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（Conserved Non-protein coding Elements，CNE）是脊椎动物中高度保守的小于1 kb的DNA序列 从斑马鱼到人类。虽然他们的作用还没有完全理解，但他们是独联体的主要候选人。 调节功能，并可以作为增强剂。因为有些与人类生物学和疾病有关， 我们开发了一种方法来鉴定携带GWAS中鉴定的风险SNP的CNE。我们的方法集中在 在脊椎动物基因组中高度保守的CNE/SNPs区域，也在它们的基因组中保留基因同线性。 以确定潜在的顺式调节基因。基于GWAS重复，我们选择了20个CNE/SNPs 对和他们的同线基因可能有助于5个人类特征（睡眠/昼夜活动，皮肤 色素沉着，心血管系统，眼睛生物学，身体大小和形态学），可以在斑马鱼中建模。 两种CNE（1和19）的独立和深入的体内表征表明：（i）人CNE特异性 转录增强子活性可以在活的斑马鱼中显示，（ii）SNP消除这种活性的风险，（iii）SNP的转录增强子活性可以在活的斑马鱼中显示，（ii）SNP消除这种活性的风险， 可以发现与人类性状相关的真正的顺式调节基因，以及（iv）人类性状的基础基因。 生物学可以通过模拟斑马鱼的遗传缺陷来识别和研究。基于这些成功的 验证和令人兴奋的承诺，揭示了支持人类的分子和细胞生物学， 生物学性状，我们建议测试中心假设，即高度保守的非编码SNPs是调节 遗传变异导致基因表达和功能差异，影响人类健康。这 将通过以下具体目标对假设进行检验。目的1将决定转录活性的 其余18个保守的人类CNE和相关的风险SNP在体内，并建立mRNA模式， 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节基因： CNE的系统性CRISPR/Cas9编辑和mRNA（dys）调控分析。最后，目标3将确定 破坏CNE的遗传和生物学后果（缺失，引入风险SNP）及其顺式 调节基因（indels）。目的1将使用斑马鱼的转基因来证明人类CNE是 增强子，其功能被风险SNP破坏。Aim 2将使用基于CRISPR/Cas9的基因组编辑 在斑马鱼中删除所有18个CNE（DCNE）或在斑马鱼基因组中引入风险SNP（CNE*）以鉴定 顺式（dys）调节的同线相邻基因。目标3将比较增强子突变体的后果 (DCNE，CNE*）与顺式调节基因突变体，以揭示人类生物学的基础机制 和特质在斑马鱼中使用高通量CRISPR/Cas9介导的基因组编辑的方法 揭示人类CNE/SNPs的功能相关性是创新的。预计这项研究将在 意义重大，因为它将确定非编码遗传变异对人类性状/疾病的功能影响 并将揭示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.