DNA methylation and epigenetic mechanisms

Genes are essential for the transmission of genetic information from generation to generation, and this mechanism of inheritance is fully understood. Genes are also essential for unfolding the genetic program for development, but the rules governing this process are obscure. Epigenetics comprises the study of the switching on and off of genes during development, the segregation of gene activities following somatic cell division, and the stable inheritance of a given spectrum of gene activities in specific cells. Some of these processes may be explained by DNA modification, particularly changes in the pattern of DNA methylation and the heritability of that pattern. There is strong evidence that DNA methylation plays an important role in the control of gene activity in cultured mammalian cells, and the properties of a CHO mutant strain affected in DNA methylation are described. Human diploid cells progressively lose cytosine methylation during serial subculture, and this may be related to their in vitro senescence. There is also evidence that DNA modifications can be inherited through the germ line. Classical genetics is based on the study of all types of change in DNA base sequence, but the rules governing the activity of genes by epigenetic mechanisms are necessarily different. Their elucidation will depend both on a theoretical framework for development and on experimental studies at the molecular, chromosomal, and cellular levels.     

DNA methylation and epigenetic inheritance

Mammalian cell lines silence genes at low frequency by the methylation of promoter sequences. These silent genes can be reactivated at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). The inactive and active epigenetic states of such genes are stably inherited. A method for silencing genes is now available. It involves treatment of permeabilized cells with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) which is incorporated into DNA. The methylation of promoter sequences has been confirmed using the bisulfite genomic sequencing procedure. Methylated oligonucleotides homologous to promoter sequences might be used to specifically target and silence given genes, but results so far have not been conclusive. Treatments that silence or reactivate genes by changing DNA methylation can be referred to as epimutagens, as distinct from mutagens that act by changing DNA sequences. The epimutagen 5-aza-CR reactivates genes but has little mutagenic activity, whereas standard mutagens (such as ethyl methane sulfonate and ultraviolet light) have little reactivation activity. Nevertheless, much more information is required about the effects of DNA-damaging agents in changing DNA methylation and gene activity and also about the role of epimutations in tumor progression.     

DNA methylation and epigenetic inheritance

Classical genetics has revealed the mechanisms for the transmission of genes from generation to generation, but the strategy of the genes in unfolding the developmental programme remains obscure. Epigenetics comprises the study of the mechanisms that impart temporal and spatial control on the activities of all those genes required for the development of a complex organism from the zygote to the adult. Epigenetic changes in gene activity can be studied in relation to DNA methylation in cultured mammalian cells and it is also possible to isolate and characterize mutants with altered DNA methylase activity. Although this experimental system is quite far removed from the epigenetic controls acting during development it does provide the means to clarify the rules governing the silencing of genes by specific DNA methylation and their reactivation by demethylation. This in turn will facilitate studies on the control of gene expression in somatic cells of the developing organism or the adult. The general principles of epigenetic mechanisms can be defined. There are extreme contrasts between instability or switches in gene expression, such as those in stem-line cells, and the stable heritability of a specialized pattern of gene activities. In some situations cell lineages are known to be important, whereas in others coordinated changes in groups of cells have been demonstrated. Control of numbers of cell divisions and the size of organisms, or parts of organisms, is also essential. The epigenetic determination of gene expression can be reversed or reprogrammed in the germ line. The extent to which methylation or demethylation of specific DNA sequences can help explain these basic epigenetic mechanisms is briefly reviewed.     

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.     

Gene silencing and endogenous DNA methylation in mammalian cells

It is known that transformed mammalian cells can spontaneously inactivate genes at low frequency by the de novo methylation of promoter sequences. It is usually assumed that this is due to DNA methyl transferase activity, but an alternative possibiity is that 5-methyldCTP is present in these cells and can be directly incorporated into DNA. The ongoing repair of DNA containing 5-methylcytosine will produce 5-methyldeoxycytidine monophosphate (5-methyldCMP), so the question arises whether this can be phosphorylated to 5-methyldCTP. We have tested this using three strains of CHO cells with different levels of 5-methyldCMP deaminase activity. That with the lowest enzyme activity, designated HAM⁻, has previously been shown to incorporate tritium labelled 5-methyldeoxycytidine into 5-methylcytosine in DNA, with a greater amount of label in thymine. This strain is phenotypically unstable producing cells resistant to bromodeoxyuridine (BrdU) and 6-thioguanine (6-TG) at high frequency. In contrast, the strain with the highest 5-methyldCMP deaminease, designated HAM⁺, is extremely stable, and the starting strain K1 HAM^s1 is intermediate between the HAM⁻ and HAM⁺ phenotypes. We have also shown that human diploid fibroblast strain MRC-5 has a phenotype like HAM⁺, whereas its SV40 transformed derivative, MRC-5V2 resembles HAM⁻ in having low 5-methyl dCMP deaminase activity, and is phenotypically unstable with regard to 6-TG resistance. It seems that 5-methyldCMP deaminase can be down-regulated in transformed cells, and this can promote de novo methylation by incorporation of 5-methyldCTP derived from 5-methyldCMP.     

DNA methylation in mammalian nuclei

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.     

