Transcriptional overlap links DNA hypomethylation with DNA hypermethylation at adjacent promoters in cancer.

Tumor development involves alterations in DNA methylation patterns, which include both gains (hypermethylation) and losses (hypomethylation) in different genomic regions. The mechanisms underlying these two opposite, yet co-existing, alterations in tumors remain unclear. While studying the human MAGEA6/GABRA3 gene locus, we observed that DNA hypomethylation in tumor cells can lead to the activation of a long transcript (CT-GABRA3) that overlaps downstream promoters (GABRQ and GABRA3) and triggers their hypermethylation. Overlapped promoters displayed increases in H3K36me3, a histone mark deposited during transcriptional elongation and known to stimulate de novo DNA methylation. Consistent with such a processive mechanism, increases in H3K36me3 and DNA methylation were observed over the entire region covered by the CT-GABRA3 overlapping transcript. Importantly, experimental induction of CT-GABRA3 by depletion of DNMT1 DNA methyltransferase, resulted in a similar pattern of regional DNA hypermethylation. Bioinformatics analyses in lung cancer datasets identified other genomic loci displaying this process of coupled DNA hypo/hypermethylation, and some of these included tumor suppressor genes, e.g. RERG and PTPRO. Together, our work reveals that focal DNA hypomethylation in tumors can indirectly contribute to hypermethylation of nearby promoters through activation of overlapping transcription, and establishes therefore an unsuspected connection between these two opposite epigenetic alterations.

Epigenetic Coactivation of MAGEA6 and CT-GABRA3 Defines Orientation of a Segmental Duplication in the Human X Chromosome.

The human genome harbors many duplicated segments, which sometimes show very high sequence identity. This may complicate assignment during genome assembly. One such example is in Xq28, where the arrangement of 2 recently duplicated segments varies between genome assembly versions. The duplicated segments comprise highly similar genes, including MAGEA3 and MAGEA6, which display specific expression in testicular germline cells, and also become aberrantly activated in a variety of tumors. Recently, a new gene was identified, CT-GABRA3, the transcription of which initiates inside the segmental duplication but extends far outside. According to the latest genome annotation, CT- GABRA3 starts near MAGEA3, with which it shares a bidirectional promoter. In an earlier annotation, however, the duplicated segment was positioned in the opposite orientation, and CT-GABRA3 was instead coupled with MAGEA6. To resolve this discrepancy, and based on the contention that genes connected by a bidirectional promoter are almost always co-expressed, we decided to compare the expression profiles of CT-GABRA3, MAGEA3, and MAGEA6. We found that in tumor tissues and cell lines of different origins, the expression of CT-GABRA3 was better correlated with that of MAGEA6. Moreover, in a cellular model of experimental induction with a DNA demethylation agent, activation CT-GABRA3 was associated with that of MAGEA6, but not with that of MAGEA3. Together these results support a connection between CT-GABRA3 and MAGEA6 and illustrate how promoter-sharing genes can be exploited to resolve genome assembly uncertainties.

Oncogenic roles of DNA hypomethylation through the activation of cancer-germline genes.

Global loss of DNA methylation is frequently observed in the genome of human tumors. Although this epigenetic alteration is clearly associated with cancer progression, the way it exerts its pro-tumoral effect remains incompletely understood. A remarkable consequence of DNA hypomethylation in tumors is the aberrant activation of "cancer-germline" genes (also known as "cancer-testis" genes), which comprise a diverse group of germline-specific genes that use DNA methylation as a primary mechanism for repression in normal somatic tissues. Here we review the evidence that such cancer-germline genes contribute to key processes of tumor development. Notably, several cancer-germline genes were found to stimulate oncogenic pathways involved in cell proliferation (SSX, DDX43, MAEL, PIWIL1), angiogenesis (DDX53), immortality (BORIS/CTCFL), and metastasis (CT-GABRA3). Others appear to inhibit tumor suppressor pathways, including those controlling growth inhibition signals (MAGEA11, MAGEB2), apoptosis (MAGEA2, MAGEC2), and genome integrity (HORMAD1, NXF2). Cancer-germline genes were also implicated in the regulation of tumor metabolism (MAGEA3/MAGEA6). Together, our survey substantiates the concept that DNA hypomethylation promotes tumorigenesis via transcriptional activation of oncogenes. Importantly, considering their highly restricted pattern of expression, cancer-germline genes may represent valuable targets for the development of anti-cancer therapies with limited side effects.

