DNA甲基化及其对哺乳动物早期发育的影响

表观遗传修饰（epigenetic reprogramming）是指不改变DNA序列的可逆性修饰，在哺乳动物中其修饰的主要方式为DNA甲基化/去甲基化和组蛋白修饰。DNA甲基化主要发生在两个时期：生殖细胞发育期和植入前胚胎期，如果在此期间发生去甲基化不充分或者是过早的再甲基化，则会导致胚胎的死亡及出生后各种遗传病的发生，也是目前体细胞核移植来源的胚胎受孕率低的重要原因。     

表遗传修饰与配子形成和胚胎早期发育

DNA甲基化/去甲基化及组蛋白的共价修饰是主要的表遗传修饰(epigenetic reprogramming)方式。在配子形成和早期胚胎发育中,其与基因组印记(genomic imprinting)、染色质结构重组(chro-matin remodeling)、X-染色体失活(X-inactivation)的形成均有密切关系,它们的共同作用机制是调节基因的表达。表遗传修饰至少发生在两个关键时期——配子形成期和植入前胚胎,如果在此期间发生异常,则会导致胚胎的死亡及生后各种疾病的发生。     

基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用

基因组DNA的甲基化修饰通常使基因转录失活,去甲基化或低甲基化则使基因转录活化。但是,胚胎干细胞向各种成体细胞分化过程中相关基因的转录活化与DNA甲基化修饰水平并不呈简单的正性或负性相关。因此,甲基化修饰调节基因转录是一个复杂的过程。目前,对甲基化修饰作用的研究主要集中在基因选择性活化、改变转录因子与靶基因的结合活性、与组蛋白修饰协同作用及其基因表达的阶段特异性等方面。     

表遗传修饰与配子形成和胚胎早期发育

DNA甲基化，去甲基化及组蛋白的共价修饰是主要的表遗传修饰(epigenetic reprogramming)方式。在配子形成和早期胚胎发育中，其与基因组印记(genomic imprinting)、染色质结构重组(chromatin remodeling)、X-染色体失活(X-inactivation)的形成均有密切关系，它们的共同作用机制是调节基因的表达。表遗传修饰至少发生在两个关键时期——配子形成期和植入前胚胎，如果在此期间发生异常，则会导致胚胎的死亡及生后各种疾病的发生。     

DNA的甲基化修饰与DNA甲基转移酶

组织特异的 DNA甲基化谱是哺乳动物基因组的显著特征。DNA的甲基化修饰参与基因表达调控、发育调节、基因组印迹和 X染色体灭活等诸多重要生物学过程。大多数印迹基因可以调节胚胎的生长和发育。印迹功能的紊乱将导致发育异常及死胎。基因组范围内的广泛的去甲基化及随后发生的重新甲基化 ,可能使胚胎消除其亲本特异的甲基化谱 ,从而进入正常发育程序。DNA甲基化异常与肿瘤等疾病有关。肿瘤细胞的总体甲基化水平比正常细胞低 ,但是伴有某些 Cp G岛甲基化程度增高。抑癌基因启动子的异常甲基化和癌基因的去甲基化均影响肿瘤发生发展过程。哺乳动物 DNA甲基化谱的建立和维持需要维持 DNA甲基化的甲基转移酶和 DNA重新甲基转移酶。     

DNA的甲基化修饰与DNA甲基转移酶

组织特异的DNA甲基化谱是哺乳动物基因组的显特征。DNA的甲基化修饰参与基因表达调控、发育调节、基因组印迹和X染色体灭活等诸多重要生物学过程。大多数印迹基因可以调节胚胎的生长和发育。印迹功能的紊乱将导致发育异常及死胎。基因组范围内的广泛的去甲基化及随后发生的重新甲基化,可能使胚胎消除其亲本特异的甲基化谱,从而进入政党发育程序。DNA甲基化异常与肿瘤等疾病有关。肿瘤细胞的总体甲基化水平比正常细胞低,但是伴有某些CpG岛甲基化程度增高。抑菌基因启动子的异常甲基化和癌基因的去甲基化均影响肿瘤发生发展过程。哺乳动物DNA甲基化谱的建立和维持需要维持DNA甲基化的甲基转移酶和DNA重新甲基转移酶。     

