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（由申请人提供）：2 型糖尿病 (T2DM) 和冠状动脉疾病 (CAD) 是全世界发病和死亡的主要原因。开发新的、更有效的预防和治疗方法需要更好地了解疾病机制。最近，我们和其他人使用全基因组关联研究 (GWAS) 来识别与血脂水平密切相关的新基因位点。 CAD 和 T2DM 以及各种相关的代谢特征中最令人感兴趣的是位于 KLF14 基因上游的染色体 7q32 上的一个基因座，该基因编码一种假定的转录因子，尽管人们对该基因的生物学知之甚少。该基因座中的单核苷酸多态性 (SNP) 与高密度脂蛋白胆固醇 (HDL-C)、甘油三酯、CAD 和 T2DM 相关。对人类脂肪组织的性状位点 (eQTL) 研究已将这些相同的 SNP 与 10 个基因的表达联系起来，这些基因在数量上本身就含有与许多代谢特征相关的 SNP，包括体重指数 (BMI)、空腹胰岛素水平和空腹血糖水平基于这些数据，我们得出结论，KLF14 是一系列代谢基因表达的“主调节器”，这些基因共同影响高血糖、胰岛素抵抗、肥胖、血脂水平和 CAD——换句话说，许多代谢基因我们试图检验这一假设并更好地表征 KLF14 分子通路，基于我们的初步研究，我们建议使用尖端的聚类规则间隔短回文重复序列 (CRISPR)/CRISPR 相关的基因组编辑。 (Cas) 系统可敲除人类多能干细胞中的 KLF14 基因，敲除小鼠模型中全身或有条件代谢组织中的 Klf14 基因，并敲入 FLAG 标签我们将使用分化细胞、原代组织和整个动物来全面研究 KLF14 对基因表达和代谢表型的影响。快速有效的多物种​​方法，用于研究基因图谱实验发现的代谢基因的影响。