Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions.

Bacterial restriction endonucleases containing the dinucleotide CpG in their cleavage sequences were used to compare the methylation patterns of primarily repeated DNA sequences in (1) bovine somatic cell native DNAs vs bovine sperm cell native DNA and (2) native vs renatured bovine liver and sperm cell DNAs. The restriction patterns of sperm native DNA differ markedly from those of somatic cell native DNAs when using Hpa II, Hha I, and Ava I but not when using the enzymes Eco RI and Msp I. Digestion patterns of germ cell renatured DNA differed significantly from those of germ cell native DNA when using Hpa II but not when using Msp I or Eco RI. The results may not be due to artifacts of renaturation of the DNAs. The results are consistent with the concept that germ cell DNA may be strand asymmetrically hemimethylated. The data also suggest that methylation of the 5'-cytosine in the sequence CCGG renders this site insensitive to cleavage by Msp I.     

Analysis of the methylation pattern of c-Ha-ras oncogene in human prostatic cancer

To determine the methylation pattern of c-Ha-ras oncogene in prostatic tissue and to identify possible changes of methylation associated with cancer, high molecular weight DNA was extracted from 7 normal and 6 carcinomatous human prostates. Analysis of the samples was performed by cleaving DNA with the restriction endonucleases Msp I, Hpa II and Cfo I, and by Southern hybridizing the DNA digests with the 32P-labelled c-Ha-ras (pT24-C3) probe. Several discrete fragments were obtained with Hpa II and Cfo I digestion while the Msp I pattern showed fewer and smaller bands. Therefore, c-Ha-ras appears to be partially methylated. While a considerable polymorphism of the sequence 5'-CCGG-3' was observed at several Msp I sites in all cases, no significant differences could be evidenced in the methylation patterns of normal and neoplastic prostatic DNA samples extracted and purified from each patient.     

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode,Heterodera glycines

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.     

Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promotor and first exon is linked to expression of the gene.

The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene.     

运用MSAP技术测定谷子基因组DNA胞嘧啶甲基化水平

DNA甲基化是真核生物一种重要的表观修饰形式。为了探讨谷子基因组DNA胞嘧啶甲基化的水平和模式,以谷子Setaria italica的两个品种朝谷58号和豫谷1号为实验材料,利用Eco RⅠ和HpaⅡ/MspⅠ双酶切建立适合于谷子基因组的甲基化敏感扩增多态性(MSAP)分析体系。结果表明,从100对MSAP选扩引物中,筛选出32对MSAP引物组合,在朝谷58号和豫谷1号中分别扩增产生1 615、1 482条清晰可辨且可重复的DNA条带,其中包括3种类型的甲基化条带,朝谷58号和豫谷1号的基因组中CCGG序列胞嘧啶甲基化水平分别为6.93%和8.77%。这种谷子不同品种间甲基化水平和分布位点的差异为从表观遗传学的角度培育新品种提供了初步的理论依据和参考。     

A novel type of secondary modification of two CCGG residues in the human gamma delta beta-globin gene locus

The majority of the CCGG residues in the human gamma delta beta-globin gene locus are cleaved by Msp I, irrespective of the tissue of origin of the DNA, although these sites show differential sensitivity to Hap II as a result of methylation of the internal C residue of the cleavage site (ref 6). Two CCGG sites, at homologous positions 54 nucleotides in front of the G gamma- and A gamma-globin genes respectively, are not cleaved by Msp I in DNA from several human tissues, although DNA from placenta, foetal liver and from some established cell lines is cut at these sites. We have cloned the A gamma-globin gene from foetal blood DNA where the relevant CCGG site is not cut by Msp I. After cloning, the CCGG site can be cut by Msp I. The failure to cleave at this CCGG site in foetal blood DNA therefore, is not the result of a change in the DNA sequence of the cleavage site. Most likely the external C residue and perhaps both C residues are blocked by methylation at these two specific sites.     

Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.     

Restriction enzyme digestion of hemimethylated DNA.

Hemimethylated duplex DNA of the bacteriophage phi X 174 was synthesized using primed repair synthesis is in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded phi X DNA was used as a template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5'-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCTAGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.     

