脂肪组织KLF14蛋白的代谢调节功能及分子机制研究

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 adipose 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.

人群全基因组关联分析(GWAS)发现，位于KLF14基因组区域的单核苷酸多态性(SNPs)与2型糖尿病的发病率、血液高密度胆固醇(HDL-C)水平以及甘油三酯水平显著相关；人群脂肪组织表达谱分析进一步发现，这些SNPs不仅与脂肪组织KLF14表达水平相关(cis-eQTL)，同时与脂肪组织若干其他代谢相关基因表达水平也显著关联(trans-eQTL)，提示脂肪组织KLF14表达变化可能调节整体代谢平衡。在前期研究中，我们以及其他团队研究发现Klf14全身敲除小鼠出现2型糖尿病症状，但脂肪组织特异KLF14的功能和分子机制仍有待探讨。本项目拟综合基因敲除多能干细胞、脂肪组织条件敲除和Klf14-FLAG敲入小鼠，深入研究脂肪组织KLF14的靶向基因、功能及其对整体代谢平衡的影响。研究将对深入解析KLF14的代谢调节功能以及2型糖尿病的遗传致病因素有重要意义。

人群全基因组关联分析(GWAS)发现，位于KLF14基因组区域的单核苷酸多态性(SNPs)与2型糖尿病的发病率、血液高密度胆固醇(HDL-C)水平以及甘油三酯水平显著相关；人群脂肪组织表达谱分析进一步发现，这些SNPs不仅与脂肪组织KLF14表达水平相关(cis-eQTL)，同时与脂肪组织若干其他代谢相关基因表达水平也显著关联(trans-eQTL)，提示脂肪组织KLF14表达变化可能调节整体代谢平衡。本项目综合基因敲除多能干细胞、全身敲除和脂肪组织条件敲除和Klf14-FLAG敲入小鼠，深入研究脂肪组织KLF14的靶向基因、功能及其对整体代谢平衡的影响。研究结果发现Klf14全身敲除和脂肪组织特异小鼠出现2型糖尿病症状，而KLF14作为转录因子，在脂肪组织中通过调节多个代谢基因的表达，影响机体的能量储存（脂肪合成）和能量消耗（肌肉运动）平衡。KLF14低表达，引起能量储存增加，糖尿病、冠心病发病率增加、血液甘油三酯升高、高密度胆固醇降低；相反，高KLF14表达促进能量消耗，因而糖尿病和冠心病发病率降低，血液甘油三酯降低，HDL升高，具有保护作用。本项目从人群遗传数据分析得到的疾病易感位点角度探讨人类代谢疾病的发病机制，项目一方面发现调节人类代谢平衡的新基因KLF14和新生物学机制，进一步加深了对代谢疾病发生的生物学机制理解；另一方面也同时为如何解析人群遗传数据，尤其是日益增多的GWAS数据，提供了典型参考案例。