Changes in structure and methylation pattern in a cluster of thymidine kinase genes.

Abstract

Cell line 101 is a thymidine kinase (TK)-positive derivative of Ltk-which contains ca. 20 copies of the herpes simplex virus TK gene organized in a tandem array. DNA methylation at three sites within the gene and flanking sequences was inversely correlated with expression: the sites were unmethylated in line 101, methylated in each of 4 TK-negative derivatives of 101, and unmethylated in each of 21 TK-positive derivatives derived from them. The same three sites were affected in most of the 20 copies of the TK gene, whereas other sites between them were not affected. Although the entire gene cluster was never lost, indicating that integration into the genome was stable, internal rearrangements occurred at a high frequency. The rearrangements had no obvious correlation with the state of methylation or with the expression of the genes.There is a well-established inverse correlation between the level of DNA methylation and the expression of many genes, including globin (31), integrated viral genomes (11,27,28,32), ovalbumin (16), and the genes coding for adenine phosphoribosyltransferase and dihydrofolate reductase (26). Studies with inhibitors of DNA methylation suggest that the inhibition of DNA methylation increases the frequency of expression of methylated, inactive genes in vivo (2,4,9,20), and it has been demonstrated that methylated DNA is expressed more poorly than unmethylated DNA after transfer into mammalian cells (18,25,33, 36).Conversely, other studies have failed to detect the involvement of methylation in the expression of some genes (8, 19), indicating either that methylation does not play a role in the expression of those genes or that the sites of methylation which were important for the regulation were not easily detected by restriction endonuclease digestion. Data from a number of studies indicate that methylation of one or a few sites in specific regions (often in the 5' noncoding region) is sufficient to abolish the expression of some genes (1,22,26), so that if the particular sites of importance do not fall within a sequence recognized by one of the methylation-sensitive restriction endonucleases, they are difficult to identify and might be missed.Several laboratories have studied the modulation of expression of thymidine kinase (TK) genes which had been transferred into cultured cells (3-5, 12, 21, 24, 29), and several mechanisms by which expression of the TK gene is regulated have been identified. Robins et al. (24) demonstrated that of 40 lines which expressed the TK genes, half retained the genes after selection against TK expression, indicating that mechanisms in addition to gene loss were involved in modulating the expression of the transferred genes. Davies et al. (5) described cell lines in which changes in expression were correlated with changes in the nuclease sensitivity of the TK-containing chromatin but in which no changes in DNA methylation could be detected. Ostrander * Corresponding author. t Contribution 1239 from the Department of Biology. et al. (21) and a previous publicati...

Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency.

“…Northern hybridization analysis conclusively demonstrated that loss of transcriptional activity was more profound when sites in the LTR were methylated (lanes 3-6) than when structural, non-LTR sequences were methylated (lane 2). This observation agrees with other findings indicating that gene promoters are sensitive to transcriptional inactivation via hypermethylation (Cedar, 1988) in contrast to minimal effects observed when structural gene sequences are methylated, with the exception of the thymidine kinase gene (Christy and Scangos, 1984). Inactivation of HIV expression by in vitro methylation is virtually identical to the in vivo inactivation of Moloney murine leukemia virus (M-MuLV) observed in undifferentiated mouse embryonal carcinoma cells (Cedar, 1988).…”

Modulation of tk expression in mouse pericentromeric heterochromatin.

“…It has been associated with changes in chromatin structure (14) and also with changes in DNA methylation (21,41). In other cases, DNA rearrangements within tandem arrays of tk inserts have been observed, but they were not directly correlated with the modulation of tk gene expression (9,51). These differing results probably reflect the different chromosomal insertion sites in each transformant.…”