FUNCTIONAL ANALYSIS OF THE CARDIAC SPECIFIC HOMEOBOX GENE CSX/NKX2.5

（摘自申请人摘要）本研究的长期目标是 项目是为了进一步了解基因调控， 通过阐明心脏特异性 同源框Csx/Nkappax-2.5蛋白。在小鼠中，这种同源异型盒蛋白是 在心脏从多能性转变为 心前中胚层细胞结构成熟。a纯合子小鼠 这些基因的突变是胚胎的。小鼠 该基因突变纯合是胚胎致死的， 心脏发育在分隔前的成环阶段停止， Tanaka and Izumor，unpublished）.以前的研究表明， 与其他同源异型盒蛋白一样，Csx/Nkappax2.5是一种DNA结合蛋白 其可以反式激活来自特定启动子序列的表达， 在体外与其他转录相关。由于严重的表型 缺乏Csx/Nkappax2.5的小鼠在指导 正常的心肌生长和发育。这种持续的表达 在整个心脏结构发育和成年心脏中， 这表明Csx蛋白的靶基因也可能是基本的 出生后心肌表型的维持。 转录因子级联反应的研究 小鼠心脏结构的生长和发育可能产生重要的 先天性心脏病的病因的关键要素 在无脊椎动物如果蝇中， 脊椎动物，如鸡和老鼠。在实验室之前的研究中， 小鼠Csx/Nkappax2.5基因及其人类同源基因均被鉴定 并确定为高度同源（100%氨基酸同一性 在同源结构域、TN结构域和Nk 2结构域内）。再加上 高度保守的基因级联直接心脏 器官发生，表明Csx/Nkappax2.5蛋白功能的研究 及其下游目标可能会产生重要的见解， 人类先天性心脏病的分子基础 因此，调查人员将在以下方面实现以下具体目标： 这个提议。具体目标1：充分表征 Csx/Nkappax 2.5基因敲除小鼠的分子生物学研究 生物学、生理学和形态学分析。具体目标2： 通过体外测定Csx/Nkappax 2.5的结构-功能关系， DNA结合和转录激活试验，通过突变高度 保守的TN结构域、NK 2特异性结构域和CD-II磷酸化 网站.具体目标3：确定TN-1的生物学功能 结构域、NK-2特异性结构域和Csx-1的CKII磷酸化位点。 Nkappax 2.5，使用Cre-loxP介导的切除进行基因靶向。具体 目的4：在类似物中在鼠Csx/Nkappax 2.5中产生突变。 ASD伴AV传导阻滞患者中发现的位置 和（a）测定它们的DNA结合、反式激活和蛋白质-蛋白质结合 体外相互作用功能，和（B）建立 基因靶向治疗先天性房间隔缺损伴传导阻滞 先天性房间隔缺损伴传导阻滞基因治疗技术 靶向技术，并研究其形态，功能和 电生理异常具体目标5：确定 Csx/Nkappax2.5的转录靶点， 酵母单杂交筛选Csx靶富集基因组DNA文库 双纯合Csx/Nkappax2.5 null ES的差异显示 携带四环素应答性Csx/Nkappax 2.5转基因的细胞。

(Adapted from the Applicant's Abstract) The long-range goal of this project is to further the understanding of the genetic regulation of cardiac development by elucidating the function of the Cardiac-specific homeobox Csx/Nkappax-2.5 protein. In the mouse this homeobox protein is expressed in the heart throughout the heart's transition from pluripotent pre-cardiac mesoderm cells to structural maturity. Mice homozygous for a mutation for a mutation for a mutation for these gene are embryonic. Mice homozygous for a mutation for this gene are embryonic lethal due to an arrest in cardiac development at the looping stage prior to septation, Tanaka and Izumor, unpublished). Previous studies have demonstrated that, like other homeobox proteins, Csx/Nkappax2.5 is a DNA-binding protein which can transactivate expression from specific promoter sequences and associates with other transcription in vitro. Due to the severe phenotype of mice lacking the Csx/Nkappax2.5 play a critical role in directing normal myocardial growth and development. The continued expression of this gene throughout the heart's structural development and in the adult heart suggests that the target genes of the Csx protein may also be fundamental to the maintenance of the cardiac muscle phenotype post-natally. The investigation of the transcription factor cascade that controls cardiac structural growth and development in the mouse may yield important insights into the genesis of congenital heart diseases. The key elements of this cascade are conserved from invertebrates, such as Drosophila, to vertebrates, such as chicken and mice. In prior studies in the laboratory, both the mouse Csx/Nkappax2.5 gene and its human homologue were identified and determined to be highly homologous (100 percent amino acid identify within the homeodomain, TN domain and Nk2 domain). This, together with the high degree of conservation of the genetic cascade directing cardiac organogenesis, suggests that the studies Csx/Nkappax2.5 protein function and its downstream targets are likely to yield important insights into the molecular basis of congenital heart disease in humans. Accordingly, the investigators will address the following specific aims in this proposal. Specific Aim 1: To fully characterize the phenotype of homozygous and heterozygous Csx/Nkappax2.5 null mice by molecular biological, physiological and morphological analyses. Specific Aim 2: To determine structure-function relationships of Csx/Nkappax2.5 in vitro by DNA binding, and transcriptional activation assays by mutating the highly conserved TN domain, the NK2-specific domain, and CD-II phosphorylation sites. Specific Aim 3: To determine the biological function of the TN- domain, the NK-2 specific domain and CKII phosphorylation site of Csx- Nkappax2.5 using gene targeting with Cre-loxP mediated excision. Specific Aim 4: To create mutations in murine Csx/Nkappax2.5 in the analogous positions found in patients with ASD associated with AV conduction block and (a) to assay their DNA binding, transactivation and protein-protein interaction function in vitro, and (b) to create mouse models of congenital ASD associated with conduction block by gene-targeting technique of congenital ASD associated with conduction block by gene targeting technique and study their morphological, functional and electrophysiological abnormalities. Specific Aim 5: To identify transcriptional targets of Csx/Nkappax2.5 by combing the methods of the yeast one-hybrid screening of a Csx-target enriched genomic DNA library and differential display of double homozygous Csx/N kappax2.5 null ES cells that harbor a tetracycline responsive Csx/Nkappax2.5 transgene.