Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

Clinical roles of soluble PD-1 and PD-L1 in plasma of NSCLC patients treated with immune checkpoint inhibitors.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have significantly improved the prognosis of non-small cell lung cancer (NSCLC). However, only a limited proportion of patients can benefit from this therapy, and clinically useful predictive biomarkers remain to be elucidated. METHODS: Blood was collected from 189 patients with NSCLC before and six weeks after the initiation of ICI treatment (anti-PD-1 or anti-PD-L1 antibody). Soluble PD-1 (sPD-1) and PD-L1 (sPD-L1) in plasma before and after treatment were analyzed to evaluate their clinical significance. RESULTS: Cox regression analysis demonstrated that higher sPD-L1 levels before treatment significantly predicted unfavorable progression-free survival (PFS; HR 15.4, 95% CI 1.10-86.7, P = 0.009) and overall survival (OS; HR 11.4, 95% CI 1.19-52.3, P = 0.007) in NSCLC patients treated with ICI monotherapy (n = 122) but not in those treated with ICIs combined with chemotherapy (n = 67: P = 0.729 and P = 0.155, respectively). In addition, higher sPD-1 levels after treatment were significantly associated with better OS (HR 0.24, 95% CI 0.06-0.91, P = 0.037) in patients treated with anti-PD-1 monotherapy, whereas higher sPD-L1 levels after treatment were significantly associated with worse PFS (HR 6.09, 95% CI 1.42-21.0, P = 0.008) and OS (HR 42.6, 95% CI 6.83-226, P < 0.001). The levels of sPD-L1 at baseline closely correlated with those of other soluble factors, such as sCD30, IL-2Ra, sTNF-R1, and sTNF-R2, which are known to be released from the cell surface by zinc-binding proteases ADAM10/17. CONCLUSIONS: These findings suggest the clinical significance of pretreatment sPD-L1 as well as posttreatment sPD-1 and sPD-L1 in NSCLC patients treated with ICI monotherapy.

Activation of EHF via STAT3 phosphorylation by LMP2A in Epstein-Barr virus-positive gastric cancer.

Epstein-Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome-widely in EBV-positive GC cell lines and in vitro EBV-infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with "signaling of WNT in cancer" were significantly enriched in EBV-positive GC, showing increased active ß-catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV-positive GC compared with normal tissue and EBV-negative GC was confirmed by RNA-seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway-related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV-positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation.

DNA Methylation and Genetic Aberrations in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. GC is a pathologically and molecularly heterogeneous disease. DNA hypermethylation in promoter CpG islands causes silencing of tumor-suppressor genes and thus contributes to gastric carcinogenesis. In addition, various molecular aberrations, including aberrant chromatin structures, gene mutations, structural variants, and somatic copy number alterations, are involved in gastric carcinogenesis. SUMMARY: Comprehensive DNA methylation analyses revealed multiple DNA methylation patterns in GCs and classified GC into distinct molecular subgroups: extremely high-methylation epigenotype uniquely observed in GC associated with Epstein-Barr virus (EBV), high-methylation epigenotype associated with microsatellite instability (MSI), and low-methylation epigenotype. In The Cancer Genome Atlas classification, EBV and MSI are extracted as independent subgroups of GC, whereas the remaining GCs are categorized into genomically stable (GS) and chromosomal instability (CIN) subgroups. EBV-positive GC, exhibiting the most extreme DNA hypermethylation in the whole human malignancies, frequently shows CDKN2A silencing, PIK3CA mutations, PD-L1/2 overexpression, and lack of TP53 mutations. MSI, exhibiting high DNA methylation, often has MLH1 silencing and abundant gene mutations. GS is generally a diffuse-type GC and frequently shows CDH1/RHOA mutations or CLDN18-ARHGAP fusion. CIN is generally an intestinal-type GC and frequently has TP53 mutations and genomic amplification of receptor tyrosine kinases. Key Messages: The frequency and targets of genetic aberrations vary depending on the epigenotype. Aberrations in the genome and epigenome are expected to synergistically interact and contribute to gastric carcinogenesis and comprehensive analyses of those in GCs may help elucidate the mechanism of carcinogenesis.

Epstein-Barr virus-positive gastric cancer involves enhancer activation through activating transcription factor 3.

Epstein-Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde-assisted isolation of regulatory elements (FAIRE) sequencing (-seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3- enhancers in EBV-infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV-determined nuclear antigen 1 (EBNA1), EBV-encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein-Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation-seq analysis on ATF3 binding sites and RNA-seq analysis on ATF3 knocked-down MKN7_EB revealed 96 genes targeted by ATF3-activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV-positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV-infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV-positive GC.

Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway.

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 < 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-ß 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.

Single cell visualization of yeast gene expression shows correlation of epigenetic switching between multiple heterochromatic regions through multiple generations.

Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR, HML, and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR-left and HMR-right regions, as well as the HMR-right and HML-right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR-left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity.

