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

(改编自申请人的摘要)这一长期目标 该项目的目的是加深对基因调控的理解 通过阐明心脏特异的功能来实现心脏发育 同源框CSx/Nkappax-2.5蛋白。在小鼠体内，这种同源框蛋白是 在心脏从多能性转变的整个过程中表达于心脏 心前中胚层细胞向结构成熟。纯合子的小鼠 突变为突变这些基因的突变是胚胎。老鼠 这种基因突变的纯合子是胚胎致命的，因为 心脏发育停滞在分流前的循环阶段， Tanaka和Izumor，未出版)。之前的研究已经证明， 与其他同源框蛋白一样，CSX/Nkappax2.5是一种DNA结合蛋白 它可以反式激活特定启动子序列的表达，并 与其他体外转录相关。由于严重的表型 缺乏CSX/Nkappax2.5的小鼠在指导 心肌生长发育正常。这一点的继续表达 贯穿心脏结构发育和成人心脏的基因 提示CSX蛋白的靶基因可能也是 对保持出生后的心肌表型有重要意义。 转录因子级联调控的研究进展 小鼠心脏结构的生长和发育可能产生重要的 对先天性心脏病起源的洞察。关键要素 从无脊椎动物，如果蝇，到 脊椎动物，如鸡和老鼠。在之前的实验室研究中， 鉴定了小鼠CSX/Nkappax2.5基因及其人类同源物 并被确定为高度同源(100%氨基酸鉴定 在同源结构域、Tn结构域和Nk2结构域内)。这一点，连同 引导心脏功能的遗传级联基因高度保守 器官发生，提示研究CSX/Nkappax2.5蛋白的功能 而它的下游目标可能会对 人类先天性心脏病的分子基础。 因此，调查员将在#年处理以下具体目标 这项提议。具体目标1：充分描述表型 CSX/Nkappax2.5缺失纯合子和杂合子小鼠的分子生物学研究 生物学、生理学和形态分析。具体目标2： 体外测定CSX/Nkappax2.5的构效关系 高度突变的DNA结合和转录激活分析 保守的TN结构域、NK2特异性结构域和CD-II磷酸化 网站。具体目标3：确定TN-的生物学功能 结构域、NK-2特异性结构域和CKII磷酸化位点。 Nkappax2.5使用基因打靶和Cre-loxP介导的切除。特定的 目的4：诱导小鼠CSX/Nkappax2.5基因突变 房间隔缺损合并房室传导阻滞患者的体位分析 和(A)检测它们的DNA结合、反式激活和蛋白质-蛋白质 体外相互作用功能，以及(B)建立小鼠模型 基因打靶治疗先天性ASD伴发传导阻滞 先天性房间隔缺损伴发基因传导阻滞的治疗技术 靶向技术并对其形态、功能和功能进行研究 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靶向富集基因组DNA文库 和双纯合子CSX/N kappax2.5空ES的差异显示 携带四环素响应型CSX/Nkappax2.5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embryonic lethal due to an arrest in cardiac development at the looping stage prior to septation, Tanaka and Izumor, unpublished).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.