Studies on mechanisms by which methylation on the cytosine residue of CpG dinucleotides suppress the tissue specific gene expression of hepatitis B virus

There is ample evidence suggesting that DNA methylation at cytosine residue of CpG dinucleotide is associated with the suppression of gene expression in vertebrate cells. The molecular mechanism which counts for the suppression of gene expression is still not clear. To study in detail of the mechanism for inhibition of tissue specific gene expression by DNA methylation, we construct a plasmid (pHBV-CAT) containing multiple HBV promoters and two HBV enhancers, and a reporter gene chloramphinical acetyl transferase (CAT). In transient CAT expression assays we demonstrated that the CAT gene expression can be inhibited by methylation on CpG dinucleotide, but not methylation on CpC. The methylation at CpG can affect expression of all genes in pHBV-CAT regardless the methylation status of each individual promoter. The further studies on HpaII methylation of deletion clone suggests that no single HpaII site in HBV can be solely responsible for the inhibition effect by HpaII methylation. Our in vitro and in vivo footprinting by ligation mediated PCR on intact pHBV-CAT reveals that (1) the protein-DNA interaction at region 1260-1280 in enhancer I can be altered by SssI methylation. (2) The accessibility of dimethylsulfate (DMS) to the down stream region of enhancer I on both HpaII and SssI methylated plasmid is reduced in vivo. These results suggest that the methylated DNA may form a special complex "inactive complex" in vivo in which at least two different kinds of so called "mediators" may be involved: the mCpG binding mediators and general mediators.