Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement—the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.

Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensivehypermethylaton 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.

[Backgrounds] POEMS syndrome is a rare plasma cell dyscrasia and its pathogenesis including the significance of monoclonal plasma cells in disease progression is poorly understood. Monoclonal plasma cells only produce λ immunoglobulins, and genes encoding immunoglobulin λ light chain (IGL) V regions are derived from IGLV1-44 or IGLV1-40 germline sequences. Here we analyzed the clonality in IGLV gene rearrangements using next generation sequencing (NGS) to evaluate the significance of monoclonal plasma cell clone size and follow its changes in clinical course to understand the pathogenesis of POEMS syndrome. [Methods] Patients who were diagnosed with POEMS syndrome between November 2006 and October 2015 at Chiba University Hospital were included in the study. As positive controls, 3 multiple myeloma (MM) patients with λ-type monoclonal light chain, and 9 negative control patients were also analyzed. NGS libraries were constructed from genomic DNA samples, extracted from bone marrow mononuclear cells. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction (PCR) using a 5' primer for the IGLV1/2 framework 3 (FR3) region and 3ʹ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were carried out using an Illumina MiSeq sequencer. The closest matched germline sequences were determined using the ImMunoGeneTics database. Subsequently, frequencies of each clonotype that were characterized by a unique V-J rearrangement, conserved complementarity determining region 3 (CDR3) anchors and a unique CDR3 amino acid sequence, were calculated. [Results] Twenty-eight patients with POEMS syndrome were enrolled. All the patients had λ-type M protein. The median follow up time of the patients was 24.4 months (range, 3.7 - 113.9). Firstly we analyzed the usage of IGLV germline genes in each case. In 8 cases, the POEMS syndrome-specific germline sequences, IGLV1-40 or IGLV1-44, were dominant; accounting for more than 40% of all germline sequence usage. However, other samples showed minimal or no differences from controls, indicating that the clonal expansion of monoclonal plasma cells is generally low in patients with POEMS syndrome. Analyzing frequencies of the most dominant rearrangement in each germline, the clonal IGLV gene rearrangements of POEMS syndrome-specific germline sequences were significantly increased in 10 POEMS patients (35.7%; IGLV1-44: n = 8, IGLV1-40: n = 2). Significant increase of clone sizes were not directly linked to the initial disease status (vascular endothelial growth factor [VEGF] level and percentage of plasma cells in the bone marrow), overall survival and progression-free survival of POEMS patients. In 12 patients that we were able to follow their clinical courses, the clone size of IGLVgene rearrangements correlated with disease course assessed with serum VEGF level in most cases (n = 8), as they decreased with serum VEGF levels in disease remission and increased with re-elevation of serum VEGF in relapse cases. Clone sizes without significant increase at diagnosis were constantly flat even after achieving disease remission (n = 3). In one case, clone size with significant increase at diagnosis was unchanged even after serum VEGF level decreased (n = 1). Further observation is needed in this case. [Discussion] Considering the cases with significant increase of IGLV rearrangement clones, it was confirmed that clonal light chain gene expression is restricted to the IGLV1-44 and IGLV1-40 germline sequences, as previously reported. IGLV gene rearrangement clone was not detected as significant increase in some cases. Therefore, we speculated that there are certain numbers of patients with POEMS syndrome with extremely low frequency of clone cells. On the other hand, significant increase of clone size did not reflect disease status, suggesting that disease status is not regulated by tumor burden alone. By contrast, in cases with significantly increased clones, clone sizes changed dependiing on the disease status. These data do demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome. [Conclusions] Our analysis of IGLV gene rearrangements has demonstrated the association between the size of IGLV gene rearrangement clones and the clinical courses in POEMS syndrome. Disclosures No relevant conflicts of interest to declare.