RESUMO
Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Mitocondrial/genética , Regulação da Expressão Gênica/genética , Animais , Ilhas de CpG/genética , Humanos , Mitocôndrias/genética , DNA Metiltransferase 3BRESUMO
Hypomethylating agents reactivate tumor suppressor genes that are epigenetically silenced in cancer. Inevitably these genes are resilenced, leading to drug resistance. Using the MLH1 tumor suppressor gene as a model, we showed that decitabine-induced re-expression was dependent upon demethylation and eviction of promoter nucleosomes. Following decitabine withdrawal, MLH1 was rapidly resilenced despite persistent promoter demethylation. Single molecule analysis at multiple time points showed that gene resilencing was initiated by nucleosome reassembly on demethylated DNA and only then was followed by remethylation and stable silencing. Taken together, these data establish the importance of nucleosome positioning in mediating resilencing of drug-induced gene reactivation and suggest a role for therapeutic targeting of nucleosome assembly as a mechanism to overcome drug resistance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Nucleares/genética , Nucleossomos/genética , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Proteína 1 Homóloga a MutL , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
BACKGROUND: DNA methylation may have a regulatory role in monogenic sensorineural hearing loss and complex, polygenic phenotypic forms of hearing loss, including age-related hearing impairment or Meniere disease. The purpose of this systematic review is to critically assess the evidence supporting a functional role of DNA methylation in phenotypes associated with hearing loss. RESULTS: The search strategy yielded a total of 661 articles. After quality assessment, 25 records were selected (12 human DNA methylation studies, 5 experimental animal studies and 8 studies reporting mutations in the DNMT1 gene). Although some methylation studies reported significant differences in CpG methylation in diverse gene promoters associated with complex hearing loss phenotypes (ARHI, otosclerosis, MD), only one study included a replication cohort that supported a regulatory role for CpG methylation in the genes TCF25 and POLE in ARHI. Conversely, several studies have independently confirmed pathogenic mutations within exon 21 of the DNMT1 gene, which encodes the DNA (cytosine-5)-methyltransferase 1 enzyme. This methylation enzyme is strongly associated with a rare disease defined by autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Of note, rare variants in DNMT1 and DNMT3A genes have also been reported in noise-induced hearing loss. CONCLUSIONS: Evidence supporting a functional role for DNA methylation in hearing loss is limited to few genes in complex disorders such as ARHI. Mutations in the DNMT1 gene are associated with ADCA-DN, suggesting the CpG methylation in hearing loss genes deserves further attention in hearing research.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Humanos , Metilação de DNA/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Animais , Ilhas de CpG/genética , Epigênese Genética/genética , Perda Auditiva/genética , Mutação , Fenótipo , Regiões Promotoras Genéticas , Perda Auditiva Neurossensorial/genética , Narcolepsia/genéticaRESUMO
The systematic mutation of histone 3 (H3) genes in model organisms has proven to be a valuable tool to distinguish the functional role of histone residues. No system exists in mammalian cells to directly manipulate canonical histone H3 due to a large number of clustered and multi-loci histone genes. Over the years, oncogenic histone mutations in a subset of H3 have been identified in humans, and have advanced our understanding of the function of histone residues in health and disease. The oncogenic mutations are often found in one allele of the histone variant H3.3 genes, but they prompt severe changes in the epigenetic landscape of cells, and contribute to cancer development. Therefore, mutation approaches using H3.3 genes could be relevant to the determination of the functional role of histone residues in mammalian development without the replacement of canonical H3 genes. In this review, we describe the key findings from the H3 mutation studies in model organisms wherein the genetic replacement of canonical H3 is possible. We then turn our attention to H3.3 mutations in human cancers, and discuss H3.3 substitutions in the N-terminus, which were generated in order to explore the specific residue or associated post-translational modification.
Assuntos
Cromatina/genética , Histonas/genética , Mutação , Animais , Cromatina/química , Cromatina/metabolismo , Epigênese Genética , Proteínas Fúngicas/metabolismo , Engenharia Genética , Variação Genética , Humanos , Mamíferos , Camundongos , Mutagênese , Neoplasias/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-TraducionalRESUMO
In mammalian genomes, the methylation of cytosine residues within CpG dinucleotides is crucial to normal development and cell differentiation. However, methylation of cytosines in the contexts of CpA, CpT, and CpC (non-CpG methylation) has been reported for decades, yet remains poorly understood. In recent years, whole genome bisulphite sequencing (WGBS) has confirmed significant levels of non-CpG methylation in specific tissues and cell types. Non-CpG methylation has several properties that distinguish it from CpG methylation. Here we review the literature describing non-CpG methylation in mammalian cells, describe the important characteristics that distinguish it from CpG methylation, and discuss its functional importance.