REGULATION OF THE CHICKEN OVALBUMIN GENE BY ESTROGEN AND CORTICOSTERONE REQUIRES A NOVEL DNA ELEMENT THAT BINDS A LABILE PROTEIN, CHIRP-I

Because induction of the chicken ovalbumin (Ov) gene by steroid hormon es requires concomitant protein synthesis, efforts have focused on def ining the binding site in the Ov gene for a labile transcription facto r, 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 t his labile protein, genomic footprinting of the Ov gene was done by tr eating primary oviduct cell cultures with dimethyl sulfate. Several al terations that include steroid-dependent protection of guanine residue s -889 and -885 and hypersensitivity of adenine residues -892 and -865 were observed, Of particular importance, the in vivo footprinting dat a are corroborated by two functional studies, one with linker-scanning mutations and the other with point mutations, Ten-base-pair linker-sc anning mutations between -900 and -878 severely reduced the induction by estrogen and glucocorticoid, Likewise, point mutations of the prote cted guanine residues profoundly attenuated the response to these ster oid hormones, in addition, in vitro binding activity correlated with i n vivo functional activity, For example, mutant A4e shows no transcrip tional activity in response to steroid hormones, and a corresponding o ligomer does not bind protein in vitro, In contrast, mutant A4e is ful ly active in both contexts. These data support the contention that the ovalbumin gene is regulated by a steroid hormone-induced transcriptio nal cascade that culminates in the binding of chicken ovalbumin induce d regulatory protein or protein complex (Chirp-I) to a DNA element fro m -891 to -878 in the SDRE.