A gene expression signature identifying transient DNMT1 depletion as a causal factor of cancer-germline gene activation in melanoma.

BACKGROUND: Many human tumors show aberrant activation of a group of germline-specific genes, termed cancer-germline (CG) genes, several of which appear to exert oncogenic functions. Although activation of CG genes in tumors has been linked to promoter DNA demethylation, the mechanisms underlying this epigenetic alteration remain unclear. Two main processes have been proposed: awaking of a gametogenic program directing demethylation of target DNA sequences via specific regulators, or general deficiency of DNA methylation activities resulting from mis-targeting or down-regulation of the DNMT1 methyltransferase. RESULTS: By the analysis of transcriptomic data, we searched to identify gene expression changes associated with CG gene activation in melanoma cells. We found no evidence linking CG gene activation with differential expression of gametogenic regulators. Instead, CG gene activation correlated with decreased expression of a set of mitosis/division-related genes (ICCG genes). Interestingly, a similar gene expression signature was previously associated with depletion of DNMT1. Consistently, analysis of a large set of melanoma tissues revealed that DNMT1 expression levels were often lower in samples showing activation of multiple CG genes. Moreover, by using immortalized melanocytes and fibroblasts carrying an inducible anti-DNMT1 small hairpin RNA (shRNA), we demonstrate that transient depletion of DNMT1 can lead to long-term activation of CG genes and repression of ICCG genes at the same time. For one of the ICCG genes (CDCA7L), we found that its down-regulation in melanoma cells was associated with deposition of repressive chromatin marks, including H3K27me3. CONCLUSIONS: Together, our observations point towards transient DNMT1 depletion as a causal factor of CG gene activation in vivo in melanoma.

A novel cancer-germline transcript carrying pro-metastatic miR-105 and TET-targeting miR-767 induced by DNA hypomethylation in tumors.

Genome hypomethylation is a common epigenetic alteration in human tumors, where it often leads to aberrant activation of a group of germline-specific genes, commonly referred to as "cancer-germline" genes. The cellular functions and tumor promoting potential of these genes remain, however, largely uncertain. Here, we report identification of a novel cancer-germline transcript (CT-GABRA3) displaying DNA hypomethylation-dependent activation in various tumors, including melanoma and lung carcinoma. Importantly, CT-GABRA3 harbors a microRNA (miR-105), which has recently been identified as a promoter of cancer metastasis by its ability to weaken vascular endothelial barriers following exosomal secretion. CT-GABRA3 also carries a microRNA (miR-767) with predicted target sites in TET1 and TET3, two members of the ten-eleven-translocation family of tumor suppressor genes, which are involved in the conversion of 5-methylcytosines to 5-hydroxymethylcytosines (5hmC) in DNA. Decreased TET activity is a hallmark of cancer; here, we provide evidence that aberrant activation of miR-767 contributes to this phenomenon. We demonstrate that miR-767 represses TET1/3 mRNA and protein expression and regulates genomic 5hmC levels. Additionally, we show that high CT-GABRA3 transcription correlates with reduced TET1 mRNA levels in vivo in lung tumors. Together, our study identified a cancer-germline gene that produces microRNAs with oncogenic potential. Moreover, our data indicate that DNA hypomethylation in tumors can contribute to reduced 5hmC levels via activation of a TET-targeting microRNA.