Genome-Wide Resources for Transcriptional Enhancers Active in the Human Heart

描述（由申请人提供）：心脏病是全球死亡的主要原因，但先天性心脏缺陷和成人心脏疾病的遗传风险因素尚不完全清楚。远距离调控序列（增强子）的变异可能通过基因表达调控对心脏发育和疾病有重要影响。然而，人类基因组中心脏增强子的基因组位置和体内功能在很大程度上仍然未知，这阻碍了建立增强子与心脏发育和临床心脏表型之间的机制联系的努力。我们的实验室率先采用ChIP-seq技术进行全基因组增强子鉴定，并通过转基因小鼠实验进行体内功能增强子鉴定。在这里，这些技术将被用来解决对人类基因组中心脏增强子位置和心脏发育和疾病状态活动的准确注释的迫切需要。尽管小鼠模型和人类细胞系的体外研究具有普遍价值，但我们已经表明，人类和小鼠心脏增强子在序列和功能保护方面存在显著差异，并且从特定细胞系生成的增强子图谱捕获了在异质组织（如心脏）中活跃的调控元件的有限片段。本提案旨在直接从代表胎儿和成人心脏不同发育阶段和亚区域的人体组织中表征心脏增强子和相关基因表达，目的是生成对基础和临床心脏研究具有重要价值的数据集。我们还建议研究心脏增强子活性和基因表达的个体间差异，以表征健康受试者和心脏病患者之间的差异。在原理证明研究中，我们已经证明了这种方法的一般可行性和科学影响。本提案的具体目的包括：1)我们将执行ChIP-seq靶向增强子相关的表观基因组标记，直接从人类心脏组织的比较中识别体内增强子，包括产前和产后发育阶段、心脏亚区和主要成人心脏病状态。我们还将在相同的样品中通过RNA-seq进行转录组分析。2)我们将进行至少250个转基因小鼠实验，以表征候选发育和疾病相关心脏增强子的体内活性模式，并研究假定的有害非编码序列变异的功能影响。测试的基因座将根据生物医学兴趣选择，并将包括社区提名的心脏基因座和风险变异。3)我们将把这些结果作为心脏遗传学的综合社区资源，提供一个数据浏览和下载的门户网站，以及分子试剂和转基因小鼠，以实现心脏发育和疾病调控序列的下游研究。这项研究将揭示人类心脏发育、功能和疾病的调控格局，并将解决国家对基因组资源的迫切需求，以实现和加速心脏研究的进展。（摘要结束）

DESCRIPTION (provided by applicant): Heart disease is the leading cause of death worldwide, but the genetic risk factors for congenital heart defects and adult cardiac disorders are incompletely understood. Variation in distant-acting regulatory sequences (enhancers) is likely to contribute significantly to heart development and disease via regulation of gene expression. However, the genomic location and in vivo function of heart enhancers in the human genome remains largely unknown, hindering efforts to establish mechanistic links between enhancers and heart development and clinical cardiac phenotypes. Our laboratories have pioneered techniques for genome-wide enhancer identification by ChIP-seq and in vivo functional enhancer characterization via transgenic mouse experiments. Here, these techniques will be leveraged to address the pressing need for accurate annotation of heart enhancer location in the human genome and activity across heart developmental and disease states. Despite the general value of mouse models and in vitro studies of human cell lines, we have shown that there is significant divergence in sequence and functional conservation between human and mouse heart enhancers and that enhancer maps generated from specific cell lines capture a limited slice of the regulatory elements active in heterogeneous tissues such as the heart. The present proposal is aimed at characterizing heart enhancers and associated gene expression directly from human tissues representing different developmental stages and subregions of the fetal and adult heart, with the goal of generating datasets of significant value to basic and clinical cardiac research. We additionally propose to examine inter-individual variation in heart enhancer activity and gene expression, characterizing differences across healthy subjects and individuals with heart disease. In proof-of-principle studies, we have demonstrated the general feasibility and scientific impact of this approach. The specific aims of this proposal include: 1) we will perform ChIP-seq targeting enhancer- associated epigenomic marks to identify in vivo enhancers directly from comparisons of human heart tissues, including pre- and postnatal developmental stages, cardiac subregions, and major adult cardiac disease states. We will also perform transcriptome profiling by RNA-seq in the same samples. 2) We will perform at least 250 transgenic mouse assays to characterize in vivo activity patterns of candidate developmental and disease-relevant heart enhancers and study functional effects of putative deleterious non-coding sequence variation. Tested loci will be selected based on biomedical interest and will include community-nominated cardiac loci and risk variants. 3) We will present the results as an integrated community resource for heart genetics, providing a web portal for data browsing and download and molecular reagents and transgenic mice to enable downstream studies of regulatory sequences in heart development and disease. This research will reveal the regulatory landscape of human cardiac development, function, and disease, and will address the pressing national need for genomic resources to enable and accelerate the advancement of cardiac research. (End of Abstract)