Enhancing Mass spectrometry-based tumor immunopeptide identification: machine learning filter leveraging HLA binding affinity, aliphatic index and retention time deviation.

Accurately identifying neoantigens is crucial for developing effective cancer vaccines and improving tumor immunotherapy. Mass spectrometry-based immunopeptidomics has emerged as a promising approach to identifying human leukocyte antigen (HLA) peptides presented on the surface of cancer cells, but false-positive identifications remain a significant challenge. In this study, liquid chromatography-tandem mass spectrometry-based proteomics and next-generation sequencing were utilized to identify HLA-presenting neoantigenic peptides resulting from non-synonymous single nucleotide variations in tumor tissues from 18 patients with renal cell carcinoma or pancreatic cancer. Machine learning was utilized to evaluate Mascot identifications through the prediction of MS/MS spectral consistency, and four descriptors for each candidate sequence: the max Mascot ion score, predicted HLA binding affinity, aliphatic index and retention time deviation, were selected as important features in filtering out identifications with inadequate fragmentation consistency. This suggests that incorporating rescoring filters based on peptide physicochemical characteristics could enhance the identification rate of MS-based immunopeptidomics compared to the traditional Mascot approach predominantly used for proteomics, indicating the potential for optimizing neoantigen identification pipelines as well as clinical applications.

Prognostic Significance of Plasma Neutrophil Extracellular Trap Levels in Patients with Non-Small Cell Lung Cancer Treated with Immune Checkpoint Inhibitors.

Neutrophil extracellular traps (NETs) released from neutrophils are related to cancer progression. However, the relationship between the therapeutic effects of immune checkpoint inhibitors (ICIs) such as anti-PD-1 and anti-PD-L1 antibodies and plasma NET concentration in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, concentrations of citrullinated histone H3 (CitH3), a surrogate marker of NETs, in plasma before/after treatment were examined in patients with advanced or recurrent NSCLC undergoing ICI treatment (n = 185). The clinical significances of NET levels before/after treatment and posttreatment changes were statistically evaluated. As a result, multivariate Cox analysis showed that high NET levels before treatment were statistically significant predictors of unfavorable overall survival (OS; p < 0.001, HR 1.702, 95% CI 1.356-2.137) and progression-free survival (PFS; p < 0.001, HR 1.566, 95% CI 1.323-1.855). The Kaplan-Meier curves showed significant separation between the high- and low-NET groups in OS (p = 0.002) and PFS (p < 0.001). Additionally, high NET levels after treatment were also significantly associated with worse OS (p < 0.001) and PFS (p < 0.001) by multivariate Cox analysis. Notably, the pretreatment NET levels were significantly correlated with the plasma levels of NET-related inflammatory cytokines, such as IL-6 and IL-8, and with NET-related gene expression and immune-suppressive profile in peripheral blood mononuclear cells. Our findings suggest that NETs released from activated neutrophils might reduce the clinical efficacy of ICIs in patients with NSCLC.

Anti-proliferating and apoptosis-inducing activity of chemical compound FTI-6D in association with p53 in human cancer cell lines.

Compounds with 3,4-fused tricyclic indole (FTI) frameworks are attractive scaffolds for drug discovery. We synthesized FTI-6D, a compound with this framework, which was cytotoxic in several human cancer cell lines. FTI-6D induced apoptosis via activation of the p53 downstream mitochondria-related apoptotic pathway, characterized by an increased ratio of pro-apoptotic Bcl-2 family members to anti-apoptotic members. This change was followed by caspase-9 and caspase-3 cleavage and activation in two cancer cell lines, RKO and AGS. The anti-proliferating effect of FTI-6D was remarkably detected in eight cancer cells with wild-type TP53 (TP53_wt), including RKO and AGS, but not in seven cancer cells with mutated TP53 (TP53_mut). Additionally, p53 protein levels increased after FTI-6D treatment in TP53_wt cancer cells, and the cytotoxic effect of FTI-6D was decreased by TP53 knockdown. Accordingly, the expression of p53 downstream genes involved in apoptotic signaling pathways, such as BBC3 and TP53INP1, and those involved in cell growth inhibition, such as CDKN1A, was upregulated in TP53_wt cancer cells. These results suggest that the anti-proliferative and apoptosis-inducing activities of FTI-6D rely on p53 and the corresponding signaling processes. This study demonstrated that FTI-6D shows anti-cancer activity against TP53_wt cancer cells. FTI-6D may have potential as a prototype compound for a new drug to utilize a functional p53 pathway in TP53_wt cancer cells.

Circulating IL-6 and not its circulating signaling components sIL-6R and sgp130 demonstrate clinical significance in NSCLC patients treated with immune checkpoint inhibitors.

