Tissue specificity and organisation of CpG methylation in calf satellite DNA I.

Examination of bovine satellite DNA I methylation within CpG dinucleotides has been made by restriction analysis. It is shown that variations in the methylation patterns occur between different tissues (brain, liver, thymus and sperm) . Some of the 8 Hpa II sites present per repeat are clearly undermethylated in sperm as compared to other tissues. Methylation is considered therefore, as a highly specific event. It is also shown that there is a spatial specificity in the methylation pattern of the 3 Hha I sites in all tissues. These results are discussed in the light of methylation and satellite DNA functions.     

In contrast to other Xenopus genes the estrogen-inducible vitellogenin genes are expressed when totally methylated

The methylation-sensitive restriction enzymes Hha I and Hpa II were used to analyze the methylation pattern of four Xenopus laevis genes in DNA of embryos, of erythrocytes, and of untreated and estrogen-treated hepatocytes. Within these four genes all sites tested are fully modified in embryonic DNA. However, the adult β1-globin gene is unmethylated in DNA of erythrocytes, where it is expressed, and the 68 kd albumin gene, active only in hepatocytes, is specifically hypomethylated in hepatic DNA. The vitellogenin genes A1 and A2, in hepatocytes simultaneously expressed upon estrogen treatment, are heavily methylated in all adult tissues, irrespective of expression. Our results reveal that specific genes can be actively transcribed even when they are fully methylated and that changes in the methylation pattern are not a general prerequisite for gene activation.     

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.     

A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA

About 1% of the mouse genome is cleaved by Hpa II to give a discrete fraction on gels. The nonmethylated fraction is present in all tested tissues, including sperm, and contains Hpa II sites at about 15 times their frequency in bulk DNA. About 80% of the fraction is composed of sequences that occur once or a few times per genome; the remainder is largely rDNA. Unlike bulk DNA, the fraction is not deficient in CpG, and this may be directly due to the lack of methylation. Genomic mapping of three nonribosomal fragments showed that they are part of islands of DNA within which nonmethylated Hpa II and Hha I sites are highly concentrated. We estimate about 30,000 islands per haploid genome and discuss evidence that many may be associated with genes.     

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.     

A possible structure for calf satellite DNA I.

Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.     

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.     

Absence of cytosine methylation at C-C-G-G and G-C-G-C sites in the rDNA coding regions and intervening sequences of Drosophila and the rDNA of other insects.

Cytosine residues in C-G dinucleotides are frequently methylated in eukaryote DNA. In DNA of the dinoflagellate C. cohnii, the sequence C-MeC-G-G apparently renders Hpa II (C-C-G-G) incapable of digesting whole cell DNA in general, and rDNA in particular. Msp I, which also recognizes C-C-G-G but cleaves irrespective of methylation, degrades C. cohnii DNA and produces rDNA segments of 10.2 to 1.4 kb. We have applied this Hpa II/Msp I test to unfractionated DNA, and to rDNA and the rDNA intervening sequence of Drosophila virilis embryos and adults. There is no evidence of C-MeC-G-G sequences in either developmental stage of this species. Absence of G-MeC-G-C from coding and intervening sequences of rDNA was shown in comparisons of Hha I (G-C-G-C) cleavage patterns of unfractionated DNA and cloned (unmodified) segments of rDNA. Comparisons of Hpa II and Msp I cleavage products of DNA from the house fly, the flesh fly and a bumblebee also revealed no internal cytosine methylation in the sequence C-C-G-G. Because amounts of McC in C-G dinucleotides vary greatly among species, from apparent nonexistence to substantial proportions, no inference may yet be drawn about the role of such base modifications in DNA.     

Methylation of human ornithine decarboxylase gene before transfection abolishes its transient expression in Chinese hamster ovary cells

Different methylations of cloned human ornithine decarboxylase gene with restriction methylases in vitro before transfection greatly reduced the transient expression of ODC in Chinese hamster ovary cells. Single methylation of the gene with Hpa II (CCGG) methylase decreased the transiently expressed peak activity by about 50%, single methylation with Hha I (CCGG) methylase by about 80% whilst a double methylation at both Hpa II and Hha I restriction sites virtually abolished any transiently expressed ornithine decarboxylase activity. These results together with our earlier circumventing evidence indicate that the expression of mammalian ornithine decarboxylase is critically influenced by the methylation state of the gene.     

