Transcriptional control of epithelial identity during pyloric border formation

描述(申请人提供)：本申请的长期目标是阐明肠道细胞特性规范背后的分子机制(S)。我们已经确定了一个先前未知的图案化步骤，该步骤发生在小鼠胚胎16.5天。这一步骤包括上调肠上皮中的&gt；1000个基因，这一过程创造了明确的上皮边界，定义了肠道和胃的身份。我们称这一过程为肠化。我们的证据也表明转录因子Tcfec是肠化的潜在调节因子。因此，这一应用的具体目的是：1)确定Tcfec是否是肠上皮细胞的激活剂，2)在肠上皮细胞分化过程中Tcfec影响下游通路的特征，3)分析Tcfec基因附近保守的非编码元件(CNE)对其在肠上皮细胞表达的时间调控的贡献。对于目标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分析来确定它们的转录活性。对于Aim 2，Tcfec的表达将在Caco-2细胞中进行操纵，Caco-2细胞是一个重现肠上皮细胞分化的体外系统。我们还将建立几个小鼠模型来测试Tcfec在肠道中的需求，以及Tcfec在胃中错误表达的影响。对于目标3，我们将产生转基因报告鼠来确定Tcfec CNE指导的表达模式。然后，将通过突变来测试对特定转录因子结合位点的要求。除了对肠化本身的理解外，这些研究很可能与胃肠化生的病理状态有关，在胃肠化生中，胃内的细胞具有肠道特征。由于肠化生被认为是某些形式的胃癌发展的早期阶段，我们的发现最终可能会影响这种通常是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.