Genomic footprinting of proteins interacting with the chicken lysozyme promoter

The functions of the promoter of the chicken-lysozyme-encoding gene (Lsz) are complex, since, on the one hand, the promoter serves to mediate the effects of five upstream regulatory elements and, on the other, it contains itself important regulatory sequences. To analyze this interplay we first monitored protein-DNA interactions at the Lsz promoter in hormonally regulated oviducts and in constitutively expressing HD11 cells by in vivo genomic footprinting. Two regions of strong protein-DNA interactions shared by both HD11 and oviduct cells were localized between nucleotides (nt) -105 and -119 and between nt -61 and -74, respectively. A third region of protein-DNA contacts was found selectively in HD11 cells from nt -199 to -204; this region corresponds in position with a previously identified HD11 cell-specific enhancer element. Furthermore, we determined the sites hypersensitive to an endogenous endonuclease in isolated nuclei using the genomic sequencing protocol. These sites fall into two regions: one encompasses the in vivo localized protein-DNA contacts between nt -105 and -119 and between nt -61 and -74; the other was found in a sequence largely devoid of in vivo protein-DNA interactions. We hypothesize that hypersensitive cleavage sites are generated by protein-induced changes in the DNA conformation.