Tissue- and cell-specific casein gene expression. II. Relationship to site-specific DNA methylation

The relationship between DNA methylation and the expression of the gamma- and beta-casein genes was investigated in both expressing and nonexpressing tissues and in isolated tumor cell subpopulations displaying differential casein gene expression. MspI/HpaII digestions of DNA isolated from liver, a totally nonexpressing tissue, indicated that specific sites of hypermethylation existed in these genes as compared to the DNA isolated from casein-producing lactating mammary gland. The positions of these sites were mapped in the gamma-casein gene by comparing total genomic DNA Southern blots to the restriction digests of several overlapping phage clones constituting the gamma-casein gene. In contrast, the methylation status of the HhaI sites in the gamma-casein gene was found to be invariant regardless of the expression status of the gene. The inverse correlation between the hypermethylation of certain MspI/HpaII restriction sites in the casein genes and their potential expressibility was further substantiated by studies in 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced mammary carcinomas, which have an attenuated casein gene expression, and in cell subpopulations isolated from the 7,12-dimethylbenz(a)-anthracene tumor which were either depleted or enriched in casein-producing cells. Analysis of total tumor DNAs indicated that the casein genes were hypermethylated at the same sites observed in liver. However, a very faint hybridization signal was observed in the HpaII digests, suggesting cell-specific methylation differences. We have confirmed the hypomethylation of at least two of these MspI/HpaII sites within the subpopulation containing the casein-producing cells at a level consistent with the relative enrichment in that fraction. These results demonstrate differential site-specific casein gene methylation not only between tissues but also between cell subpopulations within a single tissue.     

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.     

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.     

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.     

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.     

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.     

Cloning and characterization of the HpaII methylase gene.

The HpaII restriction-modification system from Haemophilus parainfluenzae recognizes the DNA sequence CCGG. The gene for the HpaII methylase has been cloned into E. coli and its nucleotide sequence has been determined. The DNA of the clones is fully protected against cleavage by the HpaII restriction enzyme in vitro, indicating that the methylase gene is active in E. coli. The clones were isolated in an McrA-strain of E. coli; attempts to isolate them in an McrA+ strain were unsuccessful. The clones do not express detectable HpaII restriction endonuclease activity, suggesting that either the endonuclease gene is not expressed well in E. coli, or that it is not present in its entirety in any of the clones that we have isolated. The derived amino acid sequence of the HpaII methylase shows overall similarity to other cytosine methylases. It bears a particularly close resemblance to the sequences of the HhaI, BsuFI and MspI methylases. When compared with three other methylases that recognize CCGG, the variable region of the HpaII methylase, which is believed to be responsible for sequence specific recognition, shows some similarity to the corresponding regions of the BsuFI and MspI methylases, but is rather dissimilar to that of the SPR methylase.     

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.     

The amino acid sequence of the CCGG recognizing DNA methyltransferase M.BsuFI: implications for the analysis of sequence recognition by cytosine DNA methyltransferases.

The Bacillus subtilis FI DNA methyltransferase (M.BsuFI) modifies the outer cytosine of the DNA sequence CCGG, causing resistance against R.BsuFI and R.MspI restriction. The M.BsuFI gene was cloned and expressed in B.subtilis and Escherichia coli. As derived from the nucleotide sequence, the M.BsuFI protein has 409 amino acids, corresponding to a molecular mass of 46,918 daltons. Including these data we have compared the nucleotide and amino acid sequences of different CCGG recognizing enzymes. These analyses showed that M.BsuFI is highly related to two other CCGG specific methyltransferases, M.MspI and M.HpaII, which were isolated from Gram-negative bacteria. Between M.BsuFI and M.MspI the sequence similarity is particularly significant in a region, which has been postulated to contain the target recognition domains (TRDs) of cytosine-specific DNA methyltransferases. Apparently M.BsuFI and M.MspI, derived from phylogenetic distant organisms, use highly conserved structural elements for the recognition of the CCGG target sequence. In contrast the very same region of M.HpaII is quite different from those of M.BsuFI and M.MspI. We attribute this difference to the different targeting of methylation within the sequence CCGG, where M.HpaII methylates the inner, M.BsuFI/M.MspI the outer cytosine. Also the CCGG recognizing TRD of the multispecific B.subtilis phage SPR Mtase is distinct from that of the host enzyme, possibly indicating different requirements for TRDs operative in mono- and multispecific enzymes.     

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.     

Multiple restriction fragment length polymorphisms in the porcine calcium release channel gene (CRC): assignment to the halothane (HAL) linkage group

Two cDNA probes for the porcine calcium release channel gene (CRC) were used in restriction fragment length polymorphism (RFLP) analysis in an attempt to develop genetic markers linked to the malignant hyperthermia (stress susceptibility) gene (HAL). Three TaqI RFLPs, denoted CRC1-CRC3, each composed of two alleles, were detected. RFLPs were also detected with MspI and PvuII, but the MspI RFLP correlated completely with CRC3 in this material and the PvuII RFLP could not be scored reliably due to a minute size difference between the two allelic fragments. The autosomal codominant inheritance of these RFLP loci was confirmed by family analyses. Significant evidence for genetic linkage between the CRC1/CRC3 loci and the A1BG locus in the HAL linkage group confirmed a previous assignment of the CRC gene to chromosome 6 in the pig.     

