Golling G, Li L, Pepling M, Stebbins M, Gergen J P
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 11794-5215, USA.
Mol Cell Biol. 1996 Mar;16(3):932-42. doi: 10.1128/MCB.16.3.932.
The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators. Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined. Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt.
果蝇的矮胖基因是转录调节因子矮胖结构域家族的创始成员。哺乳动物的矮胖结构域基因编码异源DNA结合因子PEBP2/CBF的α亚基。不相关的PEBP2/CBFβ蛋白与矮胖结构域相互作用，以增加其对DNA的亲和力。果蝇矮胖蛋白对哺乳动物PEBP2/CBFβ刺激作用作出反应的保守能力表明，果蝇可能有一个同源的β蛋白。利用酵母双杂交系统分离与矮胖蛋白相互作用的蛋白质的cDNA，我们鉴定出两个果蝇基因，分别称为兄弟基因和大兄弟基因，它们与PEBP2/CBFβ有大量的序列同源性。酵母双杂交实验以及体外DNA结合研究证实了兄弟基因、大兄弟基因和PEBP2/CBFβ蛋白的功能同源性，并表明矮胖蛋白和兄弟蛋白的保守区域是它们异二聚体相互作用所必需的。我们还研究了矮胖结构域蛋白在有和没有其伴侣存在时的DNA弯曲特性。我们的结果表明，矮胖结构域蛋白会使DNA弯曲，并且这种弯曲受到兄弟蛋白家族成员的影响，这支持了异二聚化与矮胖结构域构象变化相关的观点。对果蝇胚胎表达模式的分析表明，兄弟基因和大兄弟基因可能在体内与矮胖基因相互作用，并且进一步表明这些蛋白质的活性并不局限于它们与矮胖蛋白的相互作用。
