DNA methylation in chicken alpha-globin gene expression.

We have investigated certain specific methylation sites of the chicken alpha-globin gene cluster in DNA from embryonic and adult erythroid cells as well as from brain and sperm cells. Eight contiguous DNA fragments of the alpha-globin gene cluster were subcloned from a recombinant lambda phage. The subclones were used as probes to map all the Msp I/Hpa II and Hha I sites in the unmethylated cloned DNA and specific sites of methylation in and around the alpha-globin gene cluster in chromosomal DNA. The data show that sperm DNA is totally methylated at these restriction sites in the globin gene region, as is brain DNA, with some exceptions. Interestingly, the methylation status of specific sites 5' to the coding sequences is correlated with expression of the embryonic or adult alpha-globin genes in different stages of erythroid development. Some sites showing partial methylation, however, do not conform to the model that transcribed genes are unmethylated or undermethylated. We also find a well-defined 3.5-kilobase region of DNA 5' to the alpha-globin gene cluster in which all C-C-G-G sites are resistant to Msp I digestion in all tissues. This "Msp block" is presumably caused by 5-MeCpC methylation.     

Methylation of foreign DNA sequences in eukaryotic cells.

The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA were found to remain unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones were found to contain methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none of the inserted sequences acquired these modifications. These results are discussed in relation to various models put forth to explain the process of methylation in eukaryotic cells.     

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Previous work demonstrated an inverse correlation between methylation at the three 5'-CCGG-3' sequences in positions +24, +6, and -215 relative to the cap site of the late E2A promoter of adenovirus type 2 (Ad2) DNA and its activity. In the study presented here, we used the genomic sequencing method to detect 5-methyl-2'-deoxycytidine (m5dC) residues in 5'-CG-3' sequences other than the 5'-CCGG-3' (Hpa II) sites. The patterns of methylation in all 5'-CG-3' sequences over a region of about 180 base pairs required for gene activity in the late E2A promoter of integrated Ad2 DNA were determined in cell lines that carry this promoter in an active or inactive state. In cell lines HE1 and uc2, the late E2A promoter is active and all thirteen 5'-CG-3' sequences between positions +24 and -160 are unmethylated. In cell line HE2, the same promoter is permanently shut off and all 5'-CG-3' sequences are methylated in both strands. Thus, the inverse correlation is perfect in these cell lines over a region of about 180 base pairs in the late E2A promoter. The same promoter segment was analyzed in cell lines mc23 and mc40, in which a late E2A promoter-chloramphenicol acetyltransferase (CAT) gene construct had been genomically fixed after in vitro 5'-CCGG-3' methylation and subsequent transfection. In cell line mc23, the preimposed methylation pattern was stable and the CAT gene was inactive. Genomic sequencing confirmed the presence of m5dC in the 5'-CCGG-3' sequences and revealed the spreading of methylation to neighboring 5'-CG-3' sequences along the entire promoter. Some of these sites were hemimethylated. In cell line mc40, several of the 120 integrated copies became demethylated in positions +24 and +6, but the promoter was methylated in some of the copies upstream of position -50. Cell line mc40 expressed the CAT gene.     

Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5'' -CCGG- 3'' methylation.

The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state. We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites. Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis. Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA. In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions. The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region. By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region. This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.     

In Z-DNA the sequence G-C-G-C is neither methylated by Hha I methyltransferase nor cleaved by Hha I restriction endonuclease.

Plasmids carrying 24- or 32-base-pair inserts of alternating (dG-dC) residues were used to analyze the level of methylation of the G-C-G-C sites by Hha I DNA methyltransferase and their cleavage by Hha I endonuclease in the B-DNA or Z-DNA conformation. In supercoiled plasmids in which the inserts formed Z-DNA, the extent of methylation at the insert G-C-G-C sites was dramatically lower than the level of methylation at the G-C-G-C sites located outside the insert in the same plasmid. Similarly, cleavage by Hha I endonuclease was sharply lowered when the insert was in the Z-DNA form. In the relaxed plasmid, all its G-C-G-C sites were methylated to the same extent and the unmethylated sites were readily cleaved. After treatment with the methylase, the supercoiled plasmid was linearized and then digested with Hha I restriction endonuclease. This exposed unmethylated G-C-G-C sites from the insert that had been protected against cleavage in the Z conformation. A chemical reaction was used to study the distribution of the unmethylated cytosine residues. No accumulation of unmethylated cytosine residues was found anywhere along the entire 32-base-pair insert, which is consistent with a cooperative B-Z transition.     

Tissue-specific expression and methylation of a thyroglobulin-chloramphenicol acetyltransferase fusion gene in transgenic mice.

