In vivo footprinting of the human alpha-globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction

摘要: A major regulatory element required for expression of the human alpha-globin genes is located 40 kb upstream embryonic zeta-globin gene. To understand how this and other locus control region (LCR) elements contribute to high-level in erythroid cells, we have performed high-resolution, vivo dimethyl sulfate footprinting. In addition, modified sulfate-based ligation-mediated polymerase chain reaction footprinting procedure permit assessment interactions at guanine adenine residues, rather than guanines alone. alpha-LCR carried on chromosome 16 a mouse erythroleukemia cell environment revealed protein occupancy GATA-1, AP-1/NF-E2, CACC/GGTGG motifs, specific differences compared with vitro binding, distinct changes one upon sulfoxide-induced cellular maturation. No contacts were detected nonexpressing hepatoma cells. demonstrated that two AP-1 motifs which are occupied bind purified NF-E2 vitro. Our data suggest three proteins, NF-E2, unknown factors, minimally as DNA-binding proteins function LCR-like elements. The juxtaposition interaction these factors each other, accessory not directly contact DNA, likely account relative position independence globin