Bergad P L, Shih H M, Towle H C, Schwarzenberg S J, Berry S A
Department of Pediatrics, University of Minnesota, Minneapolis 55455, USA.
J Biol Chem. 1995 Oct 20;270(42):24903-10. doi: 10.1074/jbc.270.42.24903.
A growth hormone (GH)-inducible nuclear factor (GHINF) from rat liver has been purified to near homogeneity. On SDS-polyacrylamide gel electrophoresis and UV-cross-linking, a major band of mass approximately 93 kDa and a minor band of approximately 70 kDa are detected in the purified fraction. DNase I footprinting using purified GHINF yields a protected region of -149/-115 on the rat serine protease inhibitor 2.1 (Spi 2.1) promoter encompassed within the growth hormone response element (GHRE). Mutational analysis demonstrated that GHINF binds synergistically to two gamma-interferon-activated sites (GAS) within the GHRE, with the 3' element being the pivotal binding domain. Functional assays show that both GAS elements are necessary for full GH response. GHINF has no immunoreactivity with either a C-terminal Stat1 antibody or an N-terminal Stat3 antibody, while cross-reacting with a C-terminal Stat5 monoclonal antibody. GHINF will bind to two GAS elements from the Stat5 binding region of the beta-casein gene. These studies indicate that GHINF is a Stat5-related factor binding synergistically to two GAS elements to activate Spi 2.1 transcription.
一种来自大鼠肝脏的生长激素（GH）诱导型核因子（GHINF）已被纯化至近乎同质。在SDS-聚丙烯酰胺凝胶电泳和紫外线交联实验中，纯化组分中检测到一条主要条带，质量约为93 kDa，还有一条次要条带，约为70 kDa。使用纯化的GHINF进行DNase I足迹分析，在大鼠丝氨酸蛋白酶抑制剂2.1（Spi 2.1）启动子上产生了一个-149 / -115的受保护区域，该区域包含在生长激素反应元件（GHRE）内。突变分析表明，GHINF与GHRE内的两个γ-干扰素激活位点（GAS）协同结合，3'元件是关键的结合结构域。功能分析表明，两个GAS元件对于完整的GH反应都是必需的。GHINF与C端Stat1抗体或N端Stat3抗体均无免疫反应性，但与C端Stat5单克隆抗体发生交叉反应。GHINF将与β-酪蛋白基因Stat5结合区域的两个GAS元件结合。这些研究表明，GHINF是一种与Stat5相关的因子，它与两个GAS元件协同结合以激活Spi 2.1转录。