Decreased methylation of the major mouse long interspersed repeated DNA during aging and in myeloma cells

Sequences of DNA that hybridize on Southern blots with cloned EcoR1 1.3 kb (ER1) of long interspersed repeated sequence (L1Md) of mouse have been examined in genomic DNA of neonatal mice, livers and brains of adult mice (3, 10, 27, and 30 mo old), and the solid myeloma tumor MOPC-315. The isoschizomers Hpa II (CCGG or mCCGG) and Msp I (CCGG or CmCGG) were used to assess methylation. We found that the L1Md sequence is fully methylated in young animals but demethylated in myeloma. Demethylation of L1Md sequence also occurred in aged animals. By scanning the autoradiogram, we found that approximately 8% of the 10(4)-10(5) copies have been demethylated in 27-mo-old liver.     

The methylation status of DNA derived from potato plants recovered from slow growth

The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.     

DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis.

BACKGROUND: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. METHODS: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. RESULTS: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. CONCLUSION: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology.     

Variable methylation of the ribosomal RNA genes of the rat.

Both the pattern and level of rRNA gene methylation vary in the rat. This variation reflects stages in the maturation process and perhaps the level of gene expression in different tissues. We studied methylation at a common site, the inner cytosine of the sequence CCGG, by hybridizing 32P-rRNA to DNA digests obtained with endonuclease Msp I (which cleaves CCGG and CMCGG) and its isochizomer, HpaII (which cleaves only CCGG). In the liver, the changing pattern of rRNA gene methylation reflected the late stages of development: the rRNA genes were mostly unmethylated at 14 days gestation; by 18 days gestation, about 30% of them were methylated, and this level persisted into adulthood. In 18-day DNA, the methylation was uniform, but in adult DNA, the methylation pattern was discontinuous, because otherwise methylated genes contained a demethylated region. Similar developmental changes were observed in brain DNA. In a tissue culture cell line, the change from the continuous to the discontinuous pattern of the methylation could be induced by transformation with Kirsten sarcoma virus. And, in adult tissues, the lowest level of rRNA gene methylation was found in rapidly growing jejunal epithelium, and the highest level, in non-growing spermatozoa.     

EcoR II is an Unreliable Enzyme for Studies of CpNpG Methylation in shape Arabidopsis thaliana

Methylation patterns from cold-inducible and embryo-specific Arabidopsis thaliana gene promoter regions were investigated. Pairs of restriction enzymes sensitive and insensitive to methylation in the same recognition sequence were used to digest genomic DNA, and the methylation status was visualized by Southern hybridization. The pair BstN I/ EcoR II should detect CpNpG methylation due to the sensitivity of EcoR II to 5-methylcytosine in the second position in the recognition sequence (5-CC(A/T)GG-3). The pair Msp I/Hpa II will detect both CpNpG methylation and CpG methylation, since Msp I does not digest the recognition sequence (5-CCGG-3) when the first C residue is methylated, while Hpa II restriction is inhibited by methylation of either of the two C residues. EcoR II digestion studies suggested CpNpG methylation in all genes tested and demethylation after cold stress in all genes (including two control embryo-specific Lea genes not induced by low temperature). Control experiments indicated an unexpected pattern of methylation and low temperature demethylation in chloroplast genes. Additional control experiments, using the methylation sensitive enzyme, ScrF I (recognizing the sequence 5-CCNGG-3), disproved the presence of 5-methylcytosine in common sites not digested by EcoR II. (CpNpG-methylation was revealed in one ScrF I site in one gene and in Msp I/Hpa II sites in two genes. CpG methylation was not found in any gene tested.) Our study indicates that results obtained using EcoR II for DNA methylation studies should be interpreted with caution. The peculiarities of the EcoR II enzyme are further discussed.     

大鼠衰老过程中脑细胞DNA与c－Ha－ras原癌基因的甲基化

用ＭｓｐⅠ／ＨｐａⅡ酶解电泳法和高效液相色谱（ＨＰＬＣ）两种方法进行比较,研究了不同年龄大鼠的肝、脑细胞基因组ＤＮＡ的甲基化程度。从酶解电泳图谱可观察到,肝、脑细胞基因组ＤＮＡ甲基化在青年鼠和老年鼠之间没有差异。但用具有高分辨率的高效液相色谱测量ＤＮＡ中５－ｍＣ的含量时发现,老年鼠脑细胞ＤＮＡ甲基化程度较大年鼠的下降６２％,而肝细胞ＤＮＡ甲基化程度在老年鼠与青年鼠之间并没有显著差异。这些结果提示：（１）用常规的酶解电泳法所分析的ＤＮＡ甲基化结果并不能反映整个基因组ＤＮＡ甲基化的水平。（２）衰老过程中,不同组织ＤＮＡ甲基化的改变存在差异,引起这种差异的原因可能与组织的增殖和分化程度有关。进一步分析脑细胞原癌基因ｃ－Ｈａ－ｒａｓ的甲基化水平,无论ＭｓｐⅠ酶切图谱,还是ＨｐａⅡ酶切图谱均可观察到分子大小为１９ｋｂ、７．５ｋｂ、１．３ｋｂ、０．９ｋｂ的四条阳性带,说明该基因未发生甲基化,且与年龄无关。     

