Axelle Loriot

Myc represses transcription through recruitment of DNA methyltransferase corepressor

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc‐repressed p21Cip1 gene, whereas the expression of Myc‐activated E‐boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA‐binding factor Miz‐1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz‐1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc‐mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.

Promoter-dependent mechanism leading to selective hypomethylation within the 5 ' region of gene MAGE-A1 in tumor cells

ABSTRACT Several male germ line-specific genes, including MAGE-A1, rely on DNA methylation for their repression in normal somatic tissues. These genes become activated in many types of tumors in the course of the genome-wide demethylation process which often accompanies tumorigenesis. We show that in tumor cells expressing MAGE-A1, the 5′ region is significantly less methylated than the other parts of the gene. The process leading to this site-specific hypomethylation does not appear to be permanent in these tumor cells, since in vitro-methylated MAGE-A1 sequences do not undergo demethylation after being stably transfected. However, in these cells there is a process that inhibits de novo methylation within the 5′ region of MAGE-A1, since unmethylated MAGE-A1 transgenes undergo remethylation at all CpGs except those located within the 5′ region. This local inhibition of methylation appears to depend on promoter activity. We conclude that the site-specific hypomethylation of MAGE-A1 in tumor cells relies on a transient process of demethylation followed by a persistent local inhibition of remethylation due to the presence of transcription factors.

Five new human cancer-germline genes identified among 12 genes expressed in spermatogonia

An important class of tumor‐specific antigens is encoded by male germline‐specific genes, such as MAGE genes, that are activated in many cancers of various histological types as a result of the demethylation of their promoter region. A number of these genes were shown to be expressed exclusively during the spermatogonia stage of spermatogenesis. A recent study reported the isolation of a new set of mouse genes that are expressed in spermatogonia but not in somatic tissues. Here, we tested the tumoral expression of the human orthologs of 12 of these genes. A remarkably high proportion, i.e., 5 of 12 genes, was found to be activated in a significant fraction of tumor samples of various histological types. Expression levels of the 5 genes, namely, NXF2, TAF2Q, FTHL17, TDRD1 and TEX15, were evaluated in normal and tumoral tissues. Except for TEX15, these genes showed sufficiently high expression levels in tumors and low background transcription in normal somatic tissues to qualify them as genes that potentially code for tumor‐specific antigens. Like previously described cancer‐germline genes, the 5 genes were induced in cells treated with a demethylating agent.

DNA hypomethylation in cancer: Epigenetic scars of a neoplastic journey.

Cytosine methylation is a heritable modification of DNA in mammalian cells, and has a determinant impact on long-term gene repression and genome stability. Genomic methylation patterns, which remain generally stable in the adult, become profoundly altered in most human tumors. While discrete DNA segments become hypermethylated in cancer cells, many more sequences become hypomethylated. This review discusses our current understanding of the mechanisms that lead to DNA hypomethylation in tumors. Evidence suggests that methylation losses are not random, but rather evolve into mosaic hypomethylation patterns. It is proposed that such hypomethylation patterns result from a historical event of transient DNA demethylation, and that transcriptional regulators contribute to determining which regions escape remethylation and remain therefore unmethylated. Finally, possible stages of tumor development during which the transient DNA demethylation process may take place will be discussed.

Transient down-regulation of DNMT1 methyltransferase leads to activation and stable hypomethylation of MAGE-A1 in melanoma cells.

MAGE-A1 belongs to a group of germ line-specific genes that rely primarily on DNA methylation for repression in somatic tissues. In many types of tumors, the promoter of these genes becomes demethylated and transcription becomes activated. We showed previously that, although MZ2-MEL melanoma cells contain an active unmethylated MAGE-A1 gene, they lack the ability to induce demethylation of newly integrated MAGE-A1 transgenes that were methylated in vitro before transfection. In the same cells, unmethylated MAGE-A1 transgenes were protected against remethylation, and this appeared to depend on the level of transcriptional activity. We therefore proposed that hypomethylation of MAGE-A1 in tumors relies on a past demethylation event and on the presence of appropriate transcription factors that maintain the promoter unmethylated. Here, we tested this hypothesis further by examining whether induction of a transient demethylation phase in MZ2-MEL would suffice to convert a previously methylated MAGE-A1 transgene into a permanently hypomethylated and active one. For induction of the demethylation phase, we used antisense oligonucleotides targeting the three known human DNA methyltransferases. We found that down-regulation of DNMT1, but not of DNMT3A and DNMT3B, induces activation of the MAGE-A1 transgene, suggesting that DNMT1 has a predominant role for methylation maintenance in MZ2-MEL cells. By using a selectable MAGE-A1 transgene construct, we were able to isolate a cell population in which DNMT1 depletion had resulted in transgene activation. The promoter region of the transgene was almost completely unmethylated in these cells, and this active and unmethylated state was maintained for over 60 days after restoration of normal DNMT1 expression.

