Can DNA methylation regulate gene expression?

Abstract

The E2a region of the Ad2 genome encodes the Ad2-specific DBP. An inverse correlation between the level of DNA methylation at the 5'-CC*GG-3' sites of the E2a region and the extent of expression of DBP has been demonstrated in Ad2-transformed hamster cell lines (Vardimon et al. 1980). Four different leaders are used in the transcription of the E2a region in cells productively infected with Ad2. The leader located at coordinate 75 on the viral genome is used early after infection and the other three leaders are used late after infection (Chow et al. 1979). The analysis of the integration patterns of the viral DNA in the Ad2-transformed cell lines has revealed that the early leader is deleted in the cell lines which do not express the DBP (Vardimon and Doerfler 1981). The late leader located at coordinate 72 on the viral genome is present. The region encoding that late leader has been subcloned, and the cytoplasmic RNA from the cell line which expresses the DBP has been analyzed. It has been shown that the late leader is used in transformed cells. Hence the absence of the early leader cannot be the immediate reason for the lack of expression of the DBP. Correlations between DNA methylation and the absence of gene expression may indicate that methylation regulates gene expression or that methylation is the consequence of lacking gene expression. In order to decide between these alternatives an in vitro system has been employed. The HindIII A fragment of the Ad2 DNA which encodes the DBP has been methylated in vitro by the HpaII DNA methyltransferase. Methylated or unmethylated HindIII A fragment has been microinjected into the nuclei of Xenopus laevis oocytes. Unmethylated HindIII A fragment has been found to be expressed as specific viral RNA, whereas no viral RNA can be found in oocytes microinjected with methylated HindIII A fragment. The possibility of a nonspecific inhibitory factor in the methylated DNA preparation has been ruled out by the simultaneous microinjection of sea urchin histone gene DNA together with the methylated HindIII A fragment. Histone genes are expressed, while the expression of the methylated viral gene is blocked. By using the single-strand-specific endonuclease S1 we have shown that in Xenopus laevis oocytes initiation of transcription of the E2a region starts exactly at the same site as in Ad2 productively infected cells. These results provide direct evidence for the notion that DNA methylation at specific sites is involved in the regulation of gene expression.