Multiple DNA-protein interactions at the CpG island of the human pseudoautosomal gene MIC2

The human MIC2 gene is pseudoautosomal and in females it escapes X inactivation. At the 5' end of the gene a 1.2-kb-long CpG island has been identified that is unmethylated on the active X, the inactive X, and on the Y chromosome. We have demonstrated by 5' RACE experiments that this region contains the transcription start site of the gene. To better characterize this CpG island, we have investigated the interaction between this region and nuclear proteins in vitro by using DNA gel mobility shift and DNase I footprinting techniques. Band shift experiments with HeLa cell nuclear extract have indicated that all the island is involved in multiple interactions with nuclear proteins. Experiments with a eukaryotic purified Sp1 protein have shown that this factor specifically binds to several sites of the island. Three DNase I protected footprints have been identified in the region between nucleotides -122 and +34 with respect to the transcription initiation site. By using a recombinant Sp1 protein, we have shown that all the footprints are due to the binding of Sp1. The sequences of two footprints correspond to the decanucleotide binding site for Sp1, the sequence of the third one does not contain any published Sp1 recognition site.