Expression of BORIS in melanoma: Lack of association with MAGE-A1 activation

Several genes with specific expression in germ cells show aberrant activation in different types of tumors. These genes, termed cancer‐germline (CG) genes, encode tumor‐specific antigens, which represent potential targets for therapeutic vaccination against cancer. The germline‐specific gene BORIS (Brother Of the Regulator of Imprinted Sites), which encodes an 11‐zinc‐fingers transcriptional regulator, was recently qualified as a new CG gene, as it was found to be activated in a variety of tumor samples. Moreover, it was suggested that BORIS might be responsible for the activation of most other CG genes, including gene MAGE‐A1, in tumors. In the present study, we evaluated the frequency of BORIS activation in melanoma by quantitative RT‐PCR. BORIS activation was detected in 27% (n = 63) melanoma tissue samples. Surprisingly, many melanoma samples expressed MAGE‐A1 and other CG genes in the absence of BORIS activation, suggesting that BORIS is not an obligate factor for activation of these genes in melanoma. Consistently, forced expression of BORIS in melanoma cell lines did not induce expression of MAGE‐A1. Our results indicate that BORIS may serve as a useful target for immunotherapy of melanoma. However, it appears that BORIS is neither necessary nor sufficient for the activation of other CG genes.

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells.

Most human tumor cells acquire immortality by activating the expression of telomerase, a ribonucleoprotein that maintains stable telomere lengths at chromosome ends throughout cell divisions. Other tumors use an alternative mechanism of telomere lengthening (ALT), characterized by high frequencies of telomeric sister chromatid exchanges (T-SCEs). Mechanisms of ALT activation are still poorly understood, but recent studies suggest that DNA hypomethylation of chromosome ends might contribute to the process by facilitating T-SCEs. Here, we show that ALT/T-SCEhigh tumor cells display low DNA-methylation levels at the D4Z4 and DNF92 subtelomeric sequences. Surprisingly, however, the same sequences retained high methylation levels in ALT/T-SCEhigh SV40-immortalized fibroblasts. Moreover, T-SCE rates were efficiently reduced by ectopic expression of active telomerase in ALT tumor cells, even though subtelomeric sequences remained hypomethylated. We also show that hypomethylation of subtelomeric sequences in ALT tumor cells is correlated with genome-wide hypomethylation of Alu repeats and pericentromeric Sat2 DNA sequences. Overall, this study suggests that, although subtelomeric DNA hypomethylation is often coincident with the ALT process in human tumor cells, it is not required for T-SCE.

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.

Epigenetic Hierarchy within the MAGEA1 Cancer-Germline Gene: Promoter DNA Methylation Dictates Local Histone Modifications.

Gene MAGEA1 belongs to a group of human germline-specific genes that rely on DNA methylation for repression in somatic tissues. Many of these genes, termed cancer-germline (CG) genes, become demethylated and activated in a wide variety of tumors, where they encode tumor-specific antigens. The process leading to DNA demethylation of CG genes in tumors remains unclear. Previous data suggested that histone acetylation might be involved. Here, we investigated the relative contribution of DNA methylation and histone acetylation in the epigenetic regulation of gene MAGEA1. We show that MAGEA1 DNA hypomethylation in expressing melanoma cells is indeed correlated with local increases in histone H3 acetylation (H3ac). However, when MAGEA1-negative cells were exposed to a histone deacetylase inhibitor (TSA), we observed only short-term activation of the gene and detected no demethylation of its promoter. As a more sensitive assay, we used a cell clone harboring a methylated MAGEA1/hph construct, which confers resistance to hygromycin upon stable re-activation. TSA induced only transient de-repression of the transgene, and did not lead to the emergence of hygromycin-resistant cells. In striking contrast, transient depletion of DNA-methyltransferase-1 in the reporter cell clone gave rise to a hygromycin-resistant population, in which the re-activated MAGEA1/hph transgene displayed not only marked DNA hypomethylation, but also significant reversal of histone marks, including gains in H3ac and H3K4me2, and losses of H3K9me2. Collectively, our results indicate that DNA methylation has a dominant role in the epigenetic hierarchy governing MAGEA1 expression.

DNA Methylation-Associated Repression of Cancer-Germline Genes in Human Embryonic and Adult Stem Cells†‡

Cancer‐germline (CG) genes are a particular group of germline‐specific genes that rely primarily on DNA methylation for repression in somatic tissues. In a wide variety of tumors, the promoter of these genes is demethylated, and their transcription is activated. The mechanism underlying this tumor‐specific activation is still unclear. It was recently suggested that CG gene expression may be a hallmark of stem cells, and that expression of these genes in several tumors may reflect the expansion of constitutively expressing cancer stem cells. To clarify this issue, we carefully evaluated the expression of several CG genes in human stem cells of embryonic and adult origin. We found no or very weak expression of CG genes in these cells. Consistently, the promoter of CG genes was highly methylated in these cells. We conclude that CG genes do not qualify as “stemness” genes, and propose that their activation in cancers results from a tumor‐specific activation process. STEM CELLS 2009;27:822–824