Sirtuins及其在肿瘤发生发展过程中的作用

表观遗传是指DNA序列不发生变化但基因表达却发生了可遗传的改变,主要涉及DNA甲基化、组蛋白修饰和染色质重塑三种机制.组蛋白共价修饰包括甲基化/去甲基化、乙酰化/去乙酰化、磷酸化/去磷酸化、泛素化/去泛素化等等,发生在核心组蛋白N端尾部的这些可逆的共价修饰具有复杂的相互作用,其中,组蛋白的乙酰化/去乙酰化是组蛋白共价修饰最重要的一种机制,这种共价修饰主要由组蛋白乙酰化酶(histone acetylase, HAT)和组蛋白去乙酰化酶(histone deacetylase, HDAC)分别催化乙酰化和去乙酰化过程,这种可逆的乙酰化修饰可使染色质结构发生动态改变,并维持一个可逆而稳定的状态,同时精密调节某些基因的转录和表达,从而不但影响发育、分化和衰老等生理过程,而且与癌变密切相关.     

甲基化CpG结合蛋白家族(MeCPs)在表遗传中的作用

表遗传学（epigenetics）主要研究基因型和表型之间的关系。基因组表遗传修饰主要包括遗传物质DNA的甲基化修饰和核小体组蛋白修饰,最终结果是DNA碱基不发生改变的情况下而表型却发生了变化。甲基化CpG结合蛋白家族(methyl-CpG binding proteins,MeCPs)是能与甲基化CpG二核苷酸结合的一类核蛋白,从而有机地将DNA甲基化和组蛋白修饰耦合起来,在表遗传中发挥着中枢纽带的作用。本文简述了近年来表遗传中DNA甲基化和组蛋白修饰的最新研究进展以及MeCPs家族在表遗传中的重要作用,目的是使人们进一步了解表遗传作用规律。     

TET去甲基化酶在T细胞中的功能研究

DNA甲基化作为最重要的表观遗传修饰之一,在基因表达调控方面起重要作用。哺乳动物DNA甲基化主要发生在胞嘧啶第五位碳原子上,称为5-甲基胞嘧啶(5-methylcytosine,5mC). TET(Ten eleven translocation)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-羧基胞嘧啶的一系列DNA去甲基化过程。TET家族蛋白和DNA去甲基化修饰在T细胞中的功能也逐渐被揭示。本文主要将近年研究TET去甲基化酶在T细胞中的功能进行总结和讨论。     

表观遗传修饰剂对重构胚发育的影响

自从"Dolly"绵羊克隆成功后,先后有哺乳动物鼠、猪、牛、狗、马等克隆成功的报道,但目前哺乳动物体细胞核移植成功率仍很低,其主要与卵母细胞对供体核的不完全重编程有关.核重编程主要表现为供体核基因的表观遗传改变并指导重构胚胎的正常发育.在核移植过程中,体细胞(供核细胞)携带着其组织所特有的表观遗传修饰标志,这种标志在核重编程中必须被清除.去除供核细胞先前的表观遗传标志能够提高重构胚的体外发育率[1].在核重编程中假如表观遗传修饰标志清除或重建失败,将导致重构胚全能性消失、从而影响后期分化和发育[2].核移植后供体细胞核的重新程序化是否完全是成功产生克隆动物的先决条件[3].表观遗传修饰主要包括3个方面:DNA甲基化,RNA相关性沉寂和组蛋白翻译后修饰,其中DNA甲基化和组蛋白乙酰化在体细胞核移植中研究较多.DNA甲基化能引起染色质结构、DNA构象、DNA稳定性及DNA与蛋白质相互作用方式的改变,从而控制基因表达;组蛋白乙酰化与基因活化以及DNA复制相关,组蛋白的去乙酰化和基因的失活相关.在体细胞克隆过程中加入适量的表观遗传修饰剂,干预DNA甲基化和组蛋白乙酰化过程,将有可能影响重构胚的体内外发育.     

