线粒体DNA与DNA甲基化

线粒体是除细胞核之外唯一携带遗传物质的细胞器,其线粒体DNA（mitochondrial DNA,mtDNA）控制着线粒体一些最基本的性质,对细胞功能有着重要影响.DNA甲基化是调节基因表达的重要方式之一.研究表明mtDNA存在CpG位点的低甲基化,并且mtDNA基因的表达受核DNA（nuclear DNA,nDNA）及线粒体自身DNA甲基化的调控,mtDNA和nDNA协同作用参与机体代谢调节和疾病发生发展过程.就近年来mtDNA与DNA甲基化的关系作一综述.     

The first mitochondrial 5-methylcytosine map in a non-model teleost (Oreochromis niloticus) reveals extensive strand-specific and non-CpG methylation

DNA methylation is one of the main epigenetic mechanisms that regulate gene expression in a manner that depends on the genomic context and varies considerably across taxa. This DNA modification was first found in nuclear genomes of eukaryote several decades ago and it has also been described in mitochondrial DNA. It has recently been shown that mitochondrial DNA is extensively methylated in mammals and other vertebrates. Our current knowledge of mitochondrial DNA methylation in fish is very limited, especially in non-model teleosts. In this study, using whole-genome bisulfite sequencing, we determined methylation patterns within non-CpG (CH) and CpG (CG) contexts in the mitochondrial genome of Nile tilapia, a non-model teleost of high economic importance. Our results demonstrate the presence of mitochondrial DNA methylation in this species predominantly within a non-CpG context, similarly to mammals. We found a strand-specific distribution of methylation, in which highly methylated cytosines were located on the minus strand. The D-loop region had the highest mean methylation level among all mitochondrial loci. Our data provide new insights into the potential role of epigenetic mechanisms in regulating metabolic flexibility of mitochondria in fish, with implications in various biological processes, such as growth and development.     

同卵双胞胎在复杂性状DNA甲基化调控机制研究中的应用

同卵双胞胎来源于同一个受精卵,DNA序列基本一致,但在某些重要表型上如复杂疾病,并不完全一样。利用表型不一致的同卵双胞胎进行研究,能在遗传背景、母体效应、年龄性别效应等一致的基础上,深入研究分析复杂性状的表观调控机制。而DNA甲基化是最为稳定的一类表观遗传修饰。在人类中,利用同卵双胞胎对印记异常疾病、精神类疾病、自身免疫病及癌症等疾病的DNA甲基化调控研究已经揭示了多个致病基因,为研究疾病的表观调控以及表观遗传学药物的应用打下了基础。本文着重对同卵双胞胎DNA甲基化状态、DNA甲基化遗传力计算以及复杂性状DNA甲基化调控的研究应用及其进展展开综述,以期为复杂性状表观调控机制研究提供借鉴和参考。     

Methylation pattern of mouse mitochondrial DNA.

The pattern of methylation of mouse mitochondrial DNA (mtDNA) was studied using several techniques. By employing a sensitive analytical procedure it was possible to show that this DNA contains the modified base 5-methylcytosine (m5Cyt). This residue occurred exclusively at the dinucleotide sequence CpG at a frequency of 3 to 5%. The pattern of methylation was further investigated by determining the state of methylation of several MspI (HpaII) sites. Different sites were found to be methylated to a different extent, implying that methylation of mtDNA is nonrandom. Based on the known base composition and nucleotide sequence of mouse mtDNA, the dinucleotide sequence CpG was found to be underrepresented in this DNA. The features of mtDNA methylation (CpG methylation, partial methylation of specific sites and CpG underrepresentation) are also characteristic of vertebrate nuclear DNA. This resemblance may reflect functional relationship between the mitochondrial and nuclear genomes.     

Mitochondrial regulation of epigenetics and its role in human diseases

Most pathogenic mitochondrial DNA (mtDNA) mutations induce defects in mitochondrial oxidative phosphorylation (OXPHOS). However, phenotypic effects of these mutations show a large degree of variation depending on the tissue affected. These differences are difficult to reconcile with OXPHOS as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction. In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead to epigenetic changes causing genomic instability in the nuclear genome. We propose that “mitocheckpoint” mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role.     

The Most Redundant Sequences in Human CpG Island Library Are Derived from Mitochondrial Genome

An altered pattern of epigenetic modifications, such as DNA methylation and histone modification, is critical to many common human diseases, including cancer. Recently, mitochondrial DNA (mtDNA) was reported to be associated with tumorigenesis through epigenetic regulation of methylation patterns. One of the promising approaches to study DNA methylation and CpG islands (CGIs) is sequencing and analysis of clones derived from the physical library generated by methyl-CpG-binding domain proteins and restriction enzyme MseI. In this study, we observed that the most redundant sequences of 349 clones in a human CGI library were all generated from the human mitochondrial genome. Further analysis indicated that there was a 5,845-bp DNA transfer from mtDNA to chromosome 1, and all the clones should be the products of a 510-hp MseI fragment, which contained a putative CGI of 270 bp. The 510-bp fragment was annotated as part of cytochrome c oxidase subunit Ⅱ (COXⅡ), and phylogenetic analysis of homologous sequences containing COXII showed three DNA transfer events from mtDNA to nuclear genome, one of which underwent secondary transfer events between different chromosomes. These results may further our understanding of how the mtDNA regulates DNA methylation in the nucleus.     

