DNA Methylation In Gene Silencing Research Articles

Abstract LB-346: A novel lung tumor suppressor implicated in somatic and familial cancers

Abstract Background: Lung cancer (LC) is the most common cause of cancer death worldwide. Previous familial linkage studies have identified a tumor suppressor locus on 6q23-25. However, no single gene has yet been implicated within this 30 Mb region. Discovering the genetic and epigenetic events that affect LC risk and development will lead to better methods for risk assessment, early detection and treatment. Methods: Genome-wide genes disrupted by two-hit inactivation were identified by combining gene dosage, DNA methylation, and gene expression assays for a group of lung adenocarcinomas (AC) and adjacent non-malignant tissues. Gene expression, DNA hypermethylation and/or copy number aberrations were validated in data from AC, squamous cell carcinoma (SqCC), and pre-malignant lesions by querying other cohorts using gene-specific and whole-genome approaches. The role of DNA methylation in gene silencing was assessed using inhibition of DNMT by 5′-azacytidine. The association of allelic variants with LC risk was investigated in 193 familial LC cases and 213 controls collected by the Genetic Epidemiology of Lung Cancer Consortium (GELCC) using a Cochrane-Armitage trend test. The association of gene expression with prognosis was performed on public data using a Mantel-Cox log test. Stable mRNA knock-downs were generated using lentiviral delivery of a gene-specific shRNA, and apoptotic cells were counted using Annexin5/propidium iodide staining. Results: Integration of AC gene dosage, DNA methylation and mRNA expression showed EYA4 to be frequently affected by two-hits and significantly down-regulated. Quantitative PCR techniques confirmed that EYA4 was hypermethylated (46%) and down-regulated (72%), validating our microarray results. A direct link between EYA4 methylation and expression was verified by restoration of expression after 5′-azacytidine treatment in methylated cell lines. Congruent with EYA family member function, in vitro assays revealed that EYA4 knock-down cells displayed a decrease in the number of apoptotic cells - a hallmark of cancer. Further investigations led to the discovery of frequent EYA4 disruption in SqCC and pre-neoplastic tissue. The GELCC dataset was examined to assess EYA4 allelotype association with familial risk. Doing so revealed that numerous EYA4 variants are associated with increased risk. Finally, the association of EYA4 expression with survival was investigated along with other somatically altered genes at 6q23-25. Of these genes, low EYA4 expression was found to be the most significantly associated with poor prognosis. Conclusions: EYA4 is a frequently disrupted gene that maps to a locus previously associated with cancer risk. It is implicated in somatic as well as familial cancers, and is likely a tumor suppressor gene with apoptotic functions. The direct association of EYA4 with risk and survival underscores its relevance on a clinical level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-346.

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.

PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing.

Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human beta-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA-mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.

Epigenetic interplay between histone modifications and DNA methylation in gene silencing

Knowledge on heritable changes in gene expression that result from epigenetic events is of increasing relevance in the development of strategies for prevention, early diagnosis and treatment of cancer. Histone acetylation and DNA methylation are epigenetic modifications whose patterns can be regarded as heritable marks that ensure accurate transmission of the chromatin states and gene expression profiles over many cell generations. Importantly, patterns and levels of DNA methylation and histone acetylation are profoundly altered in human cancers. Accumulating evidence suggests that an epigenetic cross-talk, i.e. interplay between DNA methylation and histone acetylation, may be involved in the process of gene transcription and aberrant gene silencing in tumours. Although the molecular mechanism of gene activation is relatively well understood, the hierarchical order of events and dependencies leading to gene silencing in the course of cancer development remain largely unknown. While some studies suggest that DNA methylation patterns guide histone modifications (including histone acetylation and methylation) during gene silencing, other studies argue that DNA methylation takes its cues primarily from histone modification states. In this review, we summarize current knowledge on the interplay between DNA methylation and histone modifications during gene silencing and its importance in the integration of environmental and intrinsic stimuli in the control of gene expression. We also discuss the importance of an epigenetic cross-talk in the protection against genetic changes in response to environmental genotoxins as well as the implication for cancer therapy and prevention.

Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the ε-, γ-, and β-globin gene promoters in Papio anubis

The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the ε-, γ-, and β-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the ε-, γ-, and β-globin promoters occur during development. Changes in DNA methylation of the ε- and γ-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced ε-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the β-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development.