RNA expression microarray analysis in mouse prospermatogonia: identification of candidate epigenetic modifiers.

The mammalian totipotent and pluripotent lineage exhibits genome-wide dynamics with respect to DNA methylation content. The first phase of global DNA demethylation and de novo remethylation occurs during preimplantation development and gastrulation, respectively, while the second phase occurs in primordial germ cells and primary oocytes/prospermatogonia, respectively. These dynamics are indicative of a comprehensive epigenetic resetting or reprogramming of the genome in preparation for major differentiation events. To gain further insight into the mechanisms driving DNA methylation dynamics and other types of epigenetic modification, we performed an RNA expression microarray analysis of fetal prospermatogonia at the stage when they are undergoing rapid de novo DNA remethylation. We have identified a number of highly or specifically expressed genes that could be important for determining epigenetic change in prospermatogonia. These data provide a useful resource in the discovery of molecular pathways involved in epigenetic reprogramming in the mammalian germ line.     

Developmental origins of male subfertility: role of infection,inflammation, and environmental factors

Male gamete development begins with the specification of primordial cells in the epiblast of the early embryo and is not complete until spermatozoa mature in the epididymis of adult males. This protracted developmental process involves extensive alteration of the paternal germline epigenome. Initially, epigenetic reprogramming in fetal germ cells results in removal of most DNA methylation, including parent-specific epigenetic information. The germ cells then establish sex-specific epigenetic information through de novo methylation and undergo spermatogenesis. Chromatin in haploid germ cells is repackaged into protamines during spermiogenesis, providing further widespread epigenetic reorganization. Finally, after fertilization, epigenetic reprogramming in the preimplantation embryo is necessary for regaining totipotency. These events provide substantial windows during which epigenetic errors either may be corrected or may occur in the germline. There is now increasing evidence that environmental factors such as exposure to toxicants, the parents’ and individual’s diet, and even infectious and inflammatory events in the male reproductive tract may influence epigenetic reprogramming. This, together with other damage inflicted on the germline chromatin, may result in negative consequences for fertility and health. Large epidemiological birth cohort studies have yielded insight into possible causative environmental factors. Together with experimental animal studies, a clearer view of environmental impacts on fetal development and their intergenerational and even transgenerational effects on reproductive health has emerged and is reviewed in this article.     

Safeguarding parental identity: Dnmt1 maintains imprints during epigenetic reprogramming in early embryogenesis

During early mammalian embryogenesis, the genome undergoes global epigenetic reprogramming, losing most of its methylation before re-establishing it de novo at implantation. However, faithful maintenance of methylation at imprinted genes during this process is vital for embryonic development, but the DNA methyltransferase responsible for this maintenance has remained unknown. In this issue of Genes & Development, Hirasawa and colleagues (pp. 1607-1616) show that Dnmt1, and not Dnmt3a or Dnmt3b, maintains methylation at genomic imprints during preimplantation development.     

再生医学研究新进展：DNA甲基化与细胞重编程

背景：使用一些生长因子能使终分化的体细胞重编程而产生多能性干细胞。 目的：讨论表观遗传机制在细胞重编程和调节中的作用,揭示表观遗传基因调控的变化、表观遗传修饰标记的稳定性及其对基因组表达的影响。 方法：在PubMed数据库及CNKI数据库,以“DNA甲基化,细胞重编程,干细胞”为关键词检索1990/2008相关的文章。 结果与结论：尽管在体内细胞分化通常是单向和不可逆的,但这个过程可被重编程而改变。使用一些生长因子能使终分化的体细胞重编程而产生多能性干细胞。目前为止,导入转录因子、DNA去甲基化、表观基因改变已被用于诱导重编程。通过了解其分子机制,揭示表观遗传基因调控的变化、表观遗传修饰标记的稳定性及其对基因组表达的影响,对基因治疗的发展有重要意义。然而依然许多问题有待深入研究,如：是AID还是 DNA去甲基酶对DNA去甲基化起重要作用？DNA去甲基化需要什么条件？肿瘤细胞中CpG片段能否去甲基化,癌细胞是否更难重编程？     

Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance

A broad spectrum of assisted reproductive technologies has become available for couples with fertility problems. Follow-up studies of children born as a result of assisted reproduction have shown that neonatal outcome and malformation rates are not different from those of the general population, except for a low birthweight and a slight increase in chromosomal abnormalities. The safety aspect of assisted reproduction at the epigenetic level has not been well studied. Epigenetics refers to phenomena where modifications of DNA methylation and/or chromatin structure underlie changes in gene expression and phenotype characteristics. This article intends to analyse epigenetic risks related to assisted reproduction on the basis of an overview of epigenetic reprogramming events in the gamete and early embryo. Two epigenetic modifications, methylation and imprinting, are considered in more detail. The interference of in-vitro embryo culture, immature sperm cells and nuclear transfer with epigenetic reprogramming is discussed, as well as the possibility of epigenetic inheritance.     

Gradual development of a genome-wide H3-K9 trimethylation pattern in paternally derived pig pronucleus.

The cytoplasm of a mature oocyte contains many protein complexes that are programmed to restructure incoming sperm chromatins on fertilization. Of the complicated biochemical events that these functional machineries control, the most impressive and important is epigenetic reprogramming. Despite its importance in epigenetic resetting, or "de-differentiation," of gamete genomes back to an incipient status, the mechanisms of epigenetic reprogramming do not seem to be conserved among mammals. Here, we report that, unlike in the mouse, the pig sperm-derived pronucleus is markedly trimethylated at lysine 9 of histone H3 (H3-m(3)K9), which might be associated with preservation of paternally derived cytosine methylation in pig zygotes. The male H3-m(3)K9 pattern is gradually established during pronucleus development, and this process occurs independently of DNA replication. Considering these unique epigenetic features, the pig zygote is, we believe, suited to serve as another model of epigenetic reprogramming that is antithetical to the well-characterized mouse model.     

Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation

Ten-eleven translocation 1-3 (Tet1-3) proteins have recently been discovered in mammalian cells to be members of a family of DNA hydroxylases that possess enzymatic activity toward the methyl mark on the 5-position of cytosine (5-methylcytosine [5mC]), a well-characterized epigenetic modification that has essential roles in regulating gene expression and maintaining cellular identity. Tet proteins can convert 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) through three consecutive oxidation reactions. These modified bases may represent new epigenetic states in genomic DNA or intermediates in the process of DNA demethylation. Emerging biochemical, genetic, and functional evidence suggests that Tet proteins are crucial for diverse biological processes, including zygotic epigenetic reprogramming, pluripotent stem cell differentiation, hematopoiesis, and development of leukemia. Insights into how Tet proteins contribute to dynamic changes in DNA methylation and gene expression will greatly enhance our understanding of epigenetic regulation of normal development and human diseases.     

Dynamic DNA methylation reprogramming: active demethylation and immediate remethylation in the male pronucleus of bovine zygotes.

DNA methylation reprogramming (DMR) is believed to be a key process by which mammalian zygotes gain nuclear totipotency through erasing epigenetic modifications acquired during gametogenesis. Nonetheless, DMR patterns do not seem to be conserved among mammals. To identify uniform rules underlying mammalian DMRs, we explored DMRs of diverse mammalian zygotes. Of the zygotes studied, of particular interest was the bovine zygote; the paternal DNA methylation first decreased and was then rapidly restored almost to the maternal methylation level even before the two-cell stage. The 5-azadeoxycytidine treatment led to complete demethylation of the male pronucleus. The unusually dramatic changes in DNA methylation levels indicate that the bovine male pronucleus undergoes active demethylation, which is followed by de novo methylation. Our results show that, in bovine, the compound processes of active DNA demethylation and de novo DNA methylation, along with de novo H3-K9 trimethylation also, take place altogether within this very narrow window of pronucleus development.