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 s ite 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 t he status of DNA-protein interaction was different between K562 and HEL. Th ese data provide direct evidence that specific nuclear factor - DNA complex es form in vivo at functionally important sequence motifs of the HS3.5 in e rythroid cells. This indicates that HS3.5 may play an important role in the regulation of the beta -globin gene cluster. K562 is a human erythroleukem ia cell line in which the embryonic epsilon -globin gene is predominantly e xpressed, while the HEL cell line expresses predominantly the fetal beta -g lobin genes. Thus, HS3.5 might also be involved in the regulation of develo pmental stage-specific expression of beta -globin genes. Our results are al so consistent with the model that each hypersensitive site acts as a functi onal unit and HS3.5 may facilitate the formation of the HS3 functional unit .