Mutant p53 binds to estrogen receptor negative promoter via DNMT1 and HDAC1 in MDA-MB-468 breast cancer cells

DNA methylation and histone deacetylation are two epigenetic mechanisms involved in the lack of estrogen receptor (ER) expression. Our previous studies demonstrated that mutant p53 along with repression complex proteins including DNMT1, HDAC1 and MeCP2 is associated with ER-negative promoter in MDA-MB-468 cells. To elucidate the molecular mechanism of estrogen receptor 1 (ESR1) gene silencing in these cells, we down-regulated DNMT1 and HDAC1 expression using siRNAs and studied the ability of DNMT1, HDAC1, MeCP2 and p53 in binding to ESR1 promoter CpG island. Our results showed that DNMT1 or HDAC1 down-regulation disassembled the repression complex proteins and mutant p53 from ER-negative promoter. The partial demethylation of ESR1 promoter and ER re-expression in down-regulated cells supports these findings. In vivo binding studies demonstrated that mutation of p53 protein in this cell line did not affect its binding capacity to DNMT1, HDAC1 and MeCP2 proteins. Our observations suggest that not only histone deacetylase activity of HDAC1 contributes to inactivation of methylated ESR1 gene but also HDAC1 presence on ESR1 promoter is important for assembly of DNMT1 in repression complex. In addition, our data revealed that mutant p53 protein binds to the promoter of ESR1 through direct interaction with HDAC1 and indirect interaction with DNMT1, MeCP2 proteins in the ER-negative MDA-MB-468 cells.     

RANKL expression in myeloma cells is regulated by a network involving RANKL promoter methylation,DNMT1, microRNA and TNFα in the microenvironment

We studied the regulation of RANKL expression in myeloma by promoter DNA methylation. Methylation-specific polymerase chain reaction showed complete methylation of RANKL promoter in WL-2 myeloma cells but partial methylation in eight other lines. 5-AzadC treatment of WL-2 cells led to demethylation and re-expression of RANKL. Transwell and contact co-culture of WL-2 cells with normal bone marrow-derived mesenchymal stromal cells (BMSCs) resulted in comparable repression of DNA methyltransferase-1 (DNMT1) and re-expression of RANKL in WL-2 cells. Moreover, treatment of WL-2 cells with TNFα led to repression of DNMT1 and re-expression of RANKL in association with upregulation of miR-140-3p and miR-126, which are partially offset by addition of anti-TNFα antibody to transwell-coculture of WL2 with BMSC. Taken together, our results showed that TNFα in the marrow microenvironment led to RANKL demethylation and re-expression in myeloma cells through DNMT1 repression and upregulation of miR-126-3p and miR-140, both known to repress DNMT1 translation.     

The Mammalian de Novo DNA Methyltransferases DNMT3A and DNMT3B Are Also DNA 5-Hydroxymethylcytosine Dehydroxymethylases

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.     

An essential role for DNA methyltransferase DNMT3B in cancer cell survival

Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival.     

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, the molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in a gain of DNMT1 function. However, a substantial body of data suggested that RFTS is required for DNMT1 activity. Here, we demonstrate that deletion of RFTS alters DNMT1-dependent DNA methylation during malignant transformation. Compared to full-length DNMT1, ectopic expression of hyperactive DNMT1-ΔRFTS caused greater malignant transformation and enhanced promoter methylation with condensed chromatin structure that silenced DAPK and DUOX1 expression. Simultaneously, deletion of RFTS impaired DNMT1 chromatin association with pericentromeric Satellite 2 (SAT2) repeat sequences and produced DNA demethylation at SAT2 repeats and globally. To our knowledge, RFTS-deleted DNMT1 is the first single factor that can reprogram focal hypermethylation and global hypomethylation in parallel during malignant transformation. Our evidence suggests that the RFTS domain of DNMT1 is a target responsible for epigenetic changes in cancer.     

DNMT cooperativity--the developing links between methylation, chromatin structure and cancer

Controversy has reigned for some time over the biological connection between DNA methylation and cancer. For this reason, the methylation mechanism responsible for increased cancer risk has received greater attention in recent years. Tumor suppressor genes are often hypermethylated resulting in gene silencing. Although some have questioned this interpretation of the link between methylation and cancer, it appears that both hypermethylation and hypomethylation events can create epigenetic changes that can contribute to cancer development. Recent studies have shown that the methyltransferases DNMT1 and DNMT3b cooperatively maintain DNA methylation and gene silencing in human cancer cells. Disruption of the human DNMT3b only slightly reduces the overall global DNA methylation; however, demethylation was markedly potentiated when both DNMT1 and DNMT3b were simultaneously deleted. The results to these experiments provide compelling evidence towards a role for DNA methylation in cancer. This review discusses the current understanding of cancer-epigenetic information and highlights recent studies that connect the methylation machinery and chromatin remodelling with cancer susceptibility.     

Genetic variation in DNMT3B and increased global DNA methylation is associated with suicide attempts in psychiatric patients

Recently, a significant epigenetic component in the pathology of suicide has been realized. Here we investigate candidate functional SNPs in epigenetic‐regulatory genes, DNMT1 and DNMT3B, for association with suicide attempt (SA) among patients with co‐existing psychiatric illness. In addition, global DNA methylation levels [5‐methyl cytosine (5‐mC%)] between SA and psychiatric controls were quantified using the Methylflash Methylated DNA Quantification Kit. DNA was obtained from blood of 79 suicide attempters and 80 non‐attempters, assessed for DSM‐IV Axis I disorders. Functional SNPs were selected for each gene (DNMT1; n = 7, DNMT3B; n = 10), and genotyped. A SNP (rs2424932) residing in the 3′ UTR of the DNMT3B gene was associated with SA compared with a non‐attempter control group (P = 0.001; Chi‐squared test, Bonferroni adjusted P value = 0.02). Moreover, haplotype analysis identified a DNMT3B haplotype which differed between cases and controls, however this association did not hold after Bonferroni correction (P = 0.01, Bonferroni adjusted P value = 0.56). Global methylation analysis showed that psychiatric patients with a history of SA had significantly higher levels of global DNA methylation compared with controls (P = 0.018, Student's t‐test). In conclusion, this is the first report investigating polymorphisms in DNMT genes and global DNA methylation quantification in SA risk. Preliminary findings suggest that allelic variability in DNMT3B may be relevant to the underlying diathesis for suicidal acts and our findings support the hypothesis that aberrant DNA methylation profiles may contribute to the biology of suicidal acts. Thus, analysis of global DNA hypermethylation in blood may represent a biomarker for increased SA risk in psychiatric patients.