Methylation of simian virus 40 Hpa II site affects late, but not early, viral gene expression.

Abstract

DNA methylation has been correlated with reduced gene expression in a number of studies, although evidence for a causal link between the two events has been lacking. Because microinjection of simian virus 40 (SV40) DNA into the nucleus of Xenopwu laevis oocytes results in the synthesis of both early and late viral gene products, it was possible to test whether a specific methylation event can affect gene expression. The single SV40 Hpa H site at 0.72 SV40 map units was specifically methylated with Hpa H methylase. When this DNA was injected into oocytes, there was a marked reduction in the synthesis of the major late viral capsid protein VP-1, relative to the synthesis by an unmethylated control. However, production of the early proteins (the large and small tumor antigens) was not affected by Hpa II methylation. Therefore, methylation at a single site on the viral DNA located near the 5' end ofthe late region can specifically repress late gene expression. The possible mechanisms by which this repression is mediated are discussed.The only modified base in eukaryotic DNA is 5-methylcytosine (for review, see refs. 1 and 2). This modification occurs in the DNA of a wide variety of species, from bacteria through vertebrates and plants, which suggests that it may function in gene regulation. The major 5-methylcytosine residue in eukaryotic DNA is located in the dinucleotide sequence C-G. Restriction enzymes Hpa II and Msp I both cleave the sequence C-C-G-G. However, the former cleaves the sequence only if the C-G is unmethylated whereas the latter will cut the sequence in either state. By using the Southern blotting procedure to compare the restriction patterns of DNA cleaved with one or the other enzyme, it is possible to locate methylated C-C-G-G sites in genomic DNA. In this way, a strong correlation between gene activity and the state of DNA methylation has been demonstrated in various systems. For example, tissue-specific differences in the methylation patterns of chicken (3) and human (4) globin, ovalbumin (5), and viral (6, 7) DNA have been observed which suggest that actively expressed genes are under-methylated. This is supported by the observation that actively transcribing chromatin is under-methylated relative to methylated transcriptionally inactive chromatin (8).The experiments in this study were further prompted by the strikingly nonrandom fashion in which C-G residues are arranged on the simian virus 40 (SV40) genome (Fig. 1). Nineteen of the 27 C-G residues are located between 0.66 and 0.75 map units, a region comprising <10% of the viral genome. This region delineates the sequences that form the nucleosome-free "gap" region that has been identified in SV40 minichromosomes (9-11) and contains the majority of the known regulatory sequences of the viral genome including the origin of replication (reviewed in ref. 12), the early (13, 14) and late (15, 16) region promoters, and the 72-base-pair repeat sequence (17) that enhances transcription. In contrast, in the remaining 90% of the genome, whi...

Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated.

“…Equally convincing is another series of experiments in which genes were methylated in vitro before transfer into eukaryotic cells. For example, methylation of all CpG dinucleotides in the Moloney retrovirus genome virtually abolishes its expression , and in simian vims 40 (SV40) methylation of a single Hpall (CCGG) site inhibits late gene transcription when tested in the frog oocytes (Fradin et al 1982; see also Vardimon et al 1982). However, it is noteworthy that methylation affects SV40 early promoter function neither in frog oocytes nor in mammalian cells (Fradin et al 1982;Graessmann et al 1983).…”

“…For example, methylation of all CpG dinucleotides in the Moloney retrovirus genome virtually abolishes its expression , and in simian vims 40 (SV40) methylation of a single Hpall (CCGG) site inhibits late gene transcription when tested in the frog oocytes (Fradin et al 1982; see also Vardimon et al 1982). However, it is noteworthy that methylation affects SV40 early promoter function neither in frog oocytes nor in mammalian cells (Fradin et al 1982;Graessmann et al 1983). In the 7-globin gene, the methylation state of the promoter region is most critical for gene expression, whereas extensive methylation of the coding sequence is tolerated (Busslinger et al 1983;Murray and Grosveld 1987).…”

Effect of in vitro DNA methylation on beta-globin gene expression.

“…Although experiments with some genes show that DNA methylation has powerful effects at selective individual sites in the 5' region (2)(3)(4), other studies indicate that methylation may work over the entire gene domain (5), probably by interfering with the formation of a normal active chromosomal conformation (6).…”

Activation of a chicken embryonic globin gene in adult erythroid cells by 5-azacytidine and sodium butyrate.

“…5-10; refs. 5 and 6 are reviews), and several studies in which cloned eukaryotic genes were methylated in vitro have demonstrated a direct inhibitory effect on transcription (11)(12)(13)(14). In addition, a variety of studies in cell culture systems have shown that 5-azacytidine, a cytotoxic pyrimidine analog that inhibits DNA methylation, can induce cellular differentiation (15) and either directly activate (16) or allow activation of specific genes (17).…”