In vivo DNA-protein interactions at hypersensitive site 3.5 of the human beta-globin locus control region

Using ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the status of DNA-protein interactions at hypersensitive site 3.5 (HS3.5) of the locus control region in K562 and HEL cells, we found that there was protein occupancy in vivo at HS3.5 in both cell lines and the status of DNA-protein interaction was different between K562 and HEL. These data provide direct evidence that specific nuclear factor-DNA complexes form in vivo at functionally important sequence motifs of the HS3.5 in erythroid cells. This indicates that HS3.5 may play an important role in the regulation of the beta-globin gene cluster. K562 is a human erythroleukemia cell line in which the embryonic epsilon-globin gene is predominantly expressed, while the HEL cell line expresses predominantly the fetal beta-globin genes. Thus, HS3.5 might also be involved in the regulation of developmental stage-specific expression of beta-globin genes. Our results are also consistent with the model that each hypersensitive site acts as a functional unit and HS3.5 may facilitate the formation of the HS3 functional unit.