Research Project: Genome-Wide Atlas of Craniofacial Transcriptional Enhancers

描述（申请人提供）：唇裂和腭裂是最常见的颅面先天性缺陷之一。它们可以作为孟德尔障碍的一部分与其他症状共同发生，但大多数病例是非综合征性的，并且具有复杂的病因。在某些情况下，被破坏的蛋白质编码基因已被确定为导致唇腭裂风险的因素。然而，来自全基因组关联研究（GWAS）的越来越多的证据表明，非编码区域的序列变异也对包括口面部裂在内的各种临床疾病有很大的影响。虽然据推测，许多这些变异通过影响远端作用的转录增强子的功能特性来影响疾病，但只有极少数这种调节变异的孤立例子被确定。这可能是由于人类基因组中绝大多数远作用增强子的基因组位置和功能仍然未知。为了解决在基因组尺度上识别参与面部和腭发育并可能与唇裂病因相关的增强子的迫切需要，我们在这里提出了一种整合的基因组和转基因小鼠策略来识别颅面增强子并表征其活性。具体来说，我们将使用一种ChlP-seq方法来鉴定在胚胎阶段小鼠面部和腭组织中活跃的全基因组增强子，这些增强子与口面部裂有关。我们将使用转基因小鼠增强子筛选，通过确定其体内活性模式来验证和表征这些增强子预测的130个细节。此外，我们将确定与疾病相关的GWAS变异，这些变异与我们将发现的颅面增强剂有关。然后，我们将在转基因增强子试验中测试和比较变异序列和正常序列，以了解其体内活性的差异。所有的基因组和体内数据集，以及通过这些实验开发的分子试剂，将通过Face Base项目提供给其他研究人员，以最大限度地提高它们的可用性，并加快中脸和腭发育以及口面部裂的生物医学和临床研究的进展。

DESCRIPTION (provided by applicant): Clefts of the lip and palate are among the most common craniofacial birth defects. They can co-occur with other symptoms as part of Mendelian disorders, but the majority of cases are non-syndromic and have a complex etiology. In some cases disrupted protein-coding genes have been identified as contributors to orofacial clefting risk. However, accumulating evidence from genome-wide association studies (GWAS) indicates that sequence variation in non-coding regions also strongly contributes to a variety of clinical disorders including orofacial clefting. While it is speculated that many of these variants affect disease through impacting on functional properties of distant-acting transcriptional enhancers, only very few isolated examples of such regulatory variation have been identified. This is likely due to the fact that the genomic location and function of the vast majority of distant-acting enhancers in the human genome remains unknown. To address the pressing need to identify on a genomic scale enhancers that are involved in face and palate development and likely relevant for clefting etiology, we propose here an integrated genomic and transgenic mouse strategy to identify craniofacial enhancers and characterize their activities. Specifically, we will use a ChlP-seq approach to identify genome-wide sets of enhancers that are active in mouse face and palate tissues at embryonic stages that are relevant for orofacial clefting. We will use a transgenic mouse enhancer screen to validate and characterize 130 of these enhancer predictions in detail by determining their in vivo activity patterns. Furthermore, we will identify disease-associated GWAS variants that map to craniofacial enhancers that we will have discovered. We will then test and compare the variant and normal sequences in the transgenic enhancer assay for differences in their in vivo activities. All of the genomic and in vivo datasets, as well as molecular reagents developed through these experiments will be made available to other investigators through the Face Base program in order to maximize their availability and accelerate the progress of biomedical and clinical studies of mid-face and palate development and orofacial clefting.