Modulation of DNA methylation levels sensitizes doxorubicin-resistant breast adenocarcinoma cells to radiation-induced apoptosis

Chemoresistant tumors often fail to respond to other cytotoxic treatments such as radiation therapy. The mechanisms of chemo- and radiotherapy cross resistance are not fully understood and are believed to be epigenetic in nature. We hypothesize that MCF-7 cells and their doxorubicin-resistant variant MCF-7/DOX cells may exhibit different responses to ionizing radiation due to their dissimilar epigenetic status.Similar to previous studies, we found that MCF-7/DOX cells harbor much lower levels of global DNA methylation than MCF-7 cells. Furthermore, we found that MCF-7/DOX cells had lower background apoptosis levels and were less responsive to radiation than MCF-7 cells. Decreased radiation responsiveness correlated to significant global DNA hypomethylation in MCF-7/DOX cells.Here, for the first time, we show that the radiation resistance of MCF-7/DOX cells can be reversed by an epigenetic treatment - the application of methyl-donor SAM. SAM-mediated reversal of DNA methylation led to elevated radiation sensitivity in MCF-7/DOX cells. Contrarily, application of SAM on the radiation sensitive and higher methylated MCF-7 cells resulted in a decrease in their radiation responsiveness. This data suggests that a fine balance of DNA methylation is needed to insure proper radiation and drug responsiveness.     

Mitochondrial regulation of epigenetics and its role in human diseases

Most pathogenic mitochondrial DNA (mtDNA) mutations induce defects in mitochondrial oxidative phosphorylation (OXPHOS). However, phenotypic effects of these mutations show a large degree of variation depending on the tissue affected. These differences are difficult to reconcile with OXPHOS as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction. In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead to epigenetic changes causing genomic instability in the nuclear genome. We propose that “mitocheckpoint” mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role.     

Recent patents of DNA methylation biomarkers in gastrointestinal oncology

Gastrointestinal malignancies are among the most common malignancies worldwide. Advances in technology and treatment have improved diagnosis and monitoring of these tumors. As a consequence, identification of new biomarkers that can be applied at different levels of disease is urgently needed. DNA methylation is a process in which cytosines acquire a methyl group in 5' position only if they are followed by a guanine. An emerging catalog of specific genes inactivated by DNA methylation in gastrointestinal tumors has been established. In this review we will give a brief overview of the main sources of DNA used to investigate methylation biomarkers and several related patents. One of these is related to multiple genes that predict the risk of development of esophageal adenocarcinoma. Another evaluated methylation status of 24 genes to find one frequently methylated in primary tumors as well as plasma samples from gastric cancer patients. Others patented the epigenetic silencing of miR-342 as a promissory biomarker for colorectal carcinoma. Thus the new field of DNA methylation biomarkers holds the promise of better methods for screening, early detection, disease progression and outcome predictor of therapy response in gastrointestinal oncology.     

Antagonism between DNA and H3K27 methylation at the imprinted Rasgrf1 locus

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.     

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.     

Loss of CpG dinucleotides from DNA. VI. Methylation of mitochondrial and chloroplast genes

From nucleotide sequences of mitochondrial and chloroplast genes the probable frequency of the CpG----TpG + CpA substitutions was determined. These substitutions may indicate the level of prior DNA methylation. It was found that the level of this methylation is significantly lower in mitochondrial DNA (mtDNA) and chloroplast DNA (chDNA) than in nuclear DNA (nDNA) of the same species. The species (taxon) specificity of mtDNA and chDNA methylation was revealed. A correlation was found between the level of CpG methylation in nDNA, and mtDNA and chDNA in different organisms. It is shown that cytosine residues in CpG were not subjected to significant methylation in the fungi and invertebrate mtDNA and also in the algae chDNA. In contrast, the vertebrate mtDNA bears the impress of CpG-supression, which is confirmed by direct data on methylation of these DNA. Here the first data on the possible enzymatic methylation of the plant mtDNA and chDNA were obtained. It was shown that the degree of CpG-suppression in the 5S rRNA nuclear genes of lower and higher plants is significantly higher in the chloroplast genes of 4,5S and 5S rRNA. From data on pea chDNA hydrolysis with MspI and HpaII it was established that in CCGG sequences this DNA is not methylated. The role of DNA methylation in increasing the mutation rate and in accelerating the evolutionary rates of vertebrate mtDNA is discussed.     

DNA methylation and gene expression changes in monozygotic twins discordant for psoriasis: identification of epigenetically dysregulated genes

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%-80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4(+) and CD8(+) cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4(+) cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease.     

Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues

Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth. We show that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues, but was always below the error threshold modelled using negative controls. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.     

Epigenetic diagnostics of cancer — the application of DNA methylation markers

In recent years it has become apparent that epigenetic events are potentially equally responsible for cancer initiation and progression as genetic abnormalities. DNA methylation is the main epigenetic modification in humans. Two DNA methylation lesions coexist in human neoplasms: hypermethylation of promoter regions of specific genes within a context of genomic hypomethylation. Aberrant methylation is found at early stages of carcinogenesis and distinct types of cancer exhibit specific patterns of methylation changes. Tumor specific DNA is readily obtainable from different clinical samples and methylation status analysis often permits sensitive disease detection. Methylation markers may also serve for prognostic and predictive purposes as they often reflect the metastatic potential and sensitivity to therapy. As current findings show a great potential of recently characterised methylation markers, more studies in the field are needed in the future. Large clinical studies of newly developed markers are especially needed. The review describes the diagnostic potential of DNA methylation markers.