The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

Complementarity of assembly-first and mapping-first approaches for alternative splicing annotation and differential analysis from RNAseq data.

Genome-wide analyses estimate that more than 90% of multi exonic human genes produce at least two transcripts through alternative splicing (AS). Various bioinformatics methods are available to analyze AS from RNAseq data. Most methods start by mapping the reads to an annotated reference genome, but some start by a de novo assembly of the reads. In this paper, we present a systematic comparison of a mapping-first approach (FARLINE) and an assembly-first approach (KISSPLICE). We applied these methods to two independent RNAseq datasets and found that the predictions of the two pipelines overlapped (70% of exon skipping events were common), but with noticeable differences. The assembly-first approach allowed to find more novel variants, including novel unannotated exons and splice sites. It also predicted AS in recently duplicated genes. The mapping-first approach allowed to find more lowly expressed splicing variants, and splice variants overlapping repeats. This work demonstrates that annotating AS with a single approach leads to missing out a large number of candidates, many of which are differentially regulated across conditions and can be validated experimentally. We therefore advocate for the combined use of both mapping-first and assembly-first approaches for the annotation and differential analysis of AS from RNAseq datasets.

TET-Catalyzed 5-Hydroxymethylation Precedes HNF4A Promoter Choice during Differentiation of Bipotent Liver Progenitors.

Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.

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.

RNA helicases DDX5 and DDX17 dynamically orchestrate transcription, miRNA, and splicing programs in cell differentiation.

The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.

Towards incorporating epigenetic mechanisms into carcinogen identification and evaluation.

Remarkable progress in the field of epigenetics has turned academic, medical and public attention to the potential applications of these new advances in medicine and various fields of biomedical research. The result is a broader appreciation of epigenetic phenomena in the a etiology of common human diseases, most notably cancer. These advances also represent an exciting opportunity to incorporate epigenetics and epigenomics into carcinogen identification and safety assessment. Current epigenetic studies, including major international sequencing projects, are expected to generate information for establishing the 'normal' epigenome of tissues and cell types as well as the physiological variability of the epigenome against which carcinogen exposure can be assessed. Recently, epigenetic events have emerged as key mechanisms in cancer development, and while our search of the Monograph Volume 100 revealed that epigenetics have played a modest role in evaluating human carcinogens by the International Agency for Research on Cancer (IARC) Monographs so far, epigenetic data might play a pivotal role in the future. Here, we review (i) the current status of incorporation of epigenetics in carcinogen evaluation in the IARC Monographs Programme, (ii) potential modes of action for epigenetic carcinogens, (iii) current in vivo and in vitro technologies to detect epigenetic carcinogens, (iv) genomic regions and epigenetic modifications and their biological consequences and (v) critical technological and biological issues in assessment of epigenetic carcinogens. We also discuss the issues related to opportunities and challenges in the application of epigenetic testing in carcinogen identification and evaluation. Although the application of epigenetic assays in carcinogen evaluation is still in its infancy, important data are being generated and valuable scientific resources are being established that should catalyse future applications of epigenetic testing.

Dependence receptor TrkC is a putative colon cancer tumor suppressor.

The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a proto-oncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-of-proapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.

Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells.

Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most frequent human cancers and a major cause of cancer-related death worldwide. The major risk factors for developing HCC are infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcoholism, and aflatoxins; however, critical gene targets remain largely unknown. Herein, we sought to establish DNA methylation patterns in HCC and corresponding cirrhotic tissues and to identify DNA methylation changes associated with major risk factors. METHODS: We have established assays for quantitative analysis of DNA methylation levels in a panel of seven cancer-associated genes and repetitive elements, and combined these assays with a series of HCC tumors, associated with major risk factors, collected from two different geographical areas. RESULTS: We found a high frequency of aberrant hypermethylation of specific genes (RASSF1A, GSTP1, CHRNA3, and DOK1) in HCC tumors as compared to control cirrhotic or normal liver tissues, suggesting that aberrant hypermethylation exhibits non-random and tumor-specific patterns in HCC. Importantly, our analysis revealed an association between alcohol intake and the hypomethylation of MGMT and between hypermethylation of GSTP1 and HBV infection, indicating that hypermethylation of the genes analyzed in HCC tumors exhibits remarkably distinct patterns depending on associated risk factors. CONCLUSIONS: This study identifies aberrant DNA methylation of specific cellular genes in HCC and the major risk factors associated with these changes, providing information that could be exploited for biomarker discovery in clinics and molecular epidemiology.

Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. METHODS AND FINDINGS: We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. CONCLUSION: Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

Epigenetics and cancer, 2nd IARC meeting, Lyon, France, 6 and 7 December 2007.

It is becoming widely accepted that epigenetic alterations are universally present in human malignancies and that cancer is as much a disease of abnormal epigenetics as it is a genetic disease. The potential utility of epigenetics and epigenomics in cancer research and cancer control is highlighted by the fact that many funding agencies put cancer epigenetics on the priority list. The goal of this meeting, held at the offices of the International Agency for Research on Cancer in Lyon, France, 6 and 7 December 2007, was to discuss recent conceptual and technological advances in cancer epigenetics and epigenomics, the future research needs in the field, and their implications for early detection, risk assessment and prevention of cancer. While epigenetics has been historically linked to phenomena that do not follow normal genetic principles of heritability, recent mechanistic advances have begun to change our understanding of complex diseases including cancer, traditionally viewed as genetic in origin. It is now known that epigenetic mechanisms play critical roles in regulation of many cellular functions and their deregulation may disrupt the control of fundamental processes leading to tumour formation. A flurry of screening and functional studies revealed that most key processes found in cancer cells, such as silencing of tumour suppressor genes, activation of oncogenes, aberrant cell cycle processes, defects in DNA repair, and deregulation of cell death, can be triggered by epigenetic deregulation. Two important features that distinguish epigenetic changes from genetic alterations are the gradual appearance and reversibility of epigenetic events. These features make epigenetic alterations an attractive target for therapeutic intervention and the development of preventive strategies. For example, aberrant patterns of DNA methylation and histone acetylation and methylation can be targeted by drugs aiming to re-activate epigenetically silenced genes. Until now, most studies on epigenetic changes in cancer were generally focused on specific genes. However, this meeting also stressed the need to take advantage of recent progress in epigenomics and emergence of powerful technologies for detection of epigenetic changes in high throughput and genome-wide settings. This may further advance our capacity to evaluate the contribution of epigenetic changes induced by environmental epimutagens to human cancer. This information may prove critical for the design of efficient strategies for early diagnosis, therapy, and prevention of cancer.