DNA binding factors for the CpG-rich island containing the promoter of the human X-linked PGK gene.

The gene coding for the glycolytic enzyme phosphoglycerate kinase (PGK-1) is X-linked in mammals and has a G+C-rich 5' region characteristic of several constitutive genes. Despite the fact that PGK-1 is constitutively expressed, it is transcriptionally regulated in female cells by X chromosome inactivation. To study the expression and regulation of the PGK-1 gene, we have analyzed the binding of trans-acting factors to the 5' region of the PGK-1 gene. We detect at least three distinct binding activities that interact in a sequence-specific manner in vitro with at least six different sites in the 5' region. Two of these binding activities generate DNase I-protected footprints centered approximately 360 bp and 130 bp upstream of the transcription start point. We have examined the promoter specificity of the three binding activities in gel mobility-shift assays by competition with cloned promoter fragments of other genes. None of the binding activities interacts exclusively with X-linked promoters. However, one activity binds preferentially to G+C-rich promoters, and another activity appears to bind preferentially to only two of the promoters tested. Previous studies have demonstrated that one HpaII/MspI site, which is included within a footprinted region observed in this study, is fully methylated in the inactive X chromosome and totally unmethylated on the active X chromosome. Competition studies using synthetic oligonucleotides containing 5-methylcytosine at all CpG sites in this region demonstrate that DNA methylation does not significantly alter the affinity between the corresponding binding activity and this binding site.