DNA methylation and epigenetic defects in carcinogenesis

It is frequently assumed that DNA-damaging agents are carcinogenic because they induce mutations. However, another strong possibility is that the damage leads to heritable changes in the methylation of cytosine in DNA. Considerable evidence exists that gene expression in mammalian cells is in part controlled by methylation of specific DNA sequences. Carcinogens may act by altering the normal epigenetic controls of gene activity in specialised cells, and thereby produce aberrant heritable phenotypes. It is known that agents which inhibit DNA methylation can be carcinogenic and that tumour cells are altered in DNA methylation.     

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

RNA interference in mammalian DNA methylation

RNAi and Dicer-dependent siRNAs are required for constitutive heterochromatin formation in fission yeast and for establishing DNA methylation at repetitive elements in plants. In the mammalian male germ line, DICER1-independent piRNAs are required for the full establishment of DNA methylation of dispersed repetitive transposable elements. However, in other mammalian cell types, no clear picture has yet emerged of the role of RNAi in establishing heterochromatin and DNA methylation. In mouse embryonic stem cells, which remain viable on loss of DICER1 and ablation of RNAi, while no firm evidence has been obtained for defective heterochromatin formation, there are indications of defective DNA methylation. The latter has been attributed to an indirect effect of reduced DNA methyltransferase (DNMT) activity due to a loss of miRNA-mediated gene regulation. However, it is unclear whether the reductions in DNMT activity were sufficient to affect DNA methylation. We consider it equally likely that the defects in DNA methylation that can be observed in DICER1-deficient embryonic stem cells are the result of nonspecific effects related to RNAi loss aside from reduced DNMT activity.     

DNA methylation and cell memory

In this paper we address the question: How do replicating mammalian cells remember with high fidelity their proper state of differentiation? Several possible mechanisms for cell memory are discussed, and it is concluded that only mechanisms involving DNA methylation are supported by strong experimental evidence. This evidence is reviewed. The establishment and modulation of methylation patterns are discussed and a hemimethylation model for stem cells is presented. The overall conclusion is that, although little is yet known about the details, there should be little doubt about the existence of a methylation system functioning at least to aid cell memory.     

DNA甲基化——肿瘤产生的一种表观遗传学机制

在人类基因组中,DNA甲基化是一种表观遗传修饰,它与肿瘤的发生关系密切。抑癌基因和DNA修复基因的高甲基化、重复序列DNA的低甲基化、某些印记基因的印记丢失与多种肿瘤的发生有关。目前研究发现,基因组中甲基化的水平不仅受DNA 甲基化转移酶（DNMT）的影响,还与组蛋白甲基化、叶酸摄入、RNA干扰等多种因素有关。DNA甲基化在基因转录过程中扮有重要角色,并与组蛋白修饰、染色质构型重塑共同参与转录调控。     

Radiation-induced epigenetic DNA methylation modification of radiation-response pathways

DNA methylation can regulate gene expression and has been shown to modulate cancer cell biology and chemotherapy resistance. Therapeutic radiation results in a biological response to counter the subsequent DNA damage and genomic stress in order to avoid cell death. In this study, we analyzed DNA methylation changes at >450,000 loci to determine a potential epigenetic response to ionizing radiation in MDA-MB-231 cells. Cells were irradiated at 2 and 6 Gy and analyzed at 7 time points from 1–72 h. Significantly differentially methylated genes were enriched in gene ontology categories relating to cell cycle, DNA repair, and apoptosis pathways. The degree of differential methylation of these pathways varied with radiation dose and time post-irradiation in a manner consistent with classical biological responses to radiation. A cell cycle arrest was observed 24 h post-irradiation and DNA damage, as measured by γH2AX, resolved at 24 h. In addition, cells showed low levels of apoptosis 2–48 h post-6 Gy and cellular senescence became significant at 72 h post-irradiation. These DNA methylation changes suggest an epigenetic role in the cellular response to radiation.     

Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells.     

DNA methylation in mammalian development and disease

Epigenetic modification of the cytosine base of DNA by its methylation introduced the possibility that beyond the inherent information contained within the nucleotide sequence there was an additional layer of information added to the underlying genetic code. DNA methylation has been implicated in a wide range of biological functions, including an essential developmental role in the reprogramming of germ cells and early embryos, the repression of endogenous retrotransposons, and a generalized role in gene expression. Special functions of DNA methylation include the marking of one of the parental alleles of many imprinted genes, a group of genes essential for growth and development in mammals with a unique parent-of-origin expression pattern, a role in stabilizing X-chromosome inactivation, and centromere function. In this regard, it is not surprising that errors in establishing or maintaining patterns of methylation are associated with a diverse group of human diseases and syndromes.     

哺乳动物DNA甲基化修饰的动态调控机制

DNA甲基化是最主要的表观遗传修饰之一,主要发生在胞嘧啶第五位碳原子上,称为5-甲基胞嘧啶。哺乳动物DNA甲基化由从头DNA甲基转移酶DNMT3A/3B在胚胎发育早期建立。细胞分裂过程中甲基化模式的维持由DNA甲基转移酶DNMT1实现。TET家族蛋白氧化5-甲基胞嘧啶成为5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶,从而起始DNA的去甲基化过程。这些DNA甲基化修饰酶精确调节DNA甲基化的动态过程,在整个生命发育过程中发挥重要作用,其失调也与多种疾病发生密切相关。本文对近年来DNA甲基化修饰酶的结构与功能研究进行讨论。