Functional characterization of craniofacial enhancers at single cell and single base pair resolution

摘要 颅面畸形是世界范围内最常见的人类出生缺陷， 每年有多达20万名新生儿患有某种类型的颅面缺陷。这些缺陷可以 作为涉及多个组织和器官的复杂综合征的一部分发生。的综合征形式 这些疾病已经成功地与包括TWIST 1在内的近500个基因相关联， 颅缝早闭和IRF6用于口面裂。然而，更常见的是，身体的其他部位都不 直接受累（50%颅缝早闭，70%口面裂）。全基因组关联研究 表明这种缺陷的遗传性，但绝大多数协会落在基因之外 这表明有缺陷的基因调控是这种缺陷发生的主要原因。基因 调节元件可以位于整个基因组中并且通常具有组织特异性活性， 这使得它们很难识别和预测它们控制的基因。本报告的总体目标 应用是识别发育中的人脸中存在的细胞类型， 将重要的调控元件表征为单碱基对分辨率。目标1 我们建议使用基于单细胞的方法系统地鉴定细胞类型的群体， 测量人类颅面基因组中的转录组和活性调节位点 妊娠4至5周的组织和胚胎10.5和11.5天的小鼠。在目标2中， 我建议在这些研究中确定调控序列和靶基因之间的物理相互作用， 相同的组织类型最后，在目标3中，我们将从这些组织中鉴定出调控元件， 在颅神经嵴细胞的细胞培养模型中进行测试。将评估这些增强剂的 当基因表达被抑制或从基因组中移除时对基因表达的影响。跌幅超过抽样误差的就 将测试每一种变异对基因表达的影响，以确定其中的重要位置。 我们提出的研究将产生最全面的看法，细胞类型活跃在 发展人类的面孔，并揭示个人非编码变异对基因的贡献 表情

Abstract Craniofacial abnormalities are some of the most commonly occurring human birth defects worldwide, with up to 200,000 children born every year with some type of craniofacial defect. These defects can occur as part of complex syndromes that involve multiple tissues and organs. The syndromic forms of these disorders have been successfully linked to nearly 500 genes including TWIST1 for craniosynostosis and IRF6 for orofacial clefting. However more frequently no other part of the body is directly involved (50% of craniosynostoses, 70% of orofacial clefts). Genome wide association studies indicate heritability for such defects, however the vast majority of associations fall outside of genes suggesting defective gene regulation is a major contributor to incidence of such defects. Gene regulatory elements can be located throughout the genome and typically have tissue-specific activity, making them difficult to identify and predict what gene they control. The overall objective of this application is to identify the cell types present in the developing human face and functionally characterize important regulatory elements that specify them as single base pair resolution. In Aim 1 we propose to systematically identify populations of cell types using single-cell based methods for measuring the transcriptome and active regulatory sites across the genome in human craniofacial tissue from 4 to 5 weeks of gestation and mouse from embryonic days 10.5 and 11.5. In Aim 2 we propose to identify physical interactions between regulatory sequences and target genes in these same tissue types. Finally, in Aim 3 we will identify regulatory elements from these tissues that can be tested in a cell culture model of cranial neural crest cells. These enhancers will be assessed for effects on gene expression when repressed or removed from the genome. Those with significant effects on gene expression will be tested for every variant to identify important locations within them. Our proposed studies will generate the most comprehensive view of the cell types active in the developing human face and reveal the contributions individual noncoding variants make on gene expression.