Developmental Program and Genetic Disease by Six family genes.

本研究旨在揭示6个家族基因在发育中的作用，阐明包括6个基因在内的基因网络及其在强直性肌营养不良(DM1)发病机制中的作用。我们对6个基因缺陷的小鼠进行了分析，筛选了6个蛋白的靶基因，并对6个蛋白的协同因子Eya和Dach蛋白的分子功能进行了分析。1Six4/Six5双基因敲除小鼠出生后数小时内死亡，但没有发现任何明显的解剖异常。SIX1基因缺陷小鼠出生后不久就会死亡，表现为内耳、鼻子、肾脏和胸腺的形成缺陷。E10-11出现形态异常。SIX1基因在这些器官的形成中起重要作用。2.鉴定了Six5蛋白的靶基因。在P19细胞中，转录因子、信号分子及其受体在中胚层分化过程中的表达被确定为可能的靶标。神经系统中的转录因子和信号分子，神经递质的转运蛋白和受体蛋白。在成肌细胞中，鉴定出了骨骼肌特异的基因，包括肌生成素、肌球蛋白、肌钙蛋白、乙酰胆碱受体。在晶状体上皮细胞中，已经发现了与白内障发生有关的基因。提示这些靶基因的调控改变导致了DM1.3的某些症状。我们揭示了GAL4-EYA和DACH的协同激活是通过CBP介导的。只有在GAL4-EYA和DACH存在的情况下，CBP才能结合到固定的染色质模板上。我们还发现，无论是否存在GAL4-Eya蛋白，Dach都可以与染色质和DNA结合。与染色质的结合亲和力高于与裸露DNA的结合亲和力。DACH保守的DD1结构域是DNA结合活性的决定因素。

This study aims to reveal roles of Six family genes in development, to elucidate gene network including Six and involvement of Six genes in pathology of myotonic dystrophy (DM1). We performed analyses of Six gene defective mice, screening of target genes of Six proteins and analyses of molecular function of Eya and Dach protein that are cooperative factors of Six protein.1 Six4/Six5 double knockout mice die within several hours after birth but we could not find any apparent anatomical anomalies. Six1 gene defective mice die just after birth and showed defective formation of inner ear, nose, kidney and thymus. The morphological abnormalities were noted from E10-11. Six1 gene is suggested to be essential for the formation of these organs.2 Target genes of Six5 proteins were identified. Transcription factors, signaling molecules and its receptors that are expressed during mesoderm differentiation were identified as putative target in P19 cells. Transcription factors and signaling molecules in nervous systems, transporters and receptor proteins of neural transmitters. In myoblasts, genes including myogenin, myosin, troponin, acetylcholine receptors that arespecific to skeletal muscle were identified. In lens epithelial cells, genes that had been shown to be involved in cataractogenesis were identified. It is suggested that altered regulation of these target genes leads to some symptoms of DM1.3 We revealed that cooperative activation by GAL4-Eya and Dach is mediated through CBP. CBP bound to an immobilized chromatin template only in the presence of both GAL4-Eya and Dach. We also found that Dach can bind to chromatin as well as DNA regardless of the presence of GAL4-Eya protein. The binding affinity to chromatin was higher than that to naked DNA. The conserved DD1 domain of Dach is responsible for the DNA binding activity.