Molecular mechanisms that specify the cardiac cell lineage

在这项研究中，我们通过分析Nkx2.5（一种心脏特异性转录因子）的增强子，研究了指定心脏细胞谱系的分子机制。我们分离了覆盖60kb Nkx2.5上游序列的BAC克隆，并生成了大约70个包含不同长度上游序列的报告基因构建。我们测量了大鼠新生心肌原代培养中每个结构体的荧光素酶活性，并确定了6个荧光素酶活性显著高的区域。我们将这6个DNA片段亚克隆到LacZ报告载体中，并产生了转基因小鼠。6个片段中有3个片段在体内表现出增强子活性。特别是，Nkx2.5上游5.2 - 6.4kb的DNA片段在从心新月到直心管的心肌细胞中表现出很强的活性。该DNA片段含有串联Smad结合位点，Smad结合位点的缺失几乎消除了培养心肌细胞中的增强子活性。这一结果表明，在心脏发育的早期阶段，Smad信号通路是Nkx2.5表达所必需的，并且可能在心脏细胞谱系的规范中起关键作用。我们现在正试图用转基因方法在体内证实这一结果。在这个片段中，我们还发现了另一个具有高报告活性的区域。由于该区域不含已知的顺式作用元件，我们尝试用酵母单杂交系统分离结合蛋白。

In this study, we investigated molecular mechanisms that specify the cardiac cell lineage by analyzing enhancers of Nkx2.5, a cardiac specific transcription factor. We isolated BAC clones that cover 60kb of upstream sequence of Nkx2.5 and generated approximately 70 reporter constructs containing various length of the upstream sequence. We measured luciferase activity of each construct in primary culture of rat neonatal cardiac myoctyes and identified 6 regions exhibiting significantly high luciferase activities. We subcloned these 6 DNA fragments into a LacZ reporter vector and generated transgenic mice. Out of the 6 fragments, 3 fragments showed enhancer activities in vivo. Particularly, a DNA fragment between 5.2 and 6.4kb upstream of Nkx2.5 exhibited strong activity in cardiac myocytes from the cardiac crescent to the straight heart tube. This DNA fragment contained tandem Smad binding sites and deletion of the Smad binding sites almost abolished the enhancer activity in cultured cardiac myocytes. This result suggested that the Smad signaling pathway was required for expression of Nkx2.5 at early stages of cardiac development and may play a critical role in the specification of the cardiac cell lineage. We are now trying to confirm this result in vivo using transgenic approach. In this fragment, we also found another region having high reporter activity in vitro. Since this region did not contain known cis-acting elements, we are trying to isolate binding proteins using yeast one-hybrid system.