Transcriptional regulation of enteroendocrine cell differentiation by NeuroD

描述（由申请人提供）：哺乳动物肠道不断自我更新，肠道干细胞产生四种上皮细胞类型。肠内分泌细胞占上皮细胞总数的不到5%，但分泌的激素控制许多生理过程，包括食欲和饱腹感、胰岛素分泌和消化器官功能。转录因子Neurogenin 3启动肠内内分泌分化程序，并激活另一种基本螺旋环螺旋蛋白NeuroD的表达。NeuroD似乎协调肠内分泌细胞的末梢分化和细胞周期退出。在表达神经原素3的细胞中，Wnt信号的激活可诱导肠道神经内分泌肿瘤，而在表达神经原素3的细胞中，Wnt信号的激活则不会诱导肠道神经内分泌肿瘤，这表明神经原素3的表达代表了肠内分泌细胞分化的一个明显的后期阶段。NeuroD转录激活的机制尚不清楚，但初步结果表明与其他DNA结合蛋白CtBP和组蛋白修饰酶赖氨酸特异性去甲基化酶1 （LSD1）相互作用有关。由于在肠内分泌细胞中缺乏已确定的NeuroD靶点，因此很难理解这一重要转录因子在肠内分泌细胞分化中的作用。本提案的三个目标将解决NeuroD在肠内分泌细胞分化中的功能。目的1将研究NeuroD如何与Sp1、RREB1和LSD1在其已知靶标分泌素基因上结合，形成多蛋白共激活因子复合物。c端结合蛋白CtBP通常是一种辅助抑制因子，其矛盾的辅助激活因子功能将通过检查组蛋白修饰和CtBP和NeuroD在分泌素基因增强子上相关的蛋白质来表征。Aim 2的目标是通过确定NeuroD与Wnt/？-catenin。我们将在有条件地表达NeuroD或敲低NeuroD shRNA的转基因小鼠中研究NeuroD在体内抑制Wnt信号传导中的作用，以确定在分化的早期阶段，NeuroD的表达是否会阻止Wnt激活后神经内分泌肿瘤的发展，或者在神经d +细胞中，敲低NeuroD表达是否会消除Wnt激活后对肿瘤发展的阻碍。目的3将通过高通量测序（RNAseq）对小鼠小肠分离的NeuroD+细胞的RNA进行基因表达谱分析，鉴定正常肠内分泌细胞中的NeuroD调节基因。全基因组染色质占用研究（ChIPseq）将确定一个差异表达基因子集作为NeuroD的潜在直接靶点。

DESCRIPTION (provided by applicant): The mammalian intestine continuously renews itself as intestinal stem cells give rise to four epithelial cell types. Enteroendocrine cells represent less than 5% of the total number of epithelial cells but secrete hormones that control numerous physiological processes including appetite and satiety, insulin secretion, and digestive organ function. The transcription factor, Neurogenin 3, initiates the endocrine differentiation program in the intestine and activates expression of NeuroD, another basic helix loop helix protein. NeuroD appears to coordinate terminal differentiation of enteroendocrine cells with cell cycle exit. Activation of constitutive Wnt signaling in neurogenin 3 expressing cells induced intestinal neuroendocrine tumors whereas Wnt activation in NeuroD expressing cells did not, suggesting that NeuroD expression represents a distinct, later stage of differentiation of enteroendocrine cells. The mechanism of transcriptional activation by NeuroD is not well characterized but preliminary results indicate interactions with other DNA binding proteins, CtBP, and the histone modifying enzyme, lysine specific demethylase 1 (LSD1) are involved. The paucity of identified NeuroD targets in enteroendocrine cells, has made it difficult to understand the role of this important transcription factor in their differentiation. The three aims of this proposal will address the function of NeuroD in differentiating enteroendocrine cells. Aim 1 will examine how NeuroD associates with Sp1, RREB1, and LSD1 at one its known targets, the secretin gene to form a multiprotein coactivator complex. The paradoxical coactivator function of C-terminal binding protein, CtBP, which is generally a corepressor, will be characterized by examining histone modifications and proteins CtBP and NeuroD associate with at the secretin gene enhancer. The goal of Aim 2 will study the transcriptional mechanism of inhibition of Wnt signaling by NeuroD by determining the DNA binding proteins and coactivators/corepressor complexes that NeuroD associates with at promoters regulated by Wnt/?-catenin in vitro. The role of NeuroD in the inhibition of Wnt signaling in vivo will be examined in transgenic mice that either conditionally express NeuroD or a NeuroD knockdown shRNA to determine whether expression of NeuroD at an earlier stage of differentiation prevents development of neuroendocrine tumors following Wnt activation or whether knocking down NeuroD expression removes the block to developing tumors following Wnt activation in NeuroD+ cells. Aim 3 will identify NeuroD regulated genes in normal enteroendocrine cells by gene expression profiling by high throughput sequencing (RNAseq) of RNA from NeuroD+ cells isolated from mouse small intestine by a new method developed by the principal investigator. Genome-wide chromatin occupancy studies (ChIPseq) will identify a subset of differentially expressed genes as potential direct targets by NeuroD.