Williams Thomas M, Williams Melissa E, Heaton Joanne H, Gelehrter Thomas D, Innis Jeffrey W
Department of Human Genetics, University of Michigan Ann Arbor, MI, USA.
Nucleic Acids Res. 2005 Aug 8;33(14):4475-84. doi: 10.1093/nar/gki761. Print 2005.
Interactions with co-factors provide a means by which HOX proteins exert specificity. To identify candidate protein interactors of HOXA13, we created and screened an E11.5-E12.5, distal limb bud yeast two-hybrid prey library. Among the interactors, we isolated the BMP-signaling effector Smad5, which interacted with the paralogous HOXD13 but not with HOXA11 or HOXA9, revealing unique interaction capabilities of the AbdB-like HOX proteins. Using deletion mutants, we determined that the MH2 domain of Smad5 is necessary for HOXA13 interaction. This is the first report demonstrating an interaction between HOX proteins and the MH2 domain of Smad proteins. HOXA13 and HOXD13 also bind to other BMP and TGF-beta/Activin-regulated Smad proteins including Smad1 and Smad2, but not Smad4. Furthermore, HOXD13 could be co-immunoprecipitated with Smad1 from cells. Expression of HOXA13, HOXD13 or a HOXD13 homeodomain mutant (HOXD13(IQN>AAA)) antagonized TGF-beta-stimulated transcriptional activation of the pAdtrack-3TP-Lux reporter vector in Mv1Lu cells as well as the Smad3/Smad4-activated pTRS6-E1b promoter in Hep3B cells. Finally, using mammalian one-hybrid assay, we show that transcriptional activation by a GAL4/Smad3-C-terminus fusion protein is specifically inhibited by HOXA13. Our results identify a new co-factor for HOX group 13 proteins and suggest that HOX proteins may modulate Smad-mediated transcriptional activity through protein-protein interactions without the requirement for HOX monomeric DNA-binding capability.
与辅助因子的相互作用为HOX蛋白发挥特异性作用提供了一种方式。为了鉴定HOXA13的候选蛋白相互作用分子，我们构建并筛选了一个E11.5 - E12.5期远端肢体芽酵母双杂交猎物文库。在这些相互作用分子中，我们分离出了BMP信号效应分子Smad5，它与同源的HOXD13相互作用，但不与HOXA11或HOXA9相互作用，揭示了类AbdB型HOX蛋白独特的相互作用能力。利用缺失突变体，我们确定Smad5的MH2结构域对于与HOXA13的相互作用是必需的。这是首次报道HOX蛋白与Smad蛋白的MH2结构域之间存在相互作用。HOXA13和HOXD13还能与其他BMP和TGF-β/激活素调节的Smad蛋白结合，包括Smad1和Smad2，但不与Smad4结合。此外，HOXD13可以与细胞中的Smad1进行共免疫沉淀。HOXA13、HOXD13或HOXD13同源异型域突变体（HOXD13(IQN>AAA)）的表达拮抗了Mv1Lu细胞中TGF-β刺激的pAdtrack - 3TP - Lux报告载体的转录激活以及Hep3B细胞中Smad3/Smad4激活的pTRS6 - E1b启动子。最后，利用哺乳动物单杂交试验，我们发现HOXA13能特异性抑制GAL4/Smad3 - C末端融合蛋白的转录激活。我们的结果鉴定出了HOX第13组蛋白的一种新的辅助因子，并表明HOX蛋白可能通过蛋白质 - 蛋白质相互作用调节Smad介导的转录活性，而无需HOX单体的DNA结合能力。