Non-methylated Genomic Sites Coincidence Cloning (NGSCC): an approach to large scale analysis of hypomethylated CpG patterns at predetermined genomic loci

We have developed a new approach to the analysis of hypomethylated CpG patterns within predetermined, megabase long, genome regions. The approach, which we term Non-methylated Genomic Sites Coincidence Cloning (NGSCC), includes three main steps. First, total genomic DNA is digested with a methylation sensitive restriction endonuclease, such as Hpa II or Hha I. Then the fragments corresponding to the genomic area of interest are selected. To this end the fragmented genome DNA is hybridized with a mixture of clones (BACs, cosmids etc.) representing a given region and digested with the same restriction enzyme(s). A special version of the coincidence cloning procedure was developed to make this hybridization selection highly efficient and specific. Finally, fragments of the locus under study are mapped and sequenced. The technique proved to be efficient and specific. As a test, it was applied to the analysis of hypomethylated CpG patterns along the 1-Mb D19S208-COX7A1 (Chr 19q13.12) locus, on human chromosome 19, in normal testis and in seminoma tissues. Some differences in the distribution of hypomethylated CpGs between the two tissues were demonstrated. The methylation profiles in both tissues revealed a clear trend to clustering of non-methylated sites. We also analyzed the expression of genes located within hypomethylated clusters in both tissues. It was shown that, whereas the expression of some of the genes investigated was correlated with hypomethylation of the region, other genes were expressed regardless of their methylation status. NGSCC thus promises to be a useful approach for the analysis of the role of dynamic epigenetic factors in genome function.Communicated by G. P. Georgiev     

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.     

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.     

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.     

Genus specificity and extensive methylation of the W chromosome-specific repetitive DNA sequences from the domestic fowl,Gallus gallus domesticus

Two female-specific repeating DNA units of 0.6 kilobase pairs (kb) and 1.1 kb, produced by digesting the genomic DNA of the White Leghorn chicken with Xho I, were cloned by inserting them into the Xho I site of an Escherichia coli plasmid vector pACYC177. Two such recombinant plasmids, pAGD0601 and pAGD1101, containing a single 0.6-kb and 1.1-kb sequence, respectively, were used as molecular probes. In situ hybridization of the ³Hprobes to the metaphase chromosomes from the female White Leghorn embryos revealed their localization in the W chromosome. Semiquantitative Southern blot hybridization with ³²P-probes in excess indicated that the 0.6-kb unit and 1.1-kb unit were repeated approximately 14,000 and 6,000 times, respectively, in the W chromosome. The two units comprised about 46% of the W chromosomal DNA. These two repeating units were found in the female genomes of every line of Gallus g. domesticus tested and in the female genomes of three jungle fowl species (G. gallus, G. sonneratii, and G. varius) but not in three species belonging to other genera in the suborder Galli. Hha I sites in the 0.6-kb and 1.1-kb repeating units were shown to be extensively methylated and a significant fraction of the Hpa II sites in the 0.6-kb repeating units were also shown to be methylated in the female genome of the White Leghorn. Methylation patterns of Hpa II sites in or around the 0.6-kb repeating units examined by the Msp I digestion were similar in the various lines of domestic fowls and the two species of jungle fowls, but G. varius (black or green jungle fowl) produced a different pattern of digestion with Msp I.     

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.     

DNA methylation controls the inducibility of the mouse metallothionein-I gene in lymphoid cells

The W7 mouse thymoma cell line does not express the metallothionein-I (MT-I) gene in the presence of either cadmium or glucocorticoids, unlike most other cell lines. This cell line was therefore used as a model system for studying the role of DNA methylation on MT-I gene expression. The extent of DNA methylation within the MT-I gene and its flanking regions was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I. In W7 cells, all of the Hpa II sites in the vicinity of the MT-I gene are methylated, whereas in cells that have an expressible MT-I gene (for example, Friend erythroleukemia cells) all of these Hpa II sites are unmethylated. When W7 cells are treated for a few hours with 5-azacytidine, the MT-I gene becomes inducible by both cadmium and glucocorticoids. Addition of hydroxyurea along with 5-azacytidine prevents MT-I gene induction, suggesting that incorporation of 5-azacytidine into DNA is required before this gene can be activated. To determine whether 5-azacytidine treatment changes the methylation pattern near the MT-I gene, we treated W7 cells with 5-azacytidine and selected inducible cells in 10 μM cadmium. All of the Hpa II sites within the MT-I gene are unmethylated in these cadmium-resistant W7 cells. In addition, flanking DNA sequences are also undermethylated in a pattern similar to that seen in Friend erythroleukemia cells that express the MT-I gene. The possible significance of methylation as a mechanism of gene commitment during cell differentiation is discussed.