Functional studies of KLF14, a putative master regulator of metabolism

KLF14（假定的代谢主调节因子）的功能研究

• 批准号: 8886598
• 负责人: Kiran Musunuru
• 金额: $ 38.03万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8886598
• 关键词: Adipocytes; Adipose tissue; Animals; Apolipoproteins; Atherosclerosis; Binding Sites; Biological Assay; Biological Models; Biology; Body mass index; Cell Line; Cells; Chromosome Mapping; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Coronary Arteriosclerosis; DNA; Data; Development; Diabetes Mellitus; Disease; Disease susceptibility; Dyslipidemias; Fasting; Fatty Acids; GNB1 gene; Gene Expression; Gene Targeting; Genes; Genetic; Genetic study; Gluconeogenesis; Glycerol; Goals; Hepatocyte; High Density Lipoprotein Cholesterol; Human; Hyperglycemia; Inherited; Insulin; Insulin Resistance; Knock-in Mouse; Knock-out; Knockout Mice; Link; Liver; Metabolic; Metabolic syndrome; Metabolism; Molecular; Morbidity - disease rate; Mouse Cell Line; Mus; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organism; Pathway interactions; Phenotype; Physiological; Pluripotent Stem Cells; Prevention approach; Proteins; Quantitative Trait Loci; Single Nucleotide Polymorphism; Skeletal Muscle; System; TPMT gene; Testing; Tissues; Triglycerides; Variant; Visceral; Work; base; blood lipid; chromatin immunoprecipitation; fasting glucose; fatty acid oxidation; gene discovery; genetic variant; genome editing; genome wide association study; glucose tolerance; glucose uptake; improved; in vivo; insulin tolerance; lipid biosynthesis; mortality; mouse model; novel; prevent; public health relevance; research study; response; subcutaneous; success; trait; transcription factor; transcription factor KLF13

 DESCRIPTION (provided by applicant): Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are leading causes of morbidity and mortality worldwide. Development of new and more effective approaches to prevention and treatment requires improved understanding of disease mechanisms. Genetic mapping in humans offers an approach to identify novel genes and DNA variants underlying the inherited contribution to disease susceptibility. Recently, we and others have used genome-wide association studies (GWAS) to identify novel genetic loci with strong association with blood lipid levels, CAD, and T2DM, along with a variety of related metabolic traits. Among the most intriguing of these loci is one on chromosome 7q32 that is just upstream of the KLF14 gene, which encodes a putative transcription factor. Although little is known of this gene's biology, single nucleotide polymorphisms (SNPs) in the locus are associated with high-density lipoprotein cholesterol (HDL-C), triglycerides, CAD, and T2DM. Expression quantitative trait locus (eQTL) studies in human adipose tissue have linked these same SNPs to the expression of ten genes that themselves harbor SNPs linked to a host of metabolic traits, including body mass index (BMI), fasting insulin levels, and fasting glucose levels. Based on these data, we hypothesize that KLF14 is a "master regulator" of the expression of a host of metabolic genes that together influence hyperglycemia, insulin resistance, obesity, blood lipid levels, and CAD-in other words, many of the cardinal features of the metabolic syndrome. We seek to test this hypothesis and better characterize the KLF14 molecular pathway. Building on our preliminary studies, we propose to use genome editing with cutting-edge clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems to knock out the KLF14 gene in human pluripotent stem cells, knock out the Klf14 gene in either the whole body or conditionally in metabolic tissues in mouse models, and knock in a FLAG tag into the endogenous KLF14 protein in mice with which to perform physiological ChIP experiments. We will use differentiated cells, primary tissues, and whole animals to comprehensively study the effects of KLF14 on gene expression and metabolic phenotypes. In doing this, we seek to establish a rapid and efficient multi-species approach with which to study the effects of metabolic genes discovered by gene mapping experiments.

 描述（由申请方提供）：2型糖尿病（T2 DM）和冠状动脉疾病（CAD）是全球发病率和死亡率的主要原因。开发新的和更有效的预防和治疗方法需要更好地了解疾病机制。人类遗传图谱提供了一种方法来识别新的基因和DNA变异的遗传贡献的疾病易感性。最近，我们和其他人使用全基因组关联研究（GWAS）来鉴定与血脂水平、CAD和T2 DM以及沿着各种相关代谢特征有强关联的新的遗传位点。这些基因座中最有趣的是位于染色体7 q32上的一个，该基因位于KLF 14基因的上游，该基因编码一种假定的转录因子。尽管对该基因的生物学知之甚少，但该基因座的单核苷酸多态性（SNP）与高密度脂蛋白胆固醇（HDL-C）、甘油三酯、CAD和T2 DM相关。人类脂肪组织中的表达数量性状基因座（eQTL）研究将这些相同的SNP与10个基因的表达联系起来，这些基因本身含有与许多代谢性状相关的SNP，包括体重指数（BMI），空腹胰岛素水平和空腹葡萄糖水平。基于这些数据，我们假设KLF 14是一个“主调节器”的主机代谢基因的表达，共同影响高血糖症，胰岛素抵抗，肥胖，血脂水平，CAD换句话说，许多代谢综合征的主要特征。我们试图验证这一假设，并更好地表征KLF 14分子途径。在我们的初步研究的基础上，我们建议使用具有尖端成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）系统的基因组编辑来敲除人类多能干细胞中的KLF 14基因，敲除小鼠模型中全身或代谢组织中的Klf 14基因，并将FLAG标签敲入小鼠的内源性KLF 14蛋白中，用其进行生理ChIP实验。我们将使用分化的细胞，原代组织和整个动物来全面研究KLF 14对基因表达和代谢表型的影响。在这样做的过程中，我们寻求建立一个快速，高效的多物种的方法来研究基因定位实验发现的代谢基因的影响。