In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.     

DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes.

The restriction enzymes HhaI and HpaII, whose activity is inhibited by cytosine methylation within their recognition sites, have been utilised as probes to study methylation in the vicinity of the ovalbumin gene in DNA from various chicken tissues. This was complemented by a preliminary study of methylation in the regions of chicken ovotransferrin (conalbumin), ovomucoid and beta-globin genes. From our data we conclude that HaI or HpaII sites can be divided in 3 classes according to their pattern of methylation in different tissues. In the first class of sites (mV class) the extent of methylation varies in different tissues. The patterns obtained show that methylation at the sites located within and around the 3 genes which code for egg white proteins is in general lowest in oviduct of laying hen, where these genes are expressed. However some sites are not methylated (m- class) and others are 95 to 100% resistant (m+ class) to digestion by HhaI or HpaII in the DNAs of all the tissues which were tested. Our study has also revealed a remarkable number of allelic variants for the presence of HhaI or HpaII sites in the region of the ovalbumin gene.     

DNA methylation patterns in the 5S DNAs of Xenopus laevis

The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.     

DNA methylation diminishes bleomycin-mediated strand scission

Three DNA duplexes differing substantially in sequence were derived from pBR322 plasmid DNA and supercoiled SV40 DNA by digestion with appropriate restriction endonucleases. Following treatment with the restriction methylase HhaI (recognition sequence: GCGC) or HhaI and HpaII (CCGG), the unmethylated and methylated DNAs were compared as substrates for the antitumor agent bleomycin. Bleomycin-mediated strand scission was shown to diminish substantially at a number of sites in proximity to the methylated cytidine moieties, especially where multiple sites had been methylated within a DNA segment of limited size. Detailed analysis of the DNA substrates revealed that both strands of DNA within a methylated region became more refractory to cleavage by bleomycin and that the protective effect could extend as many as 14 base pairs in proximity to the 5-methylcytidine moieties. Among the methylated DNA segments that became more resistant to bleomycin cleavage was a HpaII site of SV40 DNA, methylation of which has previously been shown to diminish the synthesis of the major late viral capsid protein following microinjection into Xenopus laevis oocytes. Study of the cleavage reaction at varying salt levels suggested that diminished bleomycin strand scission may be due, at least in part, to local conformational changes of the DNA to Z form (or other non-B-form structures). The results are generally consistent with the hypothesis that one mechanism for the expression of selective therapeutic action by certain DNA damaging agents could involve the recognition of specific methylation patterns.     

Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methylcytosine with MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases.

The cleavage specificity of R.Cfr9I was determined to be C decreases CCGGG whereas the methylation specificity of M.Cfr9I was C4mCCGGG. The action of MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligonucleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation, in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonucleases. The cleavage of methylated substrates with restriction endonucleases is discussed.     

Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. Here, predicted changes in restriction enzyme sites following reaction of genomic DNA with bisulfite and amplification of the product by the polymerase chain reaction (PCR) were used to assess the methylation of CpG sites. This procedure differs from conventional DNA methylation analysis by methylation-sensitive restriction enzymes because it does not rely on an absence of cleavage to detect methylated sites, the two strands of DNA produce different restriction enzyme sites and may be differentially analyzed, and closely related sequences may be separately analyzed by using specific PCR primers.     

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.     

The effects of 5-azacytidine on the expression of the rat growth hormone gene. Methylation modulates but does not control growth hormone gene activity

The role of DNA methylation in the expression of the rat growth hormone (rGH) gene was assessed by using a hypomethylating agent, 5-azacytidine, and the iso-schizomeric restriction enzymes MspI and HpaII. 5-Azacytidine increased rGH mRNA 3-8-fold in GH3D6 cells, a subclone of rat pituitary tumor cell lines that expresses one-tenth to one-fifteenth the GH expressed by two other clones, GH3 and GC. The effect was also detected at the level of pre-mRNA. The effect was independent of glucocorticoids and thyroid hormones and was found to be inheritable. The DNA methylation pattern generated by the isoschizomeric restriction enzymes indicated that the HpaII sites in the rGH gene were mostly methylated in GH3D6 cells but mostly unmethylated in GC cells. After treatment with 5-azacytidine, about 22% of these HpaII sites in GH3D6 cells became unmethylated. Thus, DNA methylation correlates inversely with the expression of the rGH gene in these cell lines. However, three other observations indicate that factors in addition to DNA methylation control rGH expression. First, in GC cells, even though most of the HpaII sites are unmethylated, the gene is not fully expressed. Second, in rat hepatoma cells, which do not express GH at all, the GH gene is less methylated than that in GH3D6 cells. Third, within the sensitivities of the assay methods, 5-azacytidine has no effect on the GH gene when it is completely silent. Taken together, the findings indicate that DNA methylation modulates but does not control GH gene expression. It is tempting to speculate that DNA methylation can influence expression only when the gene is committed to express.     