Molecular characterization of the Spirometra mansonoides genome: renaturation kinetics, methylation, and hybridization to human cDNA probes

High molecular weight DNA from pleroceroid larvae of the tapeworm Spirometra mansonoides was purified from isolated nuclei by conventional techniques. The DNA so isolated has a melting temperature (Tm) of 87 degrees C and a guanine plus cytosine (G/C) content of 44%. 5-Methyl cytosine could not be detected in plerocercoid DNA by HPLC analysis of DNA hydrolysates, by radiolabeling 5'-termini of MspI digests with polynucleotide kinase, or by comparing restriction patterns generated by MspI and HpaII. Renaturation kinetics demonstrated that the genome of S. mansonoides contains repetitive as well as single copy sequences and has a genome size estimated at approx. 1.6 X 10(9) bp. Hybridization was carried out between plerocercoid DNA and cDNAs for human beta-actin, alpha-tubulin and growth hormone (hGH). Rationale for this analysis was based on known homologies among actin and tubulin genes in numerous species and on apparent similarities between hGH and a plerocercoid growth factor that may be reflected in similar DNA sequence. Scanning densitometry of dot blots demonstrated that the hGH probe annealed to the same extent at low stringency (1 M NaCl, 55 degrees C) to DNA from plerocercoids, rat liver and chicken erythrocytes; but this interaction was less than to DNA from human lymphocytes, calf thymus and mouse skin. Similar results were obtained when restriction endonuclease digests of these DNAs were analyzed by Southern transfer. Little or no hybridization of the growth hormone probe to plerocercoid DNA was evident at higher stringency (1 M NaCl, 65 degrees C). In contrast, human tubulin and actin probes showed extensive hybridization to pleroceroid restriction fragments under the high stringency conditions.     

Functional consequences of haem orientational disorder in sperm-whale and yellow-fin-tuna myoglobins.

With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.     

Mechanism of induction of human chorionic gonadotropin in lung tumor cells in culture. Increased levels of alpha-human chorionic gonadotropin-specific mRNA sequences and benzo(a)pyrene-induced hypomethylation

Human lung cells (ChaGo) derived from a bronchogenic carcinoma produce human chorionic gonadotropin (hCG), predominantly the alpha subunit of the glycoprotein hormone, under culture conditions. Treatment of the cells with the polycyclic aromatic hydrocarbons, benzo(a)pyrene (BaP) or dimethylbenzanthracene, at concentrations which do not affect cell growth or macromolecular synthesis, stimulates the production of hCG in these cells. The levels of alpha hCG-specific mRNA (mRNA alpha hCG) sequences in total poly(A)+ RNA isolated from control and drug-treated ChaGo cells are determined by the dot hybridization technique using 32P-labeled, cloned cDNA alpha hCG probe. A concentration-dependent increase in the levels of mRNA alpha hCG sequences in BaP or dimethylbenzanthracene-treated ChaGo cells has been observed. The increase in the level of mRNA alpha hCG sequences can be detected after treatment of the cells with either of the drugs for 24 h, and this level attains its maximum within 48-72 h following drug treatment. A comparative study of the restriction endonuclease (MspI/HpaII) digestion patterns of the control and BaP-treated cell DNA suggests that the internal "C" residues of the -CCGG- sequences in the alpha hCG gene of untreated cells are highly methylated; whereas the internal C residues of the same MspI/HpaII recognition sequences in the alpha hCG gene are comparatively less methylated in BaP-treated cell DNA.     

Identification of a DNA methylation point in the promoter region of the bovine CYP21 gene

The CYP21 (steroid 21-hydroxylase) gene is involved in the synthesis of steroid hormones. Bov-A2 is a retroposon that is common in ruminant genomes. The promoter region of bovine CYP21 contains a short interspersed nucleotide element of Bov-A2, which overlaps a putative Sp1 binding site. We looked for RFLP/HpaII polymorphism in the Bov-A2 element in bovine Zebu breeds by PCR-RFLP, and examined whether polymorphism in this element is associated with methylation. Among DNA samples from 135 Brazilian Zebu breed cattle, we identified an RFLP/HpaII polymorphism (T/C), which, based on a restriction methylation-sensitive assay employing HpaII and isoschizomer MspI enzymes (methylation-sensitive and -non-sensitive enzymes, respectively), appears to be a DNA methylation point. This is the first report of this polymorphism and on DNA methylation in the bovine CYP21 promoter region in Brazilian Zebu cattle.     

Restriction fragment length polymorphisms of three collagen genes in Russians and Buryats.

The restriction fragment length polymorphisms (RFLPs) of 3 collagen genes have been studied in two USSR populations. Four high-frequency alleles, also reported in other populations, were observed. The frequencies of alleles for the polymorphic sites MspI and EcoRI of the COL1A2 gene, HindIII of the COL2A1 gene and EcoRI of the COL3A1 gene were found to be 0.836, 0.398, 0.447 and 0.786, respectively, in Russians from Moscow (Caucasians), and 0.883, 0.519, 0.648 and 0.809, respectively, in Buryats from the Baikal region (Mongoloids). The RFLPs showed a fair agreement with the Hardy-Weinberg equilibrium. The frequencies were compared with those of other populations.     

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.     

Methylation of repetitive DNA sequences in the brain during aging of the rat

Methylation of repetitive DNA sequences (RDS) of the genomic DNA of the brain of 15- and 88-week old rats was analysed by digestion with HpaII, MspI, EcoRI + HpaII and EcoRI + MspI followed by end-labelling. mCpG doublets are present in two RDS of approximately 5 and 0.4 kb, and are also randomly distributed throughout the genome. Hemimethylated mCpC doublets also occur. Both mCpG and mCpC doublets are found more in the old than in the young. This age-related increase in DNA methylation occurs both at CCGG sites of the RDS and in the entire genome. Such increase in DNA methylation may alter chromatin conformation and gene expression in the brain as the rat ages.