DNA methylation changes during mouse spermatogenesis

Genomic imprinting in mammals is thought to be mediated by differences in the methylation level of cytosine residues in the genome. These differences in DNA methylation are thought to be generated during the development of the germ line. To characterize the profile of global methylation of the mouse genome during male gametogenesis, we have quantified the relative level of methylation in individual cells during meiosis and spermatogenesis. A decrease in the level of DNA methylation is observed from meiotic cells to elongated spermatids. The erasure of the somatic pattern of methylation during spermatogenesis suggests the existence of a subsequent mechanism generating the parental specific methylation patterns leading to genomic imprinting of specific alleles.     

Asynchronous replication timing of imprinted loci is independent of DNA methylation,but consistent with differential subnuclear localization

Genomic imprinting in mammals marks the two parental alleles resulting in differential gene expression. Imprinted loci are characterized by distinct epigenetic modifications such as differential DNA methylation and asynchronous replication timing. To determine the role of DNA methylation in replication timing of imprinted loci, we analyzed replication timing in Dnmt1- and Dnmt3L-deficient embryonic stem (ES) cells, which lack differential DNA methylation and imprinted gene expression. Asynchronous replication is maintained in these ES cells, indicating that asynchronous replication is parent-specific without the requirement for differential DNA methylation. Imprinting centers are required for regional control of imprinted gene expression. Analysis of replication fork movement and three-dimensional RNA and DNA fluorescent in situ hybridization (FISH) analysis of the Igf2-H19 locus in various cell types indicate that the Igf2-H19 imprinting center differentially regulates replication timing of nearby replicons and subnuclear localization. Based on these observations, we suggest a model in which cis elements containing nonmethylation imprints are responsible for the movement of parental imprinted loci to distinct nuclear compartments with different replication characteristics resulting in asynchronous replication timing.     

TET酶及其中间产物的研究进展

正DNA甲基化的形式主要包括以下3种:5-甲基胞嘧啶(5mC)、N~6-甲基嘌呤(N6mA)以及7-甲基鸟嘌呤(7mG)~([1])。目前,研究得最为广泛也最为透彻的就是主要存在于CpG岛中胞嘧啶的甲基化修饰,即5mC。在哺乳动物基因组中大约有70%~80%的CpG岛区域的胞嘧啶存在甲基化修饰~([2])。而CpG岛的甲基化是基因沉默的一个重要标志,在调控基因     

Paternal imprints can be established on the maternal Igf2-H19 locus without altering replication timing of DNA

Genomic imprinting in mammals marks the parental alleles in gametes, resulting in differential gene expression in offspring. A number of epigenetic features are associated with imprinted genes. These include differential DNA methylation, histone acetylation and methylation, subnuclear localization and DNA replication timing. While DNA methylation has been shown to be necessary both for establishment and maintenance of imprinting, the connections with the other types of epigenetic marking systems are not clear. Specifically, it is not known whether the other marking systems, either on their own or in conjunction with DNA methylation, are required for imprinting. Here we show that in the mouse mutant Minute (Mnt) the Igf2-H19 locus acquires a paternal methylation imprint in the maternal germline. DNA methylation of the H19 DMR is established in oogenesis, maintained during postzygotic development on the maternal allele, and erased in primordial germ cells. The fact that a paternal type methylation imprint can also be established in the maternal germline indicates that trans-acting factors that target methylation to this imprinted region are likely to be the same in both germlines. Surprisingly, however, asynchrony of DNA replication of the locus is maintained despite the altered expression and methylation imprint of Igf2 and H19. These results show clearly that replication asynchrony of this region is neither the determinant factor for, nor a consequence of, epigenetic modifications that are critical for genomic imprinting. Replication asynchrony may thus be regulated differently from methylation imprints and have a separate function.     

Epigenomic mapping in <Emphasis Type="Italic">Arabidopsis</Emphasis> using tiling microarrays

In addition to genetic information, chromosomes transmit epigenetic information from cell to cell during division, and sometimes from generation to generation. While genetic information is encoded directly in the DNA sequence, epigenetic information is not, although it is usually associated with specific chromosomal regions. Epigenetic modifications in plants include cytosine methylation as well as modification of histones and other chromosomal proteins. Small interfering RNA play major roles in targeting these modifications to specific regions. Genomic tiling microarrays are powerful tools for analysing epigenetic information, and we review their application in building epigenomic maps in the model plant, Arabidopsis.     

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.     

Age-related epigenetic changes and the immune system

The role of DNA methylation in immune function is discussed extensively in other papers in this issue. Many of these discussions assume that DNA methylation, a major mediator of epigenetic information, is fairly immutable and uniform in adult cells and tissues. There is, however, growing evidence that DNA methylation changes subtly with age. Normal aging cells and tissues show a progressive loss of 5-methylcytosine content, primarily within DNA repeated sequences, but also in potential gene regulatory areas. In parallel, selected genes show progressive age-related increases in promoter methylation, which, once a critical methylation density is reached, have the potential to permanently silence gene expression. These changes are highly mosaic within a given tissue and introduce a high degree of epigenetic variability in aging cells. Such epigenetic phenomena could impact immune response through masking/unmasking potential tissue antigens as well as by modulating the differentiation and response of immune effector cells. The contribution of epigenetic changes to the altered immune function observed in aging humans deserves careful investigation.     

