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道细胞身份规范的分子机制。我们已经确定了一个以前未被识别的图案化步骤，发生在胚胎第16.5天的小鼠。这一步骤涉及肠上皮中> 1，000个基因的上调，这一过程产生了定义肠与胃身份的独特上皮边界。我们称这个过程为非线性化。我们的证据也暗示转录因子Tcfec作为一个潜在的调节剂的再矿化。因此，本申请的具体目的是：1）确定Tcfec是否是肠上皮中的激活剂，2）表征在肠上皮细胞特化期间受Tcfec影响的下游途径，和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节的贡献。对于目标1，我们将通过RLM-RACE克隆Tcfec的肠形式，并使用基于细胞的荧光素酶报告基因测定来确定它们的转录活性。对于目标2，将在Caco-2细胞中操纵Tcfec表达，Caco-2细胞是一种重现肠上皮细胞分化的体外系统。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在胃中错误表达的影响。对于目标3，我们将产生转基因报告小鼠以确定由Tcfec CNE指导的表达模式。然后将通过诱变测试特异性转录因子结合位点的要求。这些研究除了有助于理解胃粘膜化本身外，还可能与胃肠上皮化生的病理状态有关，在这种病理状态下，胃内的细胞具有肠的特征。由于肠上皮化生被认为是某些形式胃癌发展的早期步骤，我们的研究结果最终可能会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.