Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.

Epigenetic remodelling of enhancers in response to estrogen deprivation and re-stimulation.

Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance.

Roadmap for investigating epigenome deregulation and environmental origins of cancer.

The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment.

Lowly methylated region analysis identifies EBF1 as a potential epigenetic modifier in breast cancer.

Breast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.

Unique DNA methylation signature in HPV-positive head and neck squamous cell carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited. METHODS: We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours. RESULTS: We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort. CONCLUSIONS: We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs.

DNA methylome analysis identifies accelerated epigenetic ageing associated with postmenopausal breast cancer susceptibility.

AIM OF THE STUDY: A vast majority of human malignancies are associated with ageing, and age is a strong predictor of cancer risk. Recently, DNA methylation-based marker of ageing, known as 'epigenetic clock', has been linked with cancer risk factors. This study aimed to evaluate whether the epigenetic clock is associated with breast cancer risk susceptibility and to identify potential epigenetics-based biomarkers for risk stratification. METHODS: Here, we profiled DNA methylation changes in a nested case-control study embedded in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (n = 960) using the Illumina HumanMethylation 450K BeadChip arrays and used the Horvath age estimation method to calculate epigenetic age for these samples. Intrinsic epigenetic age acceleration (IEAA) was estimated as the residuals by regressing epigenetic age on chronological age. RESULTS: We observed an association between IEAA and breast cancer risk (OR, 1.04; 95% CI, 1.007-1.076, P = 0.016). One unit increase in IEAA was associated with a 4% increased odds of developing breast cancer (OR, 1.04; 95% CI, 1.007-1.076). Stratified analysis based on menopausal status revealed that IEAA was associated with development of postmenopausal breast cancers (OR, 1.07; 95% CI, 1.020-1.11, P = 0.003). In addition, methylome-wide analyses revealed that a higher mean DNA methylation at cytosine-phosphate-guanine (CpG) islands was associated with increased risk of breast cancer development (OR per 1 SD = 1.20; 95 %CI: 1.03-1.40, P = 0.02) whereas mean methylation levels at non-island CpGs were indistinguishable between cancer cases and controls. CONCLUSION: Epigenetic age acceleration and CpG island methylation have a weak, but statistically significant, association with breast cancer susceptibility.

Targeted deep sequencing of plasma circulating cell-free DNA reveals Vimentin and Fibulin 1 as potential epigenetic biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, but is still lacking sensitive and specific biomarkers for early diagnosis and prognosis. In this study, we applied targeted massively parallel semiconductor sequencing to assess methylation on a panel of genes (FBLN1, HINT2, LAMC1, LTBP1, LTBP2, PSMA2, PSMA7, PXDN, TGFB1, UBE2L3, VIM and YWHAZ) in plasma circulating cell-free DNA (cfDNA) and to evaluate the potential of these genes as HCC biomarkers in two different series, one from France (42 HCC cases and 42 controls) and one from Thailand (42 HCC cases, 26 chronic liver disease cases and 42 controls). We also analyzed a set of HCC and adjacent tissues and liver cell lines to further compare with 'The Cancer Genome Atlas' (TCGA) data. The methylation in cfDNA was detected for FBLN1, PSMA7, PXDN and VIM, with differences in methylation patterns between cases and controls for FBLN1 and VIM. The average methylation level across analyzed CpG-sites was associated with higher odds of HCC for VIM (1.48 [1.02, 2.16] for French cases and 2.18 [1.28, 3.72] for Thai cases), and lower odds of HCC for FBLN1 (0.89 [0.76, 1.03] for French cases and 0.75 [0.63, 0.88] for Thai cases). In conclusion, our study provides evidence that changes in VIM and FBLN1 methylation levels in cfDNA are associated with HCC and could represent useful plasma-based biomarkers. Also, the potential to investigate methylation patterns in cfDNA could bring new strategies for HCC detection and monitoring high-risk groups and response to treatment.

Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the most frequent human malignancies and a major cause of cancer-related death worldwide. It is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be one of the molecular mechanisms leading to hepatocarcinogenesis. Previous studies from our group revealed that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression. Imprinted genes are mono-allelically expressed in a parent-of-origin-dependent manner and have been suggested to be more susceptible to deregulation in cancer. To test this notion, we performed a targeted analysis of DNA methylation in known imprinted genes, using HCC samples and in vitro models of carcinogenic exposure. RESULTS: Analysis of HCC DNA methylation in two independent datasets showed that differentially methylated loci are significantly enriched in imprinted genes. Most of the promoters of imprinted genes were found hypomethylated in HCC tumors compared to surrounding tissues, contrasting with the frequent promoter hypermethylation observed in tumors. We next investigated the status of methylation of the imprinting control region (ICR) of different imprinted clusters and found that the 15q11-13 ICR was significantly hypomethylated in tumors relative to their surrounding tissues. In addition, expression of imprinted genes within this cluster was frequently deregulated in a gene-specific manner, suggesting distinct mechanisms of regulation in this region. Finally, primary human hepatocytes and hepatocyte-like HepaRG cells displayed higher methylation variability in certain imprinted loci after natural hepatitis B virus (HBV) infection and after lipid accumulation, respectively. CONCLUSION: The methylation status of a large panel of imprinted genes was found deregulated in HCC, suggesting a major role of this mechanism during hepatocarcinogenesis. In vitro models support the hypothesis of imprinted gene methylation as a potential marker of environmental exposures.