Transcriptional regulation of enteroendocrine cell differentiation by NeuroD

NeuroD 对肠内分泌细胞分化的转录调控

基本信息

批准号：
8098191
负责人：
ANDREW B. LEITER
金额：
$ 39.38万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
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/8098191
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

项目摘要

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）：随着肠道干细胞产生四种上皮细胞类型，哺乳动物肠道不断自我更新。肠内分泌细胞占上皮细胞总数的不到 5%，但会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 启动肠道内的内分泌分化程序并激活 NeuroD（另一种基本螺旋环螺旋蛋白）的表达。 NeuroD 似乎可以协调肠内分泌细胞的终末分化和细胞周期退出。神经原素 3 表达细胞中组成型 Wnt 信号的激活诱导肠神经内分泌肿瘤，而 NeuroD 表达细胞中 Wnt 的激活则不然，这表明 NeuroD 表达代表了肠内分泌细胞分化的一个独特的后期阶段。 NeuroD 转录激活的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 (LSD1) 的相互作用也参与其中。由于肠内分泌细胞中缺乏确定的 NeuroD 靶标，因此很难理解这一重要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。目标 1 将研究 NeuroD 如何与 Sp1、RREB1 和 LSD1 在其已知靶标（促胰液素基因）上结合，形成多蛋白共激活复合物。 C 端结合蛋白 CtBP（通常是辅阻遏物）的矛盾共激活子功能将通过检查组蛋白修饰以及与促胰液素基因增强子相关的蛋白 CtBP 和 NeuroD 来表征。目标 2 的目标是通过确定 NeuroD 在体外与受 Wnt/β-catenin 调节的启动子相关的 DNA 结合蛋白和共激活子/辅阻遏物复合物来研究 NeuroD 抑制 Wnt 信号传导的转录机制。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 NeuroD 在分化早期的表达是否会阻止 Wnt 激活后神经内分泌肿瘤的发展，或者是否敲除 NeuroD 表达可以消除 NeuroD+ 细胞中 Wnt 激活后肿瘤形成的阻碍。目标 3 将通过主要研究者开发的新方法对从小鼠小肠分离的 NeuroD+ 细胞的 RNA 进行高通量测序 (RNAseq) 进行基因表达谱分析，从而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) 将确定差异表达基因的子集作为 NeuroD 的潜在直接目标。公共健康相关性：肠道内的少数细胞会因进餐、饥饿和其他刺激而产生激素。这些激素在控制其他重要的身体功能（如食欲、食物摄入、饱腹感和胰岛素分泌）方面发挥着重要作用。因此，肠道的正常激素分泌可能在预防或减轻肥胖和糖尿病（美国的两种主要疾病）或减轻其严重程度方面发挥重要作用。该提案将研究控制这些重要激素产生细胞发育的机制。