Transcriptional control of epithelial identity during pyloric border formation

描述（由申请人提供）：本申请的长期目标是阐明肠细胞身份规范的分子机制。我们已经确定了一个以前未被认识到的模式步骤，发生在小鼠胚胎期16.5天。这一步骤涉及肠上皮中bbbb1000基因的上调，这一过程产生了不同的上皮边界，定义了肠和胃的身份。我们称这个过程为肠道化。我们的证据也暗示转录因子Tcfec是肠化的潜在调节因子。因此，本应用程序的具体目的是：1)确定Tcfec是否是肠上皮的激活剂，2)表征肠上皮细胞分化过程中Tcfec影响的下游途径，以及3)分析Tcfec基因附近的保守非编码元件（CNE）对其在肠上皮中表达的时间调控的贡献。对于Aim 1，我们将通过RLM-RACE克隆肠道形式的Tcfec，并使用基于细胞的荧光素酶报告基因检测来确定它们的转录活性。在Aim 2中，Tcfec的表达将在Caco-2细胞中进行调控，Caco-2细胞是一个体外系统，可以再现肠上皮细胞的分化。我们还将建立几种小鼠模型来测试Tcfec在肠道中的需求，以及Tcfec在胃中的错表达的影响。在Aim 3中，我们将产生转基因报告小鼠，以确定Tcfec CNE指导的表达模式。对特定转录因子结合位点的需求将通过诱变来测试。这些研究除了有助于理解肠道化本身外，还可能与被称为胃肠化生的病理状态有关，在这种状态下，胃内的细胞具有肠道特征。由于肠化生被认为是某些形式胃癌发展的早期阶段，我们的研究结果可能最终影响这种通常致命的癌症的诊断或早期治疗。

DESCRIPTION (provided by applicant): The long-term objective of this application is to elucidate the molecular mechanism(s) underlying specification of intestinal cell identity. We have identified a previously unrecognized patterning step that takes place at embryonic day 16.5 in the mouse. This step involves the upregulation of >1,000 genes in the intestinal epithelium, a process that creates the distinct epithelial border that defines intestinal vs. stomach identity. We call this process intestinalization. Our evidence also implicates the transcription factor Tcfec as a potential regulator of intestinalization. Thus, the Specific Aims of this application are to: 1) determine if Tcfec is an activator in intestinal epithelium, 2) characterize the downstream pathways affected by Tcfec during intestinal epithelial cell specification, and 3) analyze the contribution of a conserved non-coding element (CNE) near the Tcfec gene to the temporal regulation of its expression in intestinal epithelium. For Aim 1, we will clone intestinal forms of Tcfec by RLM-RACE and determine their transcriptional activity using cell-based, luciferase reporter assays. For Aim 2, Tcfec expression will be manipulated in Caco-2 cells, an in vitro system that recapitulates intestinal epithelial cell differentiation. We will also establish several mouse models to test the requirement for Tcfec in the intestine, as well as the effect of Tcfec misexpression in the stomach. For Aim 3, we will generate transgenic reporter mice to ascertain the pattern of expression directed by the Tcfec CNE. The requirement of specific transcription factor binding sites will then be tested by mutagenesis. In addition to their contribution to the understanding of intestinalization per se, these studies may well be relevant to the pathologic state known as intestinal metaplasia of the stomach, in which cells within the stomach take on intestinal character. Since intestinal metaplasia is thought to represent an early step in the development of some forms of gastric cancer, our findings could eventually impact diagnosis or early treatment of this often fatal cancer.