Transcription Factor 7-like 2 Gene Regulatory Networks in Hypothalamic Neurons

下丘脑神经元中转录因子 7 样 2 基因调控网络

• 批准号: 9116616
• 负责人: Iriscilla Imabary Ayala
• 金额: $ 3.17万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9116616
• 关键词: Address; Adult; Affect; Binding Sites; Bioinformatics; Biological; Biological Assay; Biological Process; Birth; Brain; Cell Nucleus; ChIP-seq; Communication; Data; Data Analyses; Development; Diabetes Mellitus; Environment; Etiology; Family; Fasting; Foundations; Functional disorder; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Genetic Transcription; Genetic Variation; Glucose; Glutamates; Hepatic; Homeostasis; Hormonal; Hormones; Human; Hypoglycemia; Hypothalamic structure; In Vitro; Insulin; Intervention; Knockout Mice; Knowledge; Lead; Leptin; Measures; Mediating; Metabolic; Metabolic Pathway; Molecular Profiling; Mus; Nervous System Physiology; Neuraxis; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Organ; Pancreas; Pathogenesis; Pathway interactions; Peptides; Peripheral; Physiological; Play; Population; Prosencephalon; Proteins; Recording of previous events; Regulation; Regulator Genes; Research; Research Personnel; Research Technics; Risk; Risk Factors; Role; Scientist; Single Nucleotide Polymorphism; Small Interfering RNA; Solid; Stimulus; System; TCF7L2 gene; Tamoxifen; Technology; Testing; Time; Tissues; Training; United States; adiponectin; blood glucose regulation; career; diabetes risk; diabetic; energy balance; gastrointestinal; glucose disposal; glucose production; glucose tolerance; in vivo; in vivo Model; induced pluripotent stem cell; insulin secretion; insulin sensitivity; knock-down; male; mouse model; nerve supply; neuroregulation; protein expression; public health relevance; recombinase-mediated cassette exchange; response; stem cell technology; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): Type 2 diabetes (T2D) affects approximately 8.3% of the United States population. Family history is one of the major risk factors for T2D noncoding genetic variation in the transcription factor 7-like 2 (TCF7L2) gene is significantly associated with T2D in multiple ethnic populations, yet the biological mechanism(s) that lead to increased risk are still unclear. Evidence for involvement of the central nervous system (CNS) in regulating fasting and postprandial glucose levels continues to grow, and TCF7L2 localization in the hypothalamus suggests a potential role in glucose regulation. However, the genes and networks regulated by TCF7L2 in the hypothalamus are not known. This project will elucidate those genes and networks in human neurons using chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression profiling of induced pluripotent stem cells (iPSC)-derived neurons from adult male subjects of the San Antonio Family Diabetes Study (SAFDS). Two iPSC from each subject will be differentiated into NPY-expressing glutamatergic neurons. siRNA will be used to knockdown TCF7L2 expression. Using these human neuronal cultures, ChIP- Seq will be performed pre- and post-knockdown to identify potential target genes of TCF7L2. From the same neuronal cultures, gene expression profiles will be assayed using RNAseq in order to correlate the bound sites with changes in expression of the target genes. Bioinformatic analysis such as gene set enrichment analysis will be used to identify pathways TCF7L2 may influence. We will also investigate genes expression profiles during various environmental challenges such as high glucose, leptin and adiponectin to determine if regulation of biological networks changes in response. Regulation of glucose homeostasis by CNS and TCF7L2 will be further elucidated with an in vivo mouse model containing a conditional knockdown of TCF7L2 specific to the hypothalamus. Tamoxifen-induced knockdown will be implemented in adult mice. We will detect alterations in glucose homeostasis through several physiological measures. We shall also isolate the hypothalamus to measure changes in RNA and protein expression in the same metabolic pathways as described previously. Information regarding diabetes-related regulatory function of TCF7L2 in the hypothalamus may help to elucidate CNS pathways potentially disrupted in diabetes pathogenesis and therefore targets for intervention. This study is the first to address TCF7L2 function of glucose homeostasis in human neurons. This study also applies iPSC technology providing an ample supply of otherwise difficult to obtain neurons. The training outlined here will provide a solid foundation in research techniques, data interpretation, planning, scientific communication and professionalism to prepare me for a career as an independent research scientist.

 描述(由申请人提供)：2型糖尿病(T2D)影响大约8.3%的美国人口。家族史是T2D非编码遗传变异的主要危险因素之一，在多个民族人群中，转录因子7样蛋白2(TCF7L2)基因与T2D显著相关，但导致T2D风险增加的生物学机制(S)尚不清楚。中枢神经系统(CNS)参与调节空腹和餐后血糖水平的证据继续增加，TCF7L2在下丘脑的定位表明在血糖调节中可能发挥作用。然而，TCF7L2在下丘脑中调控的基因和网络尚不清楚。该项目将使用染色质免疫沉淀测序(ChIP-Seq)和来自圣安东尼奥家族糖尿病研究(SAFDS)成年男性受试者的诱导多能干细胞(IPSC)来源的神经元的基因表达谱来阐明人类神经元中的这些基因和网络。每个受试者的两个IPSC将被分化为表达NPY的谷氨酸能神经元。SiRNA将被用来敲除TCF7L2的表达。利用这些人类神经元培养物，将在基因敲除前后进行CHIP-SEQ，以确定TCF7L2的潜在靶基因。从相同的神经元培养中，将使用RNAseq分析基因表达谱，以便将结合部位与目标基因表达的变化相关联。生物信息学分析，如基因集浓缩分析，将被用来确定TCF7L2可能影响的途径。我们还将研究在各种环境挑战中的基因表达谱，如高糖、瘦素和脂联素，以确定生物网络的调节是否会因此而改变。中枢神经系统和TCF7L2对葡萄糖动态平衡的调节将通过含有下丘脑特异性TCF7L2的条件性敲除的体内小鼠模型进一步阐明。他莫昔芬诱导的基因敲除将在成年小鼠身上实施。我们将通过几种生理指标来检测葡萄糖稳态的变化。我们还将分离下丘脑，以测量与前面描述的代谢途径相同的RNA和蛋白质表达的变化。有关TCF7L2在下丘脑的糖尿病相关调节功能的信息可能有助于阐明糖尿病发病机制中可能被破坏的中枢神经系统通路，从而成为干预的靶点。这项研究首次研究了TCF7L2在人神经元中的葡萄糖稳态功能。这项研究还应用了IPSC技术，提供了大量原本难以获得的神经元。这里概述的培训将在研究技术、数据解释、规划、科学交流和专业精神方面提供坚实的基础，为我成为一名独立研究科学家做好准备。