BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用

Interaction among BRPF1,Its Novel Transcript BRPF2 and RHOX5 Protein

RHOX5基因是最早发现的小鼠RHOX基因簇(reproductive homeobox on the X chromosome)成员,可特异性地在生殖系统中表达。RHOX5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥途径尚不明确。在前期筛选与RHOX5蛋白相互作用的分子中初步获得一个BRPF1的新型转录本BRPF2。进一步构建pGBKT7-BRPF2质粒,酵母双杂交实验确定其与RHOX5蛋白的相互作用,GST-pull down实验确定其在体外的直接结合;PCR扩增BRPF1基因,构建pGBKT7-BRPF1和pGADT7-BRPF1质粒,酵母双杂交实验和GST-pulldown实验证明RHOX5蛋白亦可以直接结合BRPF1蛋白。BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用证实暗示了BRPF2极有可能与BRPF1竞争性结合RHOX5蛋白,为三种蛋白功能的研究提供了新的思路。

RHOX5 is the founding member of the reproductive homeobox on the X chromosome （RHOX） gene cluster. It is selectively expressed in male and female reproductive tissues. Studies based on its expression model indicated a crucial role of RHOX5 in the development of reproductive tissues and spermatogenesis. However, the molecular mechanisms underlying RHOX5 functions remained unclear. A novel transcript of BRPF1, BRPF2, was discovered interacting with RHOX5 protein during the screening of mouse 7 days embryo cDNA library using RHOX5 protein as bait. pGBKT7-BRPF2 plasmid was constructed and was used to confirm the interaction with RHOX5 using yeast two hybrid assay. In addition, the direct interaction between RHOX5 and BRPF2 was confirmed by GST-pull down assay. Moreover, BRPF1 gene was amplified and used to construct pGBKT7-BRPF1 and pGADT7-BRPF1 recombinant plasmids. The interaction between BRPF1 and RHOX5 was determined by yeast two hybrid assay and GST-pull down assay. The confirmation of the interaction between BRPF1, BRPF2 and RHOX5 suggested that BRPF2 may compete with BRPF1 for the interaction with RHOX5, which opens up the functional study on BRPF1, BRPF2 and RHOX5.