DNA甲基化对哺乳动物早期胚胎发育的影响

表遗传修饰是指不改变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甲基似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。     

Analysis of DNA methylation in human BK virus

Human BK virus may cause nephropathy due to viral replication in patients who have undergone renal transplantation. However, the mechanism regulating replication of BKV is still not clear. Previous studies have suggested that epigenetic modifications may play a crucial role in virus replication. In this study, the DNA methylation profiles of five CpG sites located within the promoter/enhancer regions and nine CpG sites located within the early and late coding regions of the replicating BKV genome were investigated. BKV genomic DNA from mature virions and from the early and late phases of replicating BKV were examined for DNA methylation by bisulfite sequencing that covered 14 CpG sites. Our results showed that none of the examined BKV DNA from the various different stages of replication was methylated. This is the first report to analyze the methylation of BKV genomic DNA during viral replication. The results seem to indicate that methylation is not involved in regulation of BKV replication.     

Methylomics in psychiatry: Modulation of gene-environment interactions may be through DNA methylation.

Fine-tuning of neuronal connections during development is regulated through environmental interactions. Some fine-tuning occurs through changes in gene expression and/or epigenetic gene-specific DNA methylation states. DNA methylation occurs by transfer of a methyl group from S-adenosyl methionine to cytosine residues in the dinucleotide sequence CpG. Although CpG sequences spread throughout the genome are usually heavily methylated, those occurring in CpG islands in the promoter regions of genes are less methylated. In most cases, the extent of DNA methylation correlates with the extent of gene inactivation. Other known epigenetic mechanisms include histone deacetylation and chromatin remodeling, RNA inhibition, RNA modification, and DNA rearrangement. Exposure memory expressed as epigenetic DNA modifications allows genomic plasticity and short-term adaptation of each generation to their environment. Environmental factors that affect DNA methylation include diet, proteins, drugs, and hormones. Induced methylation changes may produce altered gene response upon subsequent hormonal stimulation. The gene-specific DNA methylation state may be preserved upon transmission through mitosis and meiosis. An increasing amount of data implicates a role for DNA methylation in multi-factorial psychiatric disorders. For example, L-methionine treatment can exacerbate psychosis; while valproate, a drug producing hypomethylated DNA, reduces such symptoms. Hypermethylation of the promoter region of the RELN gene correlates with reduced gene expression. This gene's protein Reelin, which is necessary for neuronal migration and synaptogenesis, is reduced in schizophrenia and bipolar disorder, suggesting hypermethylation of the promoter region in these disorders. Some evidence implicates methylation of the promoter regions of the DRD2 and HTR2A genes in schizophrenia and mood disorders as well. DNA methylation usually increases with age, although hypomethylation of the promoter region of the amyloid A4 precursor gene during aging may play a role in Alzheimer's disease. More studies are needed to define the role of methylomics and other epigenetic phenomena in the nervous system.     

DNA methylation, imprinting and cancer

It is well known that a variety of genetic changes influence the development and progression of cancer. These changes may result from inherited or spontaneous mutations that are not corrected by repair mechanisms prior to DNA replication. It is increasingly clear that so called epigenetic effects that do not affect the primary sequence of the genome also play an important role in tumorigenesis. This was supported initially by observations that cancer genomes undergo changes in their methylation state and that control of parental allele-specific methylation and expression of imprinted loci is lost in several cancers. Many loci acquiring aberrant methylation in cancers have since been identified and shown to be silenced by DNA methylation. In many cases, this mechanism of silencing inactivates tumour suppressors as effectively as frank mutation and is one of the cancer-predisposing hits described in Knudson's two hit hypothesis. In contrast to mutations which are essentially irreversible, methylation changes are reversible, raising the possibility of developing therapeutics based on restoring the normal methylation state to cancer-associated genes. Development of such therapeutics will require identifying loci undergoing methylation changes in cancer, understanding how their methylation influences tumorigenesis and identifying the mechanisms regulating the methylation state of the genome. The purpose of this review is to summarise what is known about these issues.     

Genome-wide, high-resolution DNA methylation profiling using bisulfite-mediated cytosine conversion

Methylation of cytosines ((m)C) is essential for epigenetic gene regulation in plants and mammals. Aberrant (m)C patterns are associated with heritable developmental abnormalities in plants and with cancer in mammals. We have developed a genome-wide DNA methylation profiling technology employing a novel amplification step for DNA subjected to bisulfite-mediated cytosine conversion. The methylation patterns detected are not only consistent with previous results obtained with (m)C immunoprecipitation (mCIP) techniques, but also demonstrated improved resolution and sensitivity. The technology, named BiMP (for Bisulfite Methylation Profiling), is more cost-effective than mCIP and requires as little as 100 ng of Arabidopsis DNA.     

Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells

Complex organisms contain a variety of distinct cell types but only a single genome. Therefore, cellular identity must be specified by the developmentally regulated expression of a subset of genes from an otherwise static genome. In mammals, genomic DNA is modified by cytosine methylation, resulting in a pattern that is distinctive for each cell type (the epigenome). Because nucleosomal histones are subject to a wide variety of post-translational modifications (PTMs), we reasoned that an analogous "epiproteome" might exist that could also be correlated with cellular identity. Here, we show that the quantitative evaluation of nucleosome PTMs yields epiproteomic signatures that are useful for the investigation of stem cell differentiation, chromatin function, cellular identity, and epigenetic responses to pharmacologic agents. We have developed a novel enzyme-linked immunosorbent assay-based method for the quantitative evaluation of the steady-state levels of PTMs and histone variants in preparations of native intact nucleosomes. We show that epiproteomic responses to the histone deacetylase inhibitor trichostatin A trigger changes in histone methylation as well as acetylation, and that the epiproteomic responses differ between mouse embryonic stem cells and mouse embryonic fibroblasts (MEFs). ESCs subjected to retinoic acid-induced differentiation contain reconfigured nucleosomes that include increased content of the histone variant macroH2A and other changes. Furthermore, ESCs can be distinguished from embryonal carcinoma cells and MEFs based purely on their epiproteomic signatures. These results indicate that epiproteomic nucleosomal signatures are useful for the investigation of stem cell identity and differentiation, nuclear reprogramming, epigenetic regulation, chromatin dynamics, and assays for compounds with epigenetic activities. Disclosure of potential conflicts of interest is found at the end of this article.     

透明细胞肾细胞癌的表观遗传学研究进展

正肾脏肿瘤的发病率在人类泌尿系统肿瘤中排名第3,约占恶性肿瘤的3%,发病年龄主要在50~70岁,每年导致90 000多例患者死亡,且呈递增趋势~([1])。肾癌的具体发病机制不详,除遗传因素外,吸烟、肥胖、污染和辐射等也是重要因素。大多数透明细胞肾细胞癌(clear cell renal cell carcinoma,ccRCC)患者在早期无任何症状,20%~30%患者在     

The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA

The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.Maize exhibits a wealth of epigenetic phenomena, from transposon silencing, cycling, and presetting, to gene imprinting and paramutation. Furthermore, despite the complexity and sophistication of maize breeding, there is a large degree of “hidden” variation for many traits that is difficult to explain by allelic variation alone (Gottlieb et al. 2002). At least some of this unexplained variation might be due to epigenetic rather than genetic changes in the maize genome (Richards 2011). A recent study using anti-methylcytosine antibodies and microarray hybridization to detect DNA methylation demonstrated clear differences between maize inbred lines, lending support to this hypothesis (Eichten et al. 2011). Similarly, studies using genome-wide sequencing of methylation-dependent restriction fragments have revealed that most methylation is found in transposable elements, prime sources of such variation (Palmer et al. 2003; Wang et al. 2009). However, neither method had the capability to detect individual cytosines in their sequence context.In plants, cytosine methylation occurs in symmetric (CG and CHG, where H is A, C, or T) as well as asymmetric (CHH) contexts. Methylation in each context is associated with DNA replication, histone modification, and RNA interference, respectively, although these mechanisms overlap (Law and Jacobsen 2010). The maize genome comprises roughly 50,000 genes and more than 1 million transposons and related repeats (Schnable et al. 2009). Approximately 29% of the cytosine residues are methylated as 5-methylcytosine (Montero et al. 1992), mostly in transposons (Palmer et al. 2003). We have used very-high-coverage whole-genome bisulfite sequencing to explore DNA methylation at nucleotide resolution in genes, transposons, and other features of the maize genome, as well as its heritability and potential contribution to traits. We have found that methylation in different sequence contexts is guided differentially by small RNA and is correlated with transposon insertion and mRNA splicing. Heritable and predictable switches in DNA methylation were detected in recombinant inbred lines. These shifts were apparently triggered by small RNA, resembling paramutation, but then maintained by replication-dependent symmetric methylation.We present a high-resolution and high-coverage map comprising the methylation status of individual cytosines throughout the inbred maize genome. Using this resource, we demonstrate that methylome sequencing of recombinant inbred lines at much lower coverage is sufficient to detect widespread paramutation in the maize genome. Future studies using low-coverage methylome sequencing can take advantage of this resource to determine the impact of differentially methylated regions on gene expression, chromosome biology, and transgenerational inheritance. For example, this will allow breeders to determine the contribution of cytosine methylation to phenotypic variation among elite inbreds and hybrids, artificially induced chromosomal variants (such as doubled haploids), and clonally micropropagated strains, which are subject to such epigenetic variation (Richards 2011). Thus, breeders could deploy a form of “epigenomic selection,” analogous to genomic selection, by which individuals with desired epigenomic patterns could be retained in breeding programs.