MicroRNAs (miRNAs) are small regulatory non-coding RNAs with a diversity of cellular functions, and are frequently dysregulated in cancer. Using a novel computational method (ActMir) that we recently developed, the "activity" of miRNA hsa-miR-500a was implicated in estrogen receptor (ER) positive breast cancer; however its targets and functional impact remain poorly understood. Here, we performed an extensive gene expression analysis in ER+ breast cancer cell lines, to reveal the targets of miR-500a-5p after experimental modulation of its levels. We found that among mRNAs targeted by miR-500a-5p there was enrichment in oxidative stress response genes. Moreover, in vitro exposure to oxidative stress using H2O2 induces miR-500a-5p overexpression and downregulation of the oxidative stress targets TXNRD1 and NFE2L2. Finally, expression of several of the identified miR-500a-5p targets related to oxidative stress, including TXNRD1, was associated with ER+ breast cancer survival in multiple datasets. Overall, we identify miR-500a-5p as an oxidative stress response miRNA whose activity may define breast cancer progression and survival.

Epstein-Barr virus (EBV) was identified as the first human virus to be associated with a human malignancy, Burkitt's lymphoma (BL), a pediatric cancer endemic in sub-Saharan Africa. The exact mechanism of how EBV contributes to the process of lymphomagenesis is not fully understood. Recent studies have highlighted a genetic difference between endemic (EBV+) and sporadic (EBV-) BL, with the endemic variant showing a lower somatic mutation load, which suggests the involvement of an alternative virally-driven process of transformation in the pathogenesis of endemic BL. We tested the hypothesis that a global change in DNA methylation may be induced by infection with EBV, possibly thereby accounting for the lower mutation load observed in endemic BL. Our comparative analysis of the methylation profiles of a panel of BL derived cell lines, naturally infected or not with EBV, revealed that the presence of the virus is associated with a specific pattern of DNA methylation resulting in altered expression of cellular genes with a known or potential role in lymphomagenesis. These included ID3, a gene often found to be mutated in sporadic BL. In summary this study provides evidence that EBV may contribute to the pathogenesis of BL through an epigenetic mechanism.

Mammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.