GENOMIC AND TRANSGENIC RESOURCES FOR CRANIOFACIAL ENHANCER STUDIES

遗传学研究表明，嵌入人类基因组大量非编码部分的远端调控序列（增强子）在颅面发育和对颅面出生缺陷的易感性中起着重要作用。由于大多数颅面增强子的基因组位置和体内功能仍然未知，因此对这些远距离作用增强子的机制探索仍然很困难。作为FaceBase 1的成员，我们为控制颅面发育的远端增强器生成了第一组注释和功能数据。这些资源被证明对颅面研究界具有重要价值。然而，这些努力只捕获了一小部分在颅面发育过程中活跃的增强子。在这里，我们建议使用新的和互补的方法来更全面地描述颅面发育的基因调控景观。具体目标是：1)我们将通过ChIP-seq从胚胎小鼠和人类面部组织中绘制预测的增强子。在初步研究中，我们使用ChIP-seq和增强子相关蛋白p300鉴定了数千个增强子，这些增强子预计在e11.5岁时在小鼠面部和发育后期的次腭中具有活性。使用ChIP-seq对一组组蛋白修饰（H3K4me1, H3K27ac, H3K27me3），这将需要更少的组织，并将增强子发现的敏感性提高一个数量级，我们将从发育中的小鼠面部的三个发育阶段（e11.5, e13.5, e15.5）的所有亚区获得更高分辨率的数据。我们将利用人类胚胎面部组织的ChIP-seq来补充这项基于小鼠的工作，以鉴定在小鼠中不具有功能保守性的人类特异性颅面增强子。2)在最初的研究中，我们在转基因报告基因试验中鉴定了大约200种颅面增强剂。利用FaceBase 1期间建立的协议和合作，我们将继续进行急需的体内转基因试验，并辅以光学投影断层扫描，以表征由FaceBase 2研究人员和外部团体确定的新颅面基因座中的增强子。这将包括检测与颅面畸形相关的增强子变异。通过我们的努力产生的数据集、载体和转基因胚胎将作为资源提供给颅面研究界。我们坚定地致力于与Hub和其他Spoke项目的持续合作互动，为FaceBase项目提供的独特研究机会做出贡献并加以利用。

Genetic studies have shown that distant-acting regulatory sequences (enhancers) embedded in the vast non-coding portion of the human genome play important roles in craniofacial development and susceptibility to craniofacial birth defects. The mechanistic exploration of these distant-acting enhancers continues to be difficult because the genomic location and in vivo function of most craniofacial enhancers remains unknown. As members of FaceBase 1, we generated first sets of annotation and functional data for distal enhancers controlling craniofacial development. These resources proved to be of significant value to the craniofacial research community. However, these efforts captured only a small proportion of the enhancers that are active during craniofacial development in vivo. Here we propose to characterize the gene regulatory landscape of craniofacial development more comprehensively using new and complementary approaches. The specific aims are: 1) We will map predicted enhancers by ChIP-seq from embryonic mouse and human facial tissues. In preliminary studies, we used ChIP-seq with the enhancer-associated protein p300 to identify several thousand enhancers predicted to be active in the mouse face at e11.5 and in the secondary palate at later stages of development. Using ChIP-seq for a panel of histone modifications (H3K4me1, H3K27ac, H3K27me3), which will require less tissue and increase the sensitivity of enhancer discovery by an order of magnitude, we will obtain higher-resolution data from all subregions of the developing mouse face at three stages of development (e11.5, e13.5, e15.5). We will complement this mouse-based effort with ChIP-seq on human embryonic face tissue to identify human-specific craniofacial enhancers not functionally conserved in mice. 2) In initial studies we characterized ~200 craniofacial enhancers in vivo in transgenic reporter assays. Taking advantage of protocols and collaborations established during FaceBase 1, we will continue to generate critically needed in vivo transgenic assays accompanied by optical projection tomography to characterize enhancers residing in new craniofacial loci identified by FaceBase 2 investigators and outside groups. This will include testing of enhancer variants associated with craniofacial malformations. The datasets, vectors and transgenic embryos produced through our efforts will be made available as resources to the craniofacial research community. We are deeply committed to our ongoing collaborative interactions with the Hub and other Spoke projects, contributing to and taking advantage of the unique research opportunities enabled through the FaceBase program.