Functional characterization of cardiovascular disease associated cis-regulatory regions in vivo

体内心血管疾病相关顺式调控区的功能特征

• 批准号: 316478981
• 负责人: Dr. Sebastian Preißl
• 金额: --
• 依托单位: Laboratory of Gene Regulation
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/316478981?language=en
• 关键词: Functional; characterization; cardiovascular; disease; associated

Cardiovascular diseases are one of the major clinical challenges of our time. Precise spatio-temporal control of gene expression is required for proper cardiac development and heart function. Enhancers are cis-regulatory elements in non-coding regions of the genome that regulate gene expression in a cell-type specific manner. Single nucleotide polymorphisms (SNP) associated with cardiac diseases including congenital heart diseases and cardiac hypertrophy are enriched in enhancers. However, the functional role of specific enhancers is still mostly unknown. The overall aim of my project will be to characterize the molecular function of cardiomyocyte enhancers in vivo. I hypothesize that disease triggering enhancers affect gene expression of essential regulators of cardiomyocyte differentiation and function. The specific aims are:1) Characterization of the dynamics of gene regulatory promoter-enhancer networks during differentiation of cardiomyocytes from human embryonic stem cells (hESC) will be identified using high throughput analyses to study the 3D genome architecture.2) To determine enhancers and cardiac disease associated SNPs crucial for gene expression in human cardiomyocytes, I will employ the CRISPR/Cas9 genome-editing technology to manipulate the hESC genome. Resulting gene expression changes in hESC-derived cardiomyocytes will be examined to reveal functionally important sequence variants.3) To validate enhancer function in vivo, conserved regions of essential cardiomyocyte enhancers will be deleted in mouse embryonic stem cells using the CRISPR/Cas9 genome editing technology. These cells will be used for tetraploid complementation assays to generate genetic modified mouse lines. Finally, the cardiac phenotype as well as gene expression changes will be investigated.My work will uncover the molecular function of non-coding genomic elements in vivo and contribute to a better understanding of cardiac development and function. These results might pave the way to new therapeutic strategies for complex cardiac diseases by inferring with gene regulation processes.

心血管疾病是我们这个时代的主要临床挑战之一。基因表达的精确时空控制是心脏正常发育和心脏功能所必需的。增强子是基因组非编码区中的顺式调节元件，其以细胞类型特异性方式调节基因表达。单核苷酸多态性（SNP）与心脏疾病，包括先天性心脏病和心脏肥大的增强子富集。然而，特异性增强子的功能作用仍然是未知的。我的项目的总体目标将是表征心肌细胞增强剂在体内的分子功能。我假设疾病触发增强子影响心肌细胞分化和功能的重要调节因子的基因表达。具体目标是：1）使用高通量分析来研究3D基因组结构，以确定在从人胚胎干细胞（hESC）分化为心肌细胞期间基因调控启动子-增强子网络的动态特征。2）为了确定对人类心肌细胞中基因表达至关重要的增强子和心脏疾病相关SNP，我将采用CRISPR/Cas9基因组编辑技术来操纵hESC基因组。将检查hESC衍生的心肌细胞中产生的基因表达变化，以揭示功能上重要的序列变体。3）为了验证体内增强子功能，将使用CRISPR/Cas9基因组编辑技术在小鼠胚胎干细胞中删除必需心肌细胞增强子的保守区域。这些细胞将用于四倍体互补试验，以产生遗传修饰的小鼠品系。最后，研究心脏表型和基因表达的变化，揭示非编码基因组元件在体内的分子功能，有助于更好地理解心脏发育和功能。这些结果可能为复杂心脏疾病的新治疗策略铺平道路，通过推断基因调控过程。