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.     

Antiproliferative effects of DNA methyltransferase 3B depletion are not associated with DNA demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.     

A role for LSH in facilitating DNA methylation by DNMT1 through enhancing UHRF1 chromatin association

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.     

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.     

DNA hypermethylation as a chemotherapy target

Epigenetics refers to partially reversible, somatically inheritable, but DNA sequence-independent traits that modulate gene expression, chromatin structure, and cell functions such as cell cycle and apoptosis. DNA methylation is an example of a crucial epigenetic event; aberrant DNA methylation patterns are frequently found in human malignancies. DNA hypermethylation and the associated expression silencing of tumor suppressor genes represent a hallmark of neoplastic cells. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. Several small synthetic and natural molecules, are able to reverse the DNA hypermethylation through inhibition of DNA methyltransferase (DNMT). DNMT is the enzyme catalyzing the transfer of methyl groups to cytosines in genomic DNA. These reagents are studied intensively in cell cultures, animal models, and clinical trials for potential anti-cancer activities. It was found that accompanying DNA demethylation is a dramatic reactivation of the silenced genes and inhibition of cancer cell proliferation, promotion of cell apoptosis, or sensitization of cells to other chemotherapeutic reagents. During the last few decades, an increasing number of DNMT inhibitors (DNMTi) targeting DNA methylation have been developed to increase efficacy with reduced toxicity. This review provides an update on new findings on cancer epigenetic mechanisms, the development of new DNMTi, and their application in the clinical setting. Current challenges, potential solutions, and future directions concerning the development of DNMTi are also discussed in this review.     

Deregulation between miR-29b/c and DNMT3A Is Associated with Epigenetic Silencing of the CDH1 Gene,Affecting Cell Migration and Invasion in Gastric Cancer

The de-regulation of the miR-29 family and DNA methyltransferase 3A (DNMT3A) is associated with gastric cancer (GC). While increasing evidence indicates miR-29b/c could regulate DNA methylation by targeting DNMT3A, it is currently unknown if epigenetic silencing of miR-29b/c via promoter hypermethylation in GC is caused by abnormal expression of DNMT3A. Thus, we aimed to evaluate whether cross-talk regulation exists between miR-29b/c and DNMT3A and whether it is associated with a malignant phenotype in GC. First, wound healing and Transwell assays revealed that miR-29b/c suppresses tumor metastasis in GC. A luciferase reporter assay demonstrated that DNMT3A is a direct target of miR-29b/c. We used bisulfite genomic sequencing to analyze the DNA methylation status of miR-29b/c. The percentage of methylated CpGs was significantly decreased in DNMT3A-depleted cells compared to the controls. Furthermore, the involvement of DNMT3A in promoting GC cell migration was associated with the promoter methylation-mediated repression of CDH1. In 50 paired clinical GC tissue specimens, decreased miR-29b/c was significantly correlated with the degree of differentiation and invasion of the cells and was negatively correlated with DNMT3A expression. Together, our preliminary results suggest that the following process may be involved in GC tumorigenesis. miR-29b/c suppresses the downstream gene DNMT3A, and in turn, miR-29b/c is suppressed by DNMT3A in a DNA methylation-dependent manner. The de-regulation of both of miR-29b/c and DNMT3A leads to the epigenetic silencing of CDH1 and contributes to the metastasis phenotype in GC. This finding reveals that DNA methylation-associated silencing of miR-29b/c is critical for GC development and thus may be a therapeutic target.     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.     

DNA甲基化与癌症

DNA甲基化是重要的表观遗传修饰,主要发生在DNA的CpG岛. DNA的甲基化通过DNA甲基转移酶（DNA methyltransferases, DNMTs）完成. DNA甲基化参与了细胞分化、基因组稳定性、X染色体失活、基因印记等多种细胞生物学过程.单基因水平及基因组范围内的DNA甲基化改变在肿瘤发生发展中亦发挥重要作用. 抑癌基因的异常甲基化引起的表达抑制,可导致肿瘤细胞的增殖失控和侵袭转移,并参与肿瘤组织的血管生成过程.在许多肿瘤的研究中都发现了基因组整体DNA低甲基化所导致的染色体不稳定性. 本文从DNA的异常高甲基化和低甲基化两方面论述了DNA甲基化在细胞恶变发生发展过程中的改变及其影响,并阐述了DNA甲基化改变在肿瘤诊断和治疗中的作用.     

ATRA inhibits the proliferation of DU145 prostate cancer cells through reducing the methylation level of HOXB13 gene

All-trans retinoic acid (ATRA) has been widely investigated for treatments of many cancers including prostate cancer. HOXB13, silenced in androgen receptor-negative (AR(-)) prostate cancer cells, plays a role in AR(-) prostate cancer cell growth arrest. In this study we intended to elucidate the mechanisms that are involved in the proliferation inhibition of AR(-) prostate cancer cells triggered by ATRA. We discovered that ATRA was able to induce the growth arrest and to increase HOXB13 expression in AR(-) prostate cancer cells. Both EZH2 and DNMT3b participated in the repression of HOXB13 expression through an epigenetic mechanism involving DNA and histone methylation modifications. Specifically, EZH2 recruited DNMT3b to HOXB13 promoter to form a repression complex. Moreover, ATRA could upregulate HOXB13 through decreasing EZH2 and DNMT3b expressions and reducing their interactions with the HOXB13 promoter. Concurrently, the methylation level of the HOXB13 promoter was reduced upon the treatment of ATRA. Results from this study implicated a novel effect of ATRA in inhibition of the growth of AR(-) resistant human prostate cancer cells through alteration of HOXB13 expression as a result of epigenetic modifications.