Research Project: Genome-Wide Atlas of Craniofacial Transcriptional Enhancers

描述（由申请人提供）：唇腭裂是最常见的颅面出生缺陷之一。它们可以与其他症状共同出现，作为孟德尔疾病的一部分，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，蛋白质编码基因的破坏已被确定为口面裂风险的贡献者。然而，来自全基因组关联研究（GWAS）的累积证据表明，非编码区中的序列变异也对包括口面裂在内的各种临床疾病有很大贡献。虽然推测这些变体中的许多通过影响远距离作用的转录增强子的功能特性来影响疾病，但是仅鉴定了这种调节变异的非常少的孤立实例。这可能是由于人类基因组中绝大多数远距离作用增强子的基因组位置和功能仍然未知。为了解决迫切需要在基因组规模上识别参与面部和腭发育并可能与腭裂病因学相关的增强子，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ChIP-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组，这些增强子与口面裂相关。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详细验证和表征130个这些增强子预测。此外，我们将确定疾病相关的GWAS变体，这些变体映射到我们将发现的颅面增强子。然后，我们将在转基因增强子测定中测试和比较变体序列和正常序列的体内活性差异。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计划提供给其他研究人员，以最大限度地提高其可用性，并加速面中部和腭发育以及口面裂的生物医学和临床研究的进展。 公共卫生相关性：我们建议确定在面部和腭发育过程中调节单个基因活性的DNA元件，并在转基因小鼠实验中确定其功能。我们还将详细研究这些调节元件的子集，在唇腭裂患者中改变，以阐明它们在这些疾病中的作用。这项研究与NIDCR的使命直接相关，因为它有望进一步加深我们对颅面发育和疾病的遗传基础的理解。

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. PUBLIC HEALTH RELEVANCE: We propose to identify DNA elements that regulate the activity of individual genes during face and palate development and to define their function in transgenic mouse experiments. We will also examine in detail subsets of these regulatory elements that are altered in cleft lip and palate patients to elucidate their role in these disorders. This research is of direct relevance to NlDCR's mission as it is expected to further our understanding of the genetic basis of craniofacial development and disorders.