Fusion genes containing 1600 or 2000 base pairs of the bovine thyroglobulin gene 5' flanking region and the chloramphenicol acetyltransferase (CAT) coding sequence were constructed and used to generate transgenic mice. Altogether, 24 independent transgenic lines were obtained, and the expression of the transgene was assayed by measuring the CAT activity in different tissues. Depending on the transgenic lines, the fusion gene was either silent in all tissues or specifically expressed in the thyroid. The level of expression was found to be highly variable from one line to another and to be regulated by thyrotropin in a manner similar to the natural thyroglobulin gene. The methylation status of the integrated DNA was tested by digestion of DNA extracted from thyroid and other tissues with the isochizomers Msp I and Hpa II. It was found that one of the Hpa II sites was demethylated specifically in the thyroid.     

Integrated hepatitis B virus DNA sequences specifying the major viral core polypeptide are methylated in PLC/PRF/5 cells.

The methylation of various hepatitis B virus (HBV) DNA sequences was examined using the restriction endonucleases Hpa II and Msp I. HBV DNA from virions (Dane particles) and virus-infected liver tissue was digested with Hpa II or Msp I and fractionated by electrophoresis in agarose gels, and the restriction enzyme cleavage pattern was examined by Southern blot analysis. No methylation of the 5' C-C-G-G 3' recognition sequence was detected in either virion DNA or HBV DNA from infected liver tissue. The tissue culture cell line PLC/PRF/5, derived from a human hepatoma, possesses HBV DNA exclusively integrated at several sites. Digestion of PLC/PRF/5 DNA with Hpa II and Msp I revealed that the integrated HBV DNA sequences were methylated. Further analysis using probes specific for various regions of the HBV genome showed that some of the hepatitis B viral DNA sequences, including those specifying the major surface antigen polypeptide, were methylated infrequently or not at all. In contrast, the viral DNA sequences coding for the major core polypeptide were extensively methylated. Because the surface antigen is expressed in these cells while the core antigen is not, our results suggest that DNA methylation could account for the selective expression of HBV genes in this hepatoma cell line.     

Inhibition of promoter activity by methylation: possible involvement of protein mediators.

To study the relationship between DNA methylation and promoter activity we have methylated in vitro the promoters of the mouse metallothionein I gene and the herpes simplex virus thymidine kinase gene. We have transiently transfected these promoters fused to the human growth hormone in their methylated or unmethylated state into mouse L or F9 cells. Promoters methylated by methylase (M.) Hpa II and M.Hha I caused inhibition of reporter gene expression in L cells but not in F9 cells, while methylation of all CpGs by M.Sss I caused inhibition in both cell lines. Repression of promoter activity by M.Hpa II and M.Hha I methylation, but not by M.Sss I methylation, could be alleviated by cotransfection with an excess of untranscribable DNA methylated with M.Sss I. The methylated sites in nuclei isolated from the transfected L cells, but not F9 cells, were found to be protected from Msp I digestion. Taken together these results suggest that a factor present in L cells and missing in F9 cells mediates the methylation-directed inhibition of promoter activity. The ability of methylated DNA to overcome the inhibition seems to reflect competition for the mediator factor. Interestingly, treatment with Zn2+ ions brought about activation of the methylated promoter of the metallothionein gene. Similarly, butyrate could override the repression of the thymidine kinase methylated promoter. These activations were not accompanied by demethylation of the promoter or displacement of the mediator factor.     

Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation.

Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions. To assess the potential role of DNA methylation in the expression of immunoglobulin genes, which require complex rearrangements prior to expression, methylation patterns were examined in cell lines representing different stages of lymphocyte maturation. Methylation of the second cytosine in the sequence 5' C-C-G-G 3' was determined by using Hpa II/Msp I endonuclease digestion. Four CH genes (C mu, C delta, C gamma 2b, and C alpha), C kappa, V kappa, C lambda, and V lambda genes were analyzed. The results lead to the following conclusions: (i) transcribed immunoglobulin genes are undermethylated; (ii) the C gene allelic to an expressed C gene is always also undermethylated; and (iii) all immunoglobulin loci tend to become increasingly undermethylated as B cells mature.     

Clonal inheritance of the pattern of DNA methylation in mouse cells.

DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On the other hand, phi X174 replicative form DNA that was methylated in vitro at its C-C-G-G residues retains these methylations as shown by restriction enzyme analysis with Hpa II and Msp I to detect methylation at this specific site. Although these unselected methylated vectors are prone to lose 30-40% of their methyl moieties upon transfection, this demethylation appears to be random. Once established, the resulting methylation pattern is stable for at least 100 cell generations. In order to examine the specificity of methylation inheritance, fully hemimethylated duplex phi X174 DNA was synthesized in vitro from primed single-strand phi X174 DNA by using 5-methyl deoxycytidine 5'-triphosphate. This molecule was inserted into mouse L cells by cotransformation and subsequently was analyzed by a series of restriction enzymes. Only methylations located at C-G residues were conserved after many generations of cell growth. The results suggest that the inheritance of the cellular DNA methylation pattern is based on a C-G-specific methylase that operates on newly replicated hemimethylated DNA.