小鼠四倍体早期胚胎基因组甲基化模式

利用小鼠抗5-甲基胞嘧啶(5MeC)单克隆抗体检测了体外培养小鼠四倍体早期胚胎的基因组甲基化模式。结果表明: 利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程, 在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样, 呈现高度甲基化状态; 在细胞核开始融合的时候, 甲基化水平急速下降, 在细胞核完全融合的时候甲基化水平达到最低点; 随着胚胎继续分裂, 胚胎甲基化水平逐渐增加, 在桑葚胚期甲基化水平最高; 但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别, 这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。因此, 小鼠体外培养四倍体胚胎的甲基化模式是不正常的, 这可能是四倍体小鼠难以发育到妊娠足月的原因之一。这是对小鼠四倍体早期胚胎基因组甲基化模式的首次报道。     

Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.     

Regulation and function of mammalian DNA methylation patterns: a genomic perspective

Mammalian DNA methyltransferases (DNMTs) establish and maintain genomic DNA methylation patterns that are required for proper epigenetic regulation of gene expression and maintenance of genome stability during normal development. Aberrant DNA methylation patterns are implicated in a variety of pathological conditions including cancer and neurological disorders. Rapid advances in genomic technologies have allowed the generation of high resolution whole-genome views of DNA methylation and DNA methyltransferase occupancy in pluripotent stem cells and differentiated somatic cells. Furthermore, recent identification of oxidation derivatives of cytosine methylation in mammalian DNA raises the possibility that DNA methylation patterns are more dynamic than previously anticipated. Here, we review the recent progress in our understanding of the genomic function and regulatory mechanisms of mammalian DNA methylation.     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

DNA methylation in mouse gametogenesis

DNA methylation is involved in many biological processes and is particularly important for both development and germ cell differentiation. Several waves of demethylation and de novo methylation occur during both male and female germ line development. This has been found at both the gene and all genome levels, but there is no demonstrated correlation between them. During the postnatal germ line development of spermatogenesis, we found very complex and drastic DNA methylation changes that we could correlate with chromatin structure changes. Thus, detailed studies focused on localization and expression pattern of the chromatin proteins involved in both DNA methylation, histone tails modification, condensin and cohesin complex formation, should help to gain insights into the mechanisms at the origin of the deep changes occurring during this particular period.     

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.     

Different genomic DNA methylation patterns between male and female adults of white-backed planthoppers Sogatella furcifera

DNA methylation plays a key role in gene regulation and phenotype variation in many organisms. The aim of this study was to survey the frequency and variation of cytosine methylation at CCGG sequences in adult male and female planthoppers Sogatella furcifera, a major rice pest in Asia, and to determine the occurrence of methylation changes associated with sexual dimorphism using methylation-sensitive amplification polymorphism. 1131 DNA fragments including CCGG sites were amplified using 36 pairs of selective primers: about 191 methylated bands were identified. In male planthoppers, we got a total of 581 bands, including 40 fully-methylated bands, 65 hemi-methylated bands and 476 none-methylated bands, so the fully-methylated ratio, hemi-methylated ratio and total methylated ratio were 6.88%, 11.19% and 18.07%, respectively. In the female planthopper, there were a total of 550 bands, including 44 fully-methylated bands, 42 hemi-methylated bands and 464 none-methylated bands. The fully-methylated ratio was 7.64% in female planthoppers, which was slightly higher than in the male planthoppers, however, the hemi-methylated ratio was lower (8.00%) in the female compared with the male planthopper. Altogether, 46 DNA bands displayed variable cytosine methylation patterns between male and female samples: 20 of them occurred only in male samples and 26 only in female samples. Thus, the genome methylation patterns are different between male and female adults. The results suggest that DNA methylation might be related to sexual differentiation and development in S. furcifera.     

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.     

Genome-wide analysis of DNA methylation patterns

Cytosine methylation is the most common covalent modification of DNA in eukaryotes. DNA methylation has an important role in many aspects of biology, including development and disease. Methylation can be detected using bisulfite conversion, methylation-sensitive restriction enzymes, methyl-binding proteins and anti-methylcytosine antibodies. Combining these techniques with DNA microarrays and high-throughput sequencing has made the mapping of DNA methylation feasible on a genome-wide scale. Here we discuss recent developments and future directions for identifying and mapping methylation, in an effort to help colleagues to identify the approaches that best serve their research interests.     

Tissue-specific patterns of allelically-skewed DNA methylation

While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ～220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood.     

