Research Project: Genome-Wide Atlas of Craniofacial Transcriptional Enhancers

描述(申请人提供)：唇腭裂是最常见的头面部出生缺陷之一。作为孟德尔疾病的一部分，它们可以与其他症状并存，但大多数病例是非综合征的，病因复杂。在某些情况下，被破坏的蛋白质编码基因已被确定为口面部裂伤风险的贡献者。然而，来自全基因组关联研究的越来越多的证据表明，非编码区的序列变异也与包括口裂在内的各种临床疾病密切相关。虽然推测这些变异中的许多是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的，但只有极少数这种调控变异的孤立例子被识别出来。这可能是因为人类基因组中绝大多数远距离作用的增强子的基因组位置和功能尚不清楚。为了解决在基因组水平上识别与面部和腭部发育有关的增强剂的迫切需要，我们在这里提出了一种基因组和转基因小鼠相结合的策略来识别颅面增强剂并表征其活性。具体地说，我们将使用ChlP-seq方法来识别全基因组范围的增强子集，这些增强子在胚胎阶段的小鼠面部和上颚组织中活跃，与口面部分裂相关。我们将使用转基因小鼠增强子筛选来通过确定它们在体内的活动模式来详细验证和表征其中130个增强子预测。此外，我们将识别与我们将发现的颅面增强剂相对应的疾病相关GWA型变种。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，以确定它们在体内活性的差异。通过这些实验开发的所有基因组和活体数据集以及分子试剂将通过Face Base计划提供给其他研究人员，以最大限度地提高它们的可用性，并加快面中部和腭部发育和口面部裂隙的生物医学和临床研究的进展。 与公共健康相关：我们建议确定在面部和上颚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它们的功能。我们还将详细研究在唇腭裂患者中改变的这些调节元件的子集，以阐明它们在这些疾病中的作用。这项研究与NlDCR的任务直接相关，因为它有望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.