Dean D M, Jones P S, Sanders M M
Department of Biochemistry, University of Minnesota, Minneapolis 55455, USA.
Mol Cell Biol. 1996 May;16(5):2015-24. doi: 10.1128/MCB.16.5.2015.
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.
由于类固醇激素诱导鸡卵清蛋白（Ov）基因需要伴随蛋白质合成，因此研究重点集中在确定Ov基因中不稳定转录因子的结合位点。先前的凝胶迁移率变动研究在-900至-853之间的类固醇依赖性调节元件（SDRE）中确定了一个这样的位点。为了确定雌激素和糖皮质激素是否影响这种不稳定蛋白的结合，通过用硫酸二甲酯处理原代输卵管细胞培养物对Ov基因进行了基因组足迹分析。观察到了几种变化，包括鸟嘌呤残基-889和-885的类固醇依赖性保护以及腺嘌呤残基-892和-865的超敏反应。特别重要的是，体内足迹分析数据得到了两项功能研究的证实，一项是连接子扫描突变研究，另一项是点突变研究。-900至-878之间的10个碱基对连接子扫描突变严重降低了雌激素和糖皮质激素的诱导作用。同样，受保护鸟嘌呤残基的点突变极大地减弱了对这些类固醇激素的反应。此外，体外结合活性与体内功能活性相关。例如，突变体A4e对类固醇激素无转录活性，相应的寡聚物在体外不结合蛋白质。相反，突变体A4c在这两种情况下均具有完全活性。这些数据支持了这样的观点，即卵清蛋白基因受类固醇激素诱导的转录级联调节，最终导致鸡卵清蛋白诱导调节蛋白或蛋白复合物（Chirp-I）与SDRE中-891至-878的DNA元件结合。
