Liver-specific expression of a Qa-encoded class I gene is associated with DNA hypomethylation.

DNA methylation of two murine major histocompatibility complex (H-2) class I genes was examined in hybridizations to MspI and HpaII chromosomal DNA restriction digests. Q10, which exhibits liver-specific expression, and H-2Kb, a transplantation antigen gene, were examined in liver, spleen, thymus, and cell-line DNAs. Unmethylated Q10 gene sequences were detected only in the liver, whereas the H-2Kb gene was unmethylated in all tissues examined.     

Alterations in c-abl gene methylation in cells transformed by phagocyte-generated oxidants

DNA from 10T1/2 cells transformed by activated neutrophils was analyzed for restriction length polymorphisms (RFLPs) in cellular homologues of retroviral oncogenes, and consistent RFLPs were found in MspI sites of the c-abl gene of all PMN-transformed cell lines. MspI digests probed with c-myc, v-Ki-ras, v-Ha-ras or v-mos showed no RFLPs, and none were observed in EcoRI, PstI, HindIII, BamHI, SmaI, Sau3a, MboI, HhaI, or TaqI digests probed with v-abl. Analysis of HpaII digests supports the conclusion that c-abl RFLPs result from differential methylation of the CCGG HpaII/MspI recognition sequence. MspI RFLPs in the c-abl gene may provide markers for oxidant-related genetic injury.     

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.     

Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum

Prominent features of the cytosine methylation pattern of the Pisum sativum nuclear ribosomal RNA genes have been defined. Cytosine methylation within the C-C-G-G sequence was studied using the restriction enzymes HpaII and MspI and gel blot hybridizations of the restriction digests. The extent to which particular features of the methylation pattern change during seedling development has also been determined. Total cellular DNA, purified from defined sections of pea seedlings grown under different lighting conditions, was analyzed with DNA hybridization probes derived from different portions of a cloned member of the nuclear rRNA gene family. By use of an indirect end-labeling technique, a map of 23 cleavable HpaII and/or MspI sites in genomic rDNA was constructed. The map covers about 90% of the rDNA repeat including the entire non-transcribed spacer region and most of the rRNA coding sequences. One notable feature of the map is that the most prominent HpaII site, located about 800 base-pairs upstream from the 5' end of the mature 18 S rRNA, is cleaved only in one of the two most abundant rDNA length variants (the short variant). With a gel blot assay specific for cleavage at this site, we estimated the HpaII sensitivity of DNA preparations from several stages of pea seedling development. We find that, while methylation is generally low in young seedlings, DNA obtained from the apical buds of pea seedlings is highly methylated. Further, the methylation level of rDNA within the pea bud decreases as the buds are allowed to develop under continuous white light. Our data, taken together with published studies on pea seedling development, indicate that cytosine methylation levels may be related to the regulated expression of the nuclear rRNA genes in pea.     

Maintenance of milk protein gene expression in a subpopulation of 7,12-dimethylbenz (a) anthracene-induced rat mammary carcinoma cells grown on attached collagen gels

Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels. Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin (5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs. Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels. Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone) in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen gels. This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship, HD 06157.     

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.     

Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes

We have analyzed methylation of the rat albumin and alpha-fetoprotein (AFP) genes by hydridizing labeled cDNA clones to HpaII and MspI digests of DNA from different stages of development. These CCGG-cutting enzymes distinguish 5-methylcystosine in mCCGG (sensitive to HpaII) and CmCGG (sensitive to MspI). In the liver, the albumin gene is heavily methylated at 18 days gestation and uniformly demethylated in the adult. The AFP gene is also heavily methylated at 18 days gestation, and develops demethylated regions at the 3' half of the gene in the adult. These methylation changes are not observed in other embryonic or adult tissues. We also evaluated expression of these genes by measuring their corresponding mRNAs. The albumin gene is actively transcribed in 18-day fetal liver, when it is heavily methylated, as well as in adult liver, when it is unmethylated. In contrast, the AFP gene is transcribed only in fetal liver, even though it is less methylated in adult liver. These findings suggest that specific methylation changes are associated with changes in gene expression, but that this association is not adequately described by the simple hypothesis that methylation turns genes off.     

Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII

The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

MspI, an isoschizomer of hpaII which cleaves both unmethylated and methylated hpaII sites.

The cleavage of DNA by restriction endonucleases HpaII and HapII is prevented by the presence of a 5-methyl group at the internal C residue of its recognition sequence CCGG. MspI, an isoschizomer of HpaII available from New England Biolabs, cleaves DNA irrespective of the presence of a methyl group at this position. This enzyme cleaves DNA from Haemophilus parainfluenzae and Haemophilus aphrophilus readily while HpaII and HapII cannot degrade these DNAs. Practically all HpaII sites in mammalian sperm DNA are also protected by methylation at the internal C position since HpaII and HapII barely cleave this DNA (average molecular weight 40 kb). MspI, however, cleaves the DNA to an average size of about 5 kb.     

Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction

The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.     

Stage-specific DNA methylation in a fungal plant pathogen.

Significantly more 5-methylcytosine residues were found in the DNA from the dormant sclerotia of Phymatotrichum omnivorum than in the DNA from the metabolically active mycelia of the fungus, as shown by high-pressure liquid chromatography of acid-hydrolyzed DNA digests and by restriction of the DNA with the isoschizomers MspI and HpaII. N6-Methyladenine was not detected in GATC sequences in the DNA isolated from either stage.     

Specifically unmethylated cytidylic-guanylate sites in Herpesvirus saimiri DNA in tumor cells.

The restriction endonucleases MspI (CCGG), HpaII (CCGG), FnuDII (CGCG), and HaeIII (GGCC) were used to study the methylation of Herpesvirus saimiri DNA in tumor cells taken directly from tumor-bearing animals. No evidence was found for methylation of the 5' terminal C in the sequence CCGG or of the internal C in the sequence GGCC, but extensive methylation of CG was detected. Fifteen HpaII sites and 17 FnuDII sites were detected in the unique DNA region of the H. saimiri strain used. Twenty-eight of the 32 sites were methylated in greater than 90% of the viral DNA molecules in tumor cells, but the remaining 4 sites were unmethylated in greater than 95% of the viral DNA molecules in tumor cells. The locations of the four specifically unmethylated sites were mapped and appeared to be identical in the four different induced leukemias examined (one owl monkey and three white-lipped marmosets). The nonproducer 1670 tumor cell line, in continuous passage for over 7 years, contained four similar specifically unmethylated sites. Possibilities for the physiological significance of the unmethylated sites are discussed.     

Changes in methylation of tissue cultured soybean cells detected by digestion with the restriction enzymes HpaII and MspI

Each of the tandemly arranged 5S RNA genes of soybean contain two CCGG sites which, if unmethylated, can be digested by both MspI and HpaII. Methylation of the internal cytosine (CmeCGG) prevents digestion by HpaII but allows digestions by MspI.Suspension cultures were prepared from soybean plants and the DNA from these cultures was examined for the susceptibility of 5S RNA genes to digestion by MspI and HpaII. 5S genes from DNA extracted from intact plants can be partially digested with MspI but not at all by HpaII. In contrast, shortly after cells were cultured the 5S RNA could be hydrolyzed by both HpaII and MspI. After prolonged cell culture, the 5S genes from some cell lines were found to have become partially or even completely resistant to HpaII digestion. The results suggest that lack of methylation can occur when cells are cultured and that such methylation may play a role in the heritable changes observed in cell culture.Research supported by Grant 01498 from the National Institutes of Environmental Health Sciences     

DNA methylation in mouse cells in culture as measured by restriction enzymes

Methylation of DNA in normal mouse cultured 3T3 cells and in their virally or chemically transformed derivatives was studied. DNA methylation was studied by restriction with HpaII, MspI, or HpaII plus MspI. DNA from the chemically transformed cells was cleaved about twice as often with HpaII than was the DNA of normal and virally transformed cells. Digests with MspI and HpaII plus MspI were identical in all cell lines studied. Densitometry of the restriction patterns allowed an estimate of total DNA methylation from the weight average lengths. The chemically transformed cell line showed 25% reduction in methylation compared to the other cell lines. Southern blot hybridization using satellite DNA showed that these sequences followed a pattern of modification similar to that of total DNA.     

In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.     

Methylation-sensitive restriction endonuclease digestion patterns revealed in Vicia faba L. chromosomes by in situ nick-translation

Restriction endonucleases sensitive to cytosine methylation (HpaII, MspI and HhaI) and 5-azacitidine were used to study the localization of target sequences in Vicia faba metaphase chromosomes by in situ digestion and radioactive or non-radioactive nick-translation. In control experiments, neither isolated DNA nor chromosomes in situ were digested by HpaII and MspI. Pretreatment with demethylating agent, 5-azacitidine resulted both in increased effectiveness of in situ digestion and nick-translation. In 5-azacitidine-treated material, negative bands in M chromosomes appeared. HhaI cleaved isolated DNA, digested it in situ and gave positive signals as a result of nick-translation procedure in metaphase chromosomes. In S chromosomes containing heterochromatin without target sequences for HpaII and MspI, negative bands were shown after nick-translation. Such heterochromatin contains FokI sequences and in situ nick-translation driven by that restriction enzyme resulted in positive bands.