Regulation of the chicken ovalbumin gene by estrogen and corticosterone requires a novel DNA element that binds a labile protein, Chirp-1.
Because induction of the chicken ovalbumin (Ov) gene by steroid hormones requires concomitant protein synthesis, efforts have focused on defining the binding site in the Ov gene for a labile transcription factor. Previous gel mobility shift studies identified one such site in the steroid-dependent regulatory element (SDRE) between -900 and -853. To ascertain whether estrogen and glucocorticoid affect the binding of this labile protein, genomic footprinting of the Ov gene was done by treating primary oviduct cell cultures with dimethyl sulfate. Several alterations that include steroid-dependent protection of guanine residues -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed. Of particular importance, the in vivo footprinting data are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations. Ten-base-pair linker-scanning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid. Likewise, point mutations of the protected guanine residues profoundly attenuated the response to these steroid hormones. In addition, in vitro binding activity correlated with in vivo functional activity. For example, mutant A4e shows no transcriptional activity in response to steroid hormones, and a corresponding oligomer does not bind protein in vitro. In contrast, mutant A4c is fully active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptional cascade that culminates in the binding of chicken ovalbumin induced regulatory protein or protein complex (Chirp-I) to a DNA element from -891 to -878 in the SDRE.
Dean
D M
DM
Department of Biochemistry, University of Minnesota, Minneapolis 55455, USA.
Jones
P S
PS
Sanders
M M
MM
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D003345:Corticosterone / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006147:Guanine; D007328:Insulin / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D010047:Ovalbumin / Q000096:biosynthesis*; D010057:Oviducts / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D017931:DNA Primers; D004268:DNA-Binding Proteins; D004967:Estrogens; D007328:Insulin; D011994:Recombinant Proteins; D006147:Guanine; D010047:Ovalbumin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003345:Corticosterone