Inhibition of rat growth hormone promoter activity by site-specific DNA methylation

The effect of methylation on rat growth hormone (rGH) promoter activity was determined in GH3 cells by measuring rGH-Neo and rGH-CAT fusion gene expression with or without prior in vitro treatment with the site-specific DNA methyltransferases, M-BsuE and M-HhaI. To assay for rGH-promoter-specific effects of DNA methylation, RSV-Neo and RSV-CAT activities with or without M-BsuE, M-HhaI and M-HpaII treatment were measured in parallel cultures of GH3 cells. GH1-Neo and RSV-Neo fusion gene expression was inhibited by in vitro methylation from 44 to 83% as measured by the number of Geneticin-resistant GH3 cell colonies. Methylation of the GH1 promoter by M-BsuE exhibited some selective inhibition of Neo expression as determined by colony numbers, although extensive methylation of non-promoter DNA in GH1-Neo and RSV-Neo by M-HhaI and M-HpaII also inhibited Neo expression. Southern blot analysis of genomic DNA isolated from the Geneticin-resistant GH3 cells indicated that Geneticin-resistance was accompanied by demethylation of the BsuE (ThaI) sites in stably incorporated GH1-Neo DNA but not RSV-Neo DNA. Transient expression of the CAT gene in GH3 cells was selectively inhibited by 60% upon methylation of two BsuE (ThaI) sites in the GH1 promoter of GH1-CAT by M-BsuE. The data demonstrate, for the first time, to our knowledge, a direct effect of DNA methylation on the activity of the rat growth hormone promoter.     

Uneven distribution of methylation sites within the human papillomavirus la genome: possible relevance to viral gene expression.

A homogeneous preparation of human papillomavirus type 1a (HPV-1a) DNA resisted complete cleavage by the methylation-sensitive restriction endonuclease HhaI. Ten fragments additional to those predicted from the known HPV-1a DNA sequence were resolved by agarose gel electrophoresis of the HhaI-cleaved viral DNA. By determining the composite structures of the additional HhaI viral fragments, evidence was found for part-methylation of six of the thirteen HhaI sites. Two of the modified HhaI sites were localized to the 3'-end of the putative early gene region. The other four modified Hha-I sites were situated within the L1 open reading frame of the putative late gene region. Ten successive restriction endonuclease sites occurring close to and within an area of high CG density which surrounds the 5' end of the putative early gene region, were not modified detectably. The possible relevance of DNA methylation to the control of HPV-1a gene expression in epidermal cells is discussed.     

Evidence that Escherichia coli virus T1 induces a DNA methyltransferase

DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated. Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E. coli DNA adenine methylase (dam methylase) are methylated. The same methylation pattern was found for virus T1 DNA grown on an E. coli dam host, indicating a T1-specific DNA methyltransferase.     

MethylScreen: DNA methylation density monitoring using quantitative PCR

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.     

Methylation of somatic and sperm DNA in the homosporous fern Ceratopteris richardii

Plants, in general, have a high proportion of their CpG and CpNpG nucleotide motifs modified with 5-methylcytosine (5mC). Developmental changes in the proportion of 5mC are evident in mammals, particularly during gametogenesis and embryogenesis, but little information is available from flowering plants due to the intimate association of gametes with sporophytic tissues. In ferns, sperm are uninucleate and free-swimming and thus are easily isolated. We have examined 5mC in DNA isolated from fern sperm and other tissues with methylation-sensitive and -insensitive restriction enzyme isoschizomers, Southern blots probed with chloroplast and nuclear ribosomal RNA genes and end-labeled restriction fragments. We conclude that fern sperm DNA is methylated to a similar or greater degree than DNA isolated from either sporophytes or gametophytes.     

DNA methylation impacts the cleavage activity of Chlorella virus topoisomerase II

Topoisomerase II from Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) displays an extraordinarily high in vitro DNA cleavage activity that is 30-50 times higher than that of human topoisomerase IIalpha. This remarkable scission activity may reflect a unique role played by the type II enzyme during the viral life cycle that extends beyond the normal control of DNA topology. Alternatively, but not mutually exclusively, it may reflect an adaptation to some aspect of the viral environment that differs from the in vitro conditions. To this point, the genomes of many chlorella viruses contain high levels of N6-methyladenine (6mA) and 5-methylcytosine (5mC), but the DNA employed in vitro is unmodified. Therefore, to determine whether methylation impacts the ability of chlorella virus topoisomerase II to cleave DNA, the effects of 6mA and 5mC on the PBCV-1 and CVM-1 enzymes were examined. Results indicate that 6mA strongly inhibits DNA scission mediated by both enzymes, while 5mC has relatively little effect. At levels of 6mA and 5mC methylation comparable to those found in the CVM-1 genome (10% 6mA and 42% 5mC), the level of DNA cleavage decreased approximately 4-fold. As determined using a novel rapid quench pre-equilibrium DNA cleavage system in conjunction with oligonucleotide binding and ligation assays, this decrease appears to be caused primarily by a slower forward rate of DNA scission. These findings suggest that the high DNA cleavage activity of chlorella virus topoisomerase II on unmodified nucleic acid substrates may reflect, at least in part, an adaptation to act on methylated genomic DNA.