Molecular Mechanisms of Cardiomyocyte Differentiation

为了寻找与心脏同源盒转录因子Csx/Nkx 2 -5相互作用的蛋白质，我们利用酵母双杂交筛选技术，鉴定了一个新的含有LIM结构域的蛋白质Cal（CSX相关LIM蛋白）和一个含有Kruppel相关盒（KRAB）和11个锌指的蛋白质Zim 1。Csx/Nkx 2 -5和Cal通过Cal的LIM结构域和Csx/Nkx 2 -5的同源结构域彼此缔合。Cal本身具有转录促进活性，共转染Cal可增强Csx/Nkx 2 -5诱导的启动子激活。钙含有功能性核输出信号，并响应钙从细胞质穿梭到细胞核。Ca ~（2+）在P19 CL 6细胞核内的积累促进了心肌细胞的分化。Zim 1定位于心肌细胞核，并通过锌指与Csx/Nkx-2.5的N端和同源结构域结合。与Cal相反，Zim 1抑制Csx/Nkx-2.5介导的转录激活和瞬时过表达 ...更多信息 Zim 1抑制P19 CL 6细胞的心肌细胞分化。这些结果表明，Csx/Nkx 2 -5的转录活性是由微调的组合相互作用与coacitvators，如Cal和corepressors，如Zim 1。此外，通过差异显示技术，我们确定了一种新的蛋白激酶Midori（肌细胞诱导/分化发起者），这是在心脏发生早期表达，并参与P19 CL 6细胞的心肌细胞分化。为了研究Midori的发育功能，我们通过靶向诱变产生Midori缺陷小鼠。Midori的完全无效等位基因通过用核定位信号-LacZ-pA盒随后是loxP-侧翼的pgk-neo-pA盒替换其第一个编码外显子来产生。将携带突变等位基因的独立的2系ES细胞注射到来自C57 BL/6 J小鼠的胚泡中，并将雄性嵌合体与C57 BL/6 J小鼠杂交。我们将证实生殖系传播，杂合子杂交将产生后代，包括Midori缺陷小鼠。少

In search for proteins that interact with the cardiac homeobox transcription factor Csx/Nkx2-5, we identified a novel protein Cal (CSX-associated LIM protein) containing LIM domains and Zim1 containing Kruppel-associated box (KRAB) and 11 zinc fingers by using a yeast two-hybrid screening. Csx/Nkx2-5 and Cal associated with each other via the LIM domains of Cal and the homeodomain of Csx/Nkx2-5. Cal itself possessed the transcription-promoting activity, and co-transfection of Cal enhanced Csx/Nkx2-5-induced promoter activation. Cal contained a functional nuclear export signal and shuttled from the cytoplasm into the nucleus in response to calcium. Accumulation of Cal in the nucleus of P19CL6 cells promoted cardiomyocyte differentiation. Zim1 was localized at the nucleus of cardiomyocytes and was associated with N-terminal and homeodomain of Csx/Nkx-2.5 via its zinc fingers. In contrast to Cal, Zim1 suppressed Csx/Nkx-2.5-mediated transcriptional activation, and transient overexpression … More of Zim1 suppressed cardiomyocyte differentiation in P19CL6 cells. These results suggest that transcriptional activity of Csx/Nkx2-5 is regulated by fine-tuned combinatorial interactions with coacitvators such as Cal and corepressors such as Zim1. Furthermore, by differential display technique, we identified a novel protein kinase Midori (myocytic induction/differentiation originator), which is expressed early during cardiogenesis and is involved in cardiomyocyte differentiation of P19CL6 cells. To investigate the developmental function of Midori, we are generating Midori-deficient mice by targeted mutagenesis. A complete null allele of Midori was generated by replacing its first coding exon with a nuclear localization signal-LacZ -pA cassetete followed by a loxP-flanked pgk-neo-pA cassette. Independent 2 lines of ES cells carrying the mutated allele were injected into blastocysts from C57BL/6J mice and the male chimeras were crossed to C57BL/6J mice. We will confirm germ-line transmission, and hererozygous intercross will give rise to offsprings including Midori-deficient mice. Less