Flowering induced by 5-azacytidine, a DNA demethylating reagent in a short-day plant, Perilla frutescens var. crispa

Treatment with 5-azacytidine, a DNA demethylating reagent, induced flowering in Perilla frutescens (L.) Britton var. crispa (Thunb. ex Murray) Decne. ex L. H. Bailey, an absolute short-day plant under long days. The 5-azacytidine treatment induced slight suppression of vegetative growth but had no obvious effect on any other phenotypes. The Southern hybridization analysis of the genomic DNA isolated from the leaves of 5-azacytidine-treated plants and digested with restriction enzyme, methylation-insensitive Msp I or methylation-sensitive Hpa II with P. frutescens 25S-18S rDNA intergenic spacer probe indicated that the 5-azacytidine treatment caused demethylation of the genomic DNA. The 5-azacytidine-induced flowering was delayed as compared with the short day-induced flowering. Flowers were formed even at the lower nodes which had not been directly treated with 5-azacytidine. The results suggest that DNA demethylation induced flowering by inducing the production of a transmissible flowering stimulus in P. frutescens .     

Methylated and unmethylated DNA compartments in the sea urchin genome.

Sea urchin (Echinus esculentus) DNA has been separated into high and low molecular weight fractions by digestion with the mCpG-sensitive restriction endonucleases Hpa II, Hha I and Ava I. The separation was due to differences in methylation at the recognition sequences for these enzymes because an mCpG-insensitive isoschizomer of Hpa II (Msp I) digested Hpa II-resistant DNA to low molecular weight, showing that many Hpa II sites were in fact present in this fraction; and because 3H-methyl methionine administered to embryos was incorporated into the high molecular weight Hpa II-, Hha I- and Ava I-resistant fraction, but not significantly into the low molecular weight fraction. The fraction resistant to Hpa II, Hha I and Ava I amounted to about 40% of the total DNA. It consisted of long sequence tracts between 15 and well over 50 kg in length, in which many sites for each of these enzymes were methylated consecutively. The remaining 60% of the genome, (m-), was not significantly methylated. Methylated and unmethylated fractions were considered to be subfractions of the genome because enriched unique sequences from one fraction cross-reassociated poorly with the other fraction and specific sequences were found in either (m+) or (m-) but not in both (see below). Similar (m+) and (m-) compartments were found in embryos, germ cells and adult somatic tissues. Furthermor, we found no evidence for changes in the sequence composition of (m+) or (m-) between sperm, embryo or intestine DNAs, although low levels of exchange would not have been detected. Using cloned Echinus histone DNA, heterologous 5S DNA and ribosomal DNA probes, we have found that each of these gene families belongs to the unmethylated DNA compartment in all the tissues examined. In particular, there was no detectable methylation of histone DNA either in early embryos, which are thought to be actively transcribing the bulk of histone genes, or in sperm and gastrulae, in which most histone genes are not being transcribed. In contrast to these gene families, sequences complementary to an internally repetitious Echinus DNA clone were found primarily in the methylated DNA compartment.     

Extensive methylation of chloroplast DNA by a nuclear gene mutation does not affect chloroplast gene transmission in chlamydomonas

Based on analysis by high pressure liquid chromatography, greater than 35% of the cytosine residues in chloroplast DNA of vegetative cells were found to be methylated constitutively in the nuclear gene mutation (me-1) of Chlamydomonas reinhardtii, which has an otherwise wild-type phenotype. Digestion of chloroplast DNA from vegetative cells and gametes of this mutant with restriction endonucleases Hpa II and Msp I reveals that in the 5′CCGG3′ sequence, CpG is methylated extensively, whereas CpC is only methylated occasionally. Hae III (5′GGCC3′) digestion of the mutant chloroplast DNA also shows extensive methylation of the GpC sequence. In contrast to the results of Sager and colleagues, which show a correlation between methylation of chloroplast DNA and transmission of chloroplast genes in crosses, our results with crosses of the me-1 mutant suggest that extensive chloroplast DNA methylation may be insufficient to account for the pattern of inheritance of chloroplast genes in Chlamydomonas.