METHYLATION SENSITIVE PROTEIN-BINDING TO AN INTRAGENIC ACTIVE X-SPECIFIC METHYLATED REGION IN THE M-ROBUSTUS HPRT GENE

An intragenic region of the wallaroo (Macropus robustus) hypoxanthine phosphoribosyltransferase gene which contains three active X-specific methylated cytosines was examined for protein(s) binding. In vitro DNa se I footprinting of unmethylated DNA identified three footprints, one of which (footprint I) contained two of the known differentially meth ylated sites (a HpaII and a HhaI site). Methylation of the footprint I HpaII site only, abolished the formation of several, specific DNA-pro tein complexes in mobility shift assays. UV cross-linking experiments indicated that polypeptides involved in the methylation sensitive inte ractions with footprint I had molecular weights ranging from 72 to 48 kDa. Analogous results were obtained with nuclear extracts from both e utherian and metatherian cells, indicating that these proteins are con served. We suggest that the binding of these proteins to the inactive X may play some role in gene silencing, with the active gene being pro tected from this effect by methylation of the binding site.