Regulation of the Chicken Ovalbumin Gene by Estrogen and Corticosterone Requires a Novel DNA Element That Binds a Labile Protein, Chirp-I

Abstract

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.Eukaryotic genes are primarily regulated by the modulation of the initiation of transcription via the binding of proteins to DNA flanking the coding sequences of genes. Two basic models have been proposed to explain the effects that trans-acting proteins have on transcription rates when bound to their cisacting DNA elements (42). The first of these models involves altering the topology of the DNA by assembling a three-dimensional multimeric complex. The roles of several proteins that appear to be involved in rearranging chromatin structure are explained by this model. The second model invokes protein-protein interactions between the bound transcription factors and members of the general transcriptional machinery (42). These models are not mutually exclusive, and both rely on the ability of trans-acting factors to recognize a specific DNA element(s) in order to impart specificity to the response.Steroid receptors apparently affect the initiation of transcription through both of the mechanisms described above, chromatin remodeling (3) and direct protein-protein interaction (17). Steroid hormones activate transcription by binding to specific receptor proteins, which then bind to well-defined DNA sequences in specific steroid-regulated genes (21). The steroid receptor binding sites or steroid response elements (SREs) behave as true enhancers....

A Winged-Helix Family Member Is Involved in a Steroid Hormone-Triggered Regulatory Circuit**This work was supported by NIH Grants RO1-DK-40082 (to M.M.S.) and T32-DK-0703 (to D.M.D.).

“…1, in bold, and Table 1). Interestingly, this site is encompassed in the in vivo footprint for Chirp-I (8). When this binding site was further examined, a 9-out-of-10-bp identity was found to the consensus sequence for the W-H family and a 7-out-of-7-bp identity was found to the core HNF-3 binding sequence of the rat tyrosine amino transferase (TAT) gene (Table 1).…”

“…1) (6,8,9,27). In vivo footprinting (8) demonstrated steroid-dependent protection of guanine residues Ϫ889 and Ϫ885 and hypersensitivity of adenine residue Ϫ892 within site A (Fig.…”

“…Complete loss of function was observed when a double mutant was constructed: G(Ϫ885)c, G(Ϫ889)a (Fig. 2, mutant A4e) (8).…”

“…Therefore, the Ov gene presents an excellent model system for studying the indirect and possibly hierarchical regulation of transcription by steroid hormones. The steroid regulation of the chick Ov gene maps to a 112-bp hormone response unit called the steroid-dependent regulatory element (SDRE) that is nearly 1 kb upstream from the transcriptional start site (6,8). Analysis of the SDRE indicates that it binds at least three cycloheximide-sensitive nuclear proteins (9).…”

“…Analysis of the SDRE indicates that it binds at least three cycloheximide-sensitive nuclear proteins (9). One of these, chicken Ov-induced regulatory protein-I (Chirp-I), binds in vivo only in response to treatment with estrogen and glucocorticoid (8). Therefore, the steroidal induction of the Ov gene is believed to be through the regulation of the assembly of essential transcription factors on the SDRE.…”