection results in emergence of pathogen mutants not inducing the host suicide in addition to host mutants resistant to the pathogen.

Homing endonuclease genes are ``selfish&apos;&apos; mobile genetic elements whose endonuclease promotes the spread of its own gene by creating a break at a specific target site and using the host machinery to repair the break by copying and inserting the gene at this site. Horizontal transfer across the boundary of a species or population within which mating takes place has been thought to be necessary for their evolutionary persistence. This is based on the assumption that they will become fixed in a host population, where opportunities of homing will disappear, and become susceptible to degeneration. To test this hypothesis, we modeled behavior of a homing endonuclease gene that moves during meiosis through double-strand break repair. We mathematically explored conditions for persistence of the homing endonuclease gene and elucidated their parameter dependence as phase diagrams. We found that, if the cost of the pseudogene is lower than that of the homing endonuclease gene, the 2 forms can persist in a population through autonomous periodic oscillation. If the cost of the pseudogene is higher, 2 types of dynamics appear that enable evolutionary persistence: bistability dependent on initial frequency or fixation irrespective of initial frequency. The prediction of long persistence in the absence of horizontal transfer was confirmed by stochastic simulations in finite populations. The average time to extinction of the endonuclease gene was found to be thousands of meiotic generations or more based on realistic parameter values. These results provide a solid theoretical basis for an understanding of these and other extremely selfish elements. POPULATION BIOLOGY