Capillary electrophoretic analysis of genomic DNA methylation levels

Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene- specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established a method for accurate high-throughput quantification of 5-methylcytosine in genomic DNA. Our protocol requires a small amount (<1 µg) of DNA that is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then derivatized with a fluorescent marker and separated by capillary electrophoresis. After calibration of the method, we have determined cytosine methylation levels from tumor samples of 81 patients that had been diagnosed with chronic lymphocytic leukemia (CLL). These patients showed a high variability in their methylation levels with a general trend towards hypomethylation. Because of its high accuracy and throughput our method will be useful in determining the role of genomic DNA methylation levels in tumorigenesis.     

Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase 1

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.     

Alterations of DNA methylation patterns in germ cells and Sertoli cells from developing mouse testis

In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G(0)-G(1) phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function.     

Analysis of genomic DNA methylation patterns in regenerated and control plants of rye (Secale cereale L.)

Rye (Secale cereale L.) is a species that has shown high rates of somaclonal variation when plants obtained by in vitro culture were analysed using different techniques. In this study, using methylation-sensitive amplified polymorphism (MSAP) markers, we analysed the cytosine methylation status at genomic level of regenerated plants of rye that were obtained by somatic embryogenesis. Such plants were originated from three different cell lines and the results were compared with the data obtained from the control plants grown from seeds of the same cultivar and lot. A similar total number of MSAP markers was observed in the regenerated (937) and control plants (1,022), while the mean number detected per plant was significantly higher in regenerated (554.43) than in control plants (356.00). The analysis indicated conservation of the number of partially-methylated CCGG/GGCC sites for all type of plants. However the mean number of non-methylated sites was near twofold in the regenerated plants (442.48) than in control plants (248.19). Methylation changes have been detected in all the regenerated plants when compared within cell lines, with an average frequency of 9.01 % of the detected markers. We also observed that regenerated plants from one or several cell lines shared methylation changes at the same locus pointing to a non-random behaviour of the changes in genomic methylation.     

Genome-wide DNA methylation studies suggest distinct DNA methylation patterns in pediatric embryonal and alveolar rhabdomyosarcomas

Rhabdomyosarcoma is the most common soft-tissue sarcoma in children. While cytogenetic abnormalities have been well characterized in this disease, aberrant epigenetic events such as DNA hypermethylation have not been described in genome-wide studies. We have analyzed the methylation status of 25,500 promoters in normal skeletal muscle, and in cell lines and tumor samples of embryonal and alveolar rhabdomyosarcoma from pediatric patients. We identified over 1,900 CpG islands that are hypermethylated in rhabdomyosarcomas relative to skeletal muscle. Genes involved in tissue development, differentiation, and oncogenesis such as DNAJA4, HES5, IRX1, BMP8A, GATA4, GATA6, ALX3, and P4HTM were hypermethylated in both RMS cell lines and primary samples, implicating aberrant DNA methylation in the pathogenesis of rhabdomyosarcoma. Furthermore, cluster analysis revealed embryonal and alveolar subtypes had distinct DNA methylation patterns, with the alveolar subtype being enriched in DNA hypermethylation of polycomb target genes. These results suggest that DNA methylation signatures may aid in the diagnosis and risk stratification of pediatric rhabdomyosarcoma and help identify new targets for therapy.     

Genome-wide DNA methylation studies suggest distinct DNA methylation patterns in pediatric embryonal and alveolar rhabdomyosarcomas

Rhabdomyosarcoma is the most common soft-tissue sarcoma in children. While cytogenetic abnormalities have been well characterized in this disease, aberrant epigenetic events such as DNA hypermethylation have not been described in genome-wide studies. We have analyzed the methylation status of 25,500 promoters in normal skeletal muscle, and in cell lines and tumor samples of embryonal and alveolar rhabdomyosarcoma from pediatric patients. We identified over 1,900 CpG islands that are hypermethylated in rhabdomyosarcomas relative to skeletal muscle. Genes involved in tissue development, differentiation, and oncogenesis such as DNAJA4, HES5, IRX1, BMP8A, GATA4, GATA6, ALX3, and P4HTM were hypermethylated in both RMS cell lines and primary samples, implicating aberrant DNA methylation in the pathogenesis of rhabdomyosarcoma. Furthermore, cluster analysis revealed embryonal and alveolar subtypes had distinct DNA methylation patterns, with the alveolar subtype being enriched in DNA hypermethylation of polycomb target genes. These results suggest that DNA methylation signatures may aid in the diagnosis and risk stratification of pediatric rhabdomyosarcoma and help identify new targets for therapy.