Characterization of deoxyribonucleic acid-protein interactions at a growth hormone-inducible nuclease hypersensitive site in the rat insulin-like growth factor-I gene.
Many of the growth-promoting effects of GH are mediated by insulin-like growth factor-I (IGF-I), a highly conserved, 70-residue basic peptide. Previous studies have demonstrated that GH rapidly stimulates IGF-I expression in vivo, and our laboratory has identified a GH-regulated alteration in chromatin configuration, manifested as a hormonally induced deoxyribonuclease-I (DNase-I)-hypersensitive site in the second IGF-I intron. In the current study, we have used in vivo DNase-I footprinting to map this hormonally responsive chromatin domain to an approximately 350-nucleotide region and have identified DNA-protein interactions within the hypersensitive site by in vitro gel mobility shift experiments and DNase-I footprinting studies. DNA-protein binding was localized to two adjacent segments of 32 and 48 nucleotides. In 1 of these regions, protein-DNA contacts were also detected in vivo on guanine residues by dimethylsulfate footprinting. DNA-binding activity was present in GH-deficient rats, but was not modified by hormone treatment. Our results define a rapid and reversible genomic alteration in response to GH in a GH-regulated gene and delineate a target within chromatin for GH action.
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
M J
MJ
Thomas
K
K
Kikuchi
D P
DP
Bichell
P
P
Rotwein
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002874:Chromosome Mapping; D016172:DNA Fingerprinting; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D003850:Deoxyribonuclease I / Q000235:genetics; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley
D017931:DNA Primers; D004268:DNA-Binding Proteins; D007334:Insulin-Like Growth Factor I; D013006:Growth Hormone; D003850:Deoxyribonuclease I