Binding of HMG proteins to the 5'-flanking sequence of human beta-globin gene

Our previous studies have identified that there are at least three regulatory regions (two negative regions and one positive region) in the 5'-flanking sequence of human beta-globin gene (-610 to +1 bp). The binding of HMG proteins to both negative regulatory regions was examined by the gel mobility shift and DNase I protection assays. In gel mobility shift assay, we observed that HMG proteins 1 and 2 could bind to both negative regulatory regions (NCR1 and NCR2). Using the gel shift competition assay, we identified that the binding proteins between the two regions are different from each other. DNase I protection analysis shows that HMG proteins 1 and 2 only bind to one site (between -560 and -533 bp) in NCR1. However, two protected regions can be detected in NCR2, one between -272 and -252 bp relative to the cap site, the other between -306 and -329 bp. We also observed that HMG proteins 14 and 17 could not bind to both negative regions, so it seems that HMG proteins 1 and 2 may play an important role in the regulation of beta-globin expression through DNA-protein interaction or through protein-protein interaction.