Transcriptional regulation of enteroendocrine cell differentiation by NeuroD

NeuroD 对肠内分泌细胞分化的转录调控

• 批准号: 8281566
• 负责人: ANDREW B. LEITER
• 金额: $ 39.09万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8281566
• 关键词: Accounting; Address; BHLH Protein; C-terminal binding protein; Cell Cycle; Cell Differentiation process; Cell Line; Cell Proliferation; Cell secretion; Cells; Chromatin; Complex; DNA-Binding Proteins; Data; Desire for food; Development; Diabetes Mellitus; Disease; Eating; Endocrine; Enhancers; Enterocytes; Enteroendocrine Cell; Enzymes; Epigenetic Process; Epithelial Cells; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Goblet Cells; Histones; Homeostasis; Hormones; Hunger; In Vitro; Insulin; Intestinal Neoplasms; Intestinal Neuroendocrine Neoplasm; Intestines; Lysine; Methods; Modeling; Molecular; Multiprotein Complexes; Mus; Neuroendocrine Tumors; Obesity; Organ; Pancreas; Paneth Cells; Physiological Processes; Play; Population; Principal Investigator; Proliferating; Protein C; Proteins; RNA; Regulation; Repression; Rodent; Role; Satiation; Secretin; Severities; Signal Transduction; Small Intestines; Staging; Stem cells; Stimulus; Transcriptional Activation; Transcriptional Regulation; Transgenic Mice; Transgenic Organisms; Work; base; cdc Genes; cell type; genome-wide; histone modification; in vivo; insight; insulin secretion; intestinal crypt; knock-down; mouse Neurog3 protein; novel; precursor cell; prevent; programs; promoter; public health relevance; response; self-renewal; small hairpin RNA; stem cell population; transcription factor; tumor; tumorigenesis

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. PUBLIC HEALTH RELEVANCE: A small number of the cells lining the intestine produce hormones in response to meals, hunger, and other stimuli. These hormones have a major role in the control other important body functions such as appetite, food intake, satiety, and insulin secretion. Thus, normal hormone secretion from the intestine may have an important role in preventing or reducing the severity of obesity and diabetes, two major diseases in the U.S. This proposal will study mechanisms controlling the development of these important hormone-producing cells.

描述（由申请人提供）：哺乳动物的肠不断更新自身，因为肠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%，但分泌控制许多生理过程的激素，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启动肠内分泌分化程序，并激活另一种碱性螺旋环螺旋蛋白NeuroD的表达。NeuroD似乎协调肠内分泌细胞的终末分化与细胞周期退出。在神经生成素3表达细胞中组成性Wnt信号传导的激活诱导肠神经内分泌肿瘤，而在NeuroD表达细胞中Wnt激活则没有，这表明NeuroD表达代表肠内分泌细胞分化的一个独特的后期阶段。NeuroD的转录激活机制尚未得到很好的表征，但初步结果表明与其他DNA结合蛋白，CtBP和组蛋白修饰酶，赖氨酸特异性脱甲基酶1（LSD 1）的相互作用。在肠内分泌细胞中鉴定的NeuroD靶点的缺乏使得难以理解这种重要的转录因子在其分化中的作用。该提案的三个目标将解决NeuroD在分化肠内分泌细胞中的功能。目的1将研究NeuroD如何与Sp1，RREB 1和LSD 1在其已知的靶点之一，分泌素基因，形成一个多蛋白辅激活因子复合物。C-末端结合蛋白CtBP通常是一种辅阻遏物，其反常的辅激活功能将通过检查组蛋白修饰和与促胰液素基因增强子相关的蛋白CtBP和NeuroD来表征。目的2的目标是通过确定DNA结合蛋白和辅激活子/辅抑制子复合物来研究NeuroD抑制Wnt信号传导的转录机制，NeuroD与Wnt/β-启动子调控的启动子相关。体外连环蛋白。将在条件性表达NeuroD或NeuroD敲低shRNA的转基因小鼠中检查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的表达是否阻止Wnt激活后神经内分泌肿瘤的发展，或者敲低NeuroD表达是否消除了NeuroD+细胞中Wnt激活后肿瘤发展的阻断。目的3将通过高通量测序（RNAseq）对从小鼠小肠分离的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NeuroD调控的基因。全基因组染色质占有率研究（ChIPseq）将确定差异表达基因的子集作为NeuroD的潜在直接靶点。 与公共卫生的关系：肠道内的一小部分细胞会产生激素，以应对进食、饥饿和其他刺激。这些激素在控制其他重要的身体功能中起着重要作用，如食欲，食物摄入，饱腹感和胰岛素分泌。因此，肠道正常的激素分泌可能在预防或减轻肥胖和糖尿病的严重程度方面发挥重要作用，这两种疾病是美国的两种主要疾病。