In vivo characterization of CNE/SNPs and identification of cis (dys)regulated genes

项目摘要 保守的非蛋白质编码元件(CNE)是脊椎动物中高度保守的1kb DNA元件 从斑马鱼到人类的基因组。虽然他们的作用还没有被完全理解，但他们是独联体的主要候选者- 具有调节功能，并可作为增强剂。因为有些人与人类生物学和疾病有关， 我们开发了一种方法来识别GWAS中发现的含有风险SNPs的CNE。我们的方法侧重于 CNE/SNPs区域在脊椎动物基因组中高度保守，在其基因组中也保留了基因同源性 以精确定位潜在的顺式调控基因。基于GWAS的复制，我们选择了20个CNE/SNPs 配对和它们的共线基因可能影响人类的5个特征(睡眠/昼夜活动、皮肤 色素沉着、心血管系统、眼睛生物学、身体大小和形态)，可以在斑马鱼中模拟。 两个CNE(1和19)的独立和深入的体内鉴定表明：(1)人CNE特异性 可以在活斑马鱼中发现转录增强子活性，(Ii)SNP取消这种活性的风险，(Iii) 可以发现与人类特征相关的真正的顺式调控基因，以及(Iv)支撑人类的 通过对斑马鱼的遗传缺陷进行建模，可以对生物进行鉴定和研究。基于这些成功的 验证和揭示支撑人类的分子和细胞生物学的令人兴奋的前景 生物学特征，我们建议检验深度保守的非编码SNP是调节的这一中心假说 基因变异导致影响人类健康的基因表达和功能的差异。这 假设将通过以下具体目标进行检验。目标1将确定基因的转录活性 其余18个在体内保守的人CNE及其相关的风险SNPs，并建立了 34个同线相邻基因。在后者中，Aim 2将通过以下方式识别实际的顺式调控基因 系统的CRISPR/Cas9编辑CNES和mRNA(Dys)调控分析。最后，目标3将确定 破坏CNES的遗传和生物学后果(缺失、引入风险SNP)及其顺式- 受调控的基因(INDELs)。目标1将使用斑马鱼的转基因来证明人类CNES是 其功能被风险SNP扰乱的增强剂。AIM 2将使用基于CRISPR/Cas9的基因组编辑 在斑马鱼基因组中删除所有18个CNE(DCNE)或在斑马鱼基因组中引入风险SNPs(CNE*)以识别 顺式(强式)调控的同线相邻基因。目标3将比较增强子突变的后果 (DCNE，CNE*)与顺式调控的基因突变，以揭示支撑人类生物学的机制 和特点。利用高通量CRISPR/Cas9介导的斑马鱼基因组编辑方法 揭示人类CNE/SNPs的功能相关性具有创新性。拟议的研究预计将是 意义重大，因为它将确定非编码遗传变异在人类特征/疾病中的功能影响 并将阐明与斑马鱼体内遗传建模相关的人类生物学。

Project Summary Conserved Non-protein coding Elements (CNEs) are <1kb DNA elements deeply conserved across vertebrate genomes from zebrafish to human. While their role is not fully understood, they are prime candidates for cis- regulatory function and can act as enhancers. As some have been implicated in human biology and diseases, we developed a method to identify CNEs harboring risk SNPs identified in GWAS. Our method focused on CNE/SNPs regions deeply conserved across vertebrate genomes that also preserve gene synteny in their neighborhood to pinpoint potential cis regulated genes. Based on GWAS replications, we selected 20 CNE/SNPs pairs and their syntenic genes potentially contributing to 5 human traits (sleep/circadian activity, skin pigmentation, cardiovascular system, eye biology, body size and morphology) that can be modeled in zebrafish. Independent and in depth in vivo characterization of two CNEs (1 and 19) showed that (i) human CNE specific transcriptional enhancer activity can be revealed in live zebrafish, (ii) the risk SNP abolishes this activity, (iii) the genuine cis-regulated gene associated to the human trait can be discovered, and (iv) the underpinning human biology can be identified and studied by modeling the genetic defect in zebrafish. Based on these successful validations and the exciting promise of shedding light on the molecular and cellular biology underpinning human biological traits, we propose to test the central hypothesis that deeply conserved non-coding SNPs are regulatory genetic variants responsible for differences in gene expression and function that affect human health. This hypothesis will be tested via the following specific aims. Aim 1 will determine the transcriptional activity of the remaining 18 conserved human CNEs and associated risk SNPs in vivo, and establish the mRNA patterns of the 34 syntenic neighbor genes. Among the latters, Aim 2 will identify the actual cis-regulated genes via systematic CRISPR/Cas9 editing of CNEs and mRNA (dys)regulation analysis. Finally, Aim 3 will identify the genetic and biological consequences of disrupting CNEs (deletion, introduction of risk SNP) and their cis- regulated genes (indels). Aim 1 will use transgenesis in zebrafish to demonstrate that human CNEs are enhancers whose functions are disrupted by the risk SNPs. Aim 2 will use CRISPR/Cas9-based genome editing in zebrafish to delete all 18 CNEs (DCNE) or introduce risk SNPs in the zebrafish genome (CNE*) to identify the syntenic neighbor genes that are cis-(dys)regulated. Aim 3 will compare the consequences of enhancer mutants (DCNE, CNE*) with cis-regulated gene mutants to uncover the mechanisms underpinning the human biology and traits. The approach of using high-throughput CRISPR/Cas9-mediated genome editing in zebrafish to uncover the functional relevance of human CNE/SNPs is innovative. The proposed research is expected to be significant because it will establish the functional impact of non-coding genetic variants in human traits/diseases and will shed light on the associated human biology with in vivo genetic modeling in zebrafish.