Intragenic matrix attachment and DNA-protein interactions in the human X-linked Hprt gene

To investigate the possible contribution of intragenic differentially methylated cytosines to X-linked gene expression, we examined DNA-protein interactions in a region in intron 3 of the human hypoxanthine phosphoribosyltransferase gene which contains at least one HpaII site methylated specifically on the active X. In vitro DNase I footprinting experiments using unmethylated DNA and HeLa nuclear extract identified three footprints (I-III). Footprints I and III flank an Alu repeat containing the HpaII site(s), one of which is contained within footprint II. Although methylation of the HpaII site had no effect on footprint II binding interactions, methylation of nearby CpGs substantially reduced the formation of three of the specific DNA-protein complexes binding to footprint II in mobility shift assays. Additionally, an A+T rich region immediately 5' to the HpaII-containing Alu repeat was found to bind specifically to nuclear matrices in vitro. We suggest that differential methylation of CpGs may affect the binding of regulatory proteins in vivo, and that interactions between the footprint proteins and those binding to the matrix attachment region may be involved in controlling X-linked Hprt expression.