Introduction: Clinical roles of plasma IL-6 levels have been reported in patients with various cancers, including non-small cell lung cancer (NSCLC), treated with immune checkpoint inhibitors (ICIs). However, the roles of other IL-6 signaling components, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130), in the plasma have not been elucidated. Methods: Blood was collected from 106 patients with NSCLC before initiation of ICI treatment (anti-PD-1 or anti-PD-L1 antibody). Plasma levels of IL-6, sIL-6R, sgp130, and their complexes were assessed by Cox regression hazard model to evaluate their clinical significance. The clinical role of IL-6 or IL-6R genetic polymorphisms was also analyzed. Results: Cox regression analysis showed that higher plasma IL-6 levels significantly predicted unfavorable overall survival (OS; hazard ratio [HR] 1.34, 95% confidence interval [CI] 1.05-1.68, p = 0.012) in NSCLC patients treated with ICIs. However, plasma sIL-6R and sgp130 levels showed no prognostic significance (p = 0.882 and p = 0.934, respectively). In addition, the estimated concentrations of binary IL-6:sIL-6R and ternary IL-6:sIL-6R:sgp130 complexes and their ratios (binary/ternary complex) were not significantly associated with OS (p = 0.647, p = 0.727, and p = 0.273, respectively). Furthermore, the genetic polymorphisms of IL-6 (-634G>C) and IL-6R (48892A>C) showed no clinical role by Kaplan-Meier survival analysis (p = 0.908 and p = 0.639, respectively). Discussion: These findings demonstrated the clinical significance of plasma levels of IL-6, but not of other IL-6 signaling components, sIL-6R and sgp130, suggesting that classical IL-6 signaling, but not trans-signaling, may be related to anti-tumor immune responses in cancer patients treated with ICIs.

Cinobufagin inhibits proliferation of acute myeloid leukaemia cells by repressing c-Myc pathway-associated genes.

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na+/K+ ATPase and have similar effects as cardiac glycosides, such as digitoxin or ouabain derived from toxic herbs. Recently, the anti-cancer effects of bufanolides have gained attention, however the underlying molecular mechanisms remain unclear. Selecting cinobufagin as a candidate anti-leukaemia agent, we here conducted transcriptomic analyses on the effect of cinobufagin on human acute myeloid leukaemia (AML) cell lines, HL60 and Kasumi-1. Flow cytometry analysis showed that cinobufagin induced apoptosis in both cell lines. RNA-sequencing (RNA-seq) of the two cell lines treated with cinobufagin revealed commonly downregulated genes with enrichment in the term "Myc active pathway" according to Gene Ontology (GO) analysis. Gene Set Enrichment Analysis (GSEA) of genes downregulated by cinobufagin also showed "MYC_TARGETS_V2" with the highest normalised enrichment score (NES) in both cell lines. In contrast, hallmarks such as "TNFA_SIGNALING_VIA_NFKB", "APOPTOSIS", and "TGF_BETA_SIGNALING" were significantly enriched as upregulated gene sets. Epigenetic analysis using chromatin immunoprecipitation and sequencing (ChIP-seq) confirmed that genes encoding cell death-related signalling molecules were upregulated by gain of H3K27ac, whereas downregulation of c-Myc-related genes was not accompanied by H3K27ac alteration. Cinobufagin is an anti-proliferative natural compound with c-Myc-inhibiting and epigenetic-modulating activity in acute myeloid leukaemia.

Identification of AR-V7 downstream genes commonly targeted by AR/AR-V7 and specifically targeted by AR-V7 in castration resistant prostate cancer.

Primary prostate cancer (PC) progresses to castration-resistant PC (CRPC) under androgen deprivation therapy, by mechanisms e.g. expression of androgen receptor (AR) splice variant-7 (AR-V7). Here we conducted comprehensive epigenome and transcriptome analyses comparing LNCaP, primary PC cells, and LNCaP95, AR-V7-expressing CRPC cells derived from LNCaP. Of 399 AR-V7 target regions identified through ChIP-seq analysis, 377 could be commonly targeted by hormone-stimulated AR, and 22 were specifically targeted by AR-V7. Among genes neighboring to these AR-V7 target regions, 78 genes were highly expressed in LNCaP95, while AR-V7 knockdown led to significant repression of these genes and suppression of growth of LNCaP95. Of the 78 AR-V7 target genes, 74 were common AR/AR-V7 target genes and 4 were specific AR-V7 target genes; their most suppressed genes by AR-V7 knockdown were NUP210 and SLC3A2, respectively, and underwent subsequent analyses. NUP210 and SLC3A2 were significantly upregulated in clinical CRPC tissues, and their knockdown resulted in significant suppression of cellular growth of LNCaP95 through apoptosis and growth arrest. Collectively, AR-V7 contributes to CRPC proliferation by activating both common AR/AR-V7 target and specific AR-V7 target, e.g. NUP210 and SLC3A2.

Cross-species chromatin interactions drive transcriptional rewiring in Epstein-Barr virus-positive gastric adenocarcinoma.

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.

Epigenetic alteration to activate Bmp2-Smad signaling in Raf-induced senescence.

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.

TET2 functions as a resistance factor against DNA methylation acquisition during Epstein-Barr virus infection.

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.

Fub1p, a novel protein isolated by boundary screening, binds the proteasome complex.

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.