Genomic, Transgenic and Knockout Resources for Craniofacial Enhancer Studies

描述(申请人提供)：遗传学研究表明，嵌入在人类基因组的大量非编码部分的远程作用调控序列(增强子)在头面部发育和对头面部出生缺陷的易感性方面发挥着重要作用。由于大多数头面部增强剂的基因组位置和体内功能尚不清楚，因此对这些远程作用增强剂的机制探索仍然困难。作为FaceBase 1的成员，我们为控制颅面发育的远端增强剂生成了第一组注释和功能数据。事实证明，这些资源对颅面研究界具有重要价值。然而，这些努力只捕捉到了体内颅面部发育过程中活跃的一小部分增强剂。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图景。具体目标是：1)利用CHIP-SEQ从胚胎、小鼠和人类面部组织中定位预测的增强子。在初步研究中，我们使用CHIP-SEQ和增强子相关蛋白p300来鉴定几千个增强子，这些增强子预计在e11.5的小鼠面部和发育后期的次级上颚中都有活性。使用CHIP-SEQ进行组蛋白修饰(H3K4me1，H3K27ac，H3K27me3)，这将需要更少的组织，并将增强子发现的敏感度提高一个数量级，我们将从发育中的小鼠面部的三个阶段(e11.5，e13.5，e15.5)获得更高分辨率的数据。我们将用人类胚胎面部组织上的芯片序列来补充这一基于老鼠的努力，以识别在老鼠身上没有功能保守的人类特有的颅面增强剂。2)在最初的研究中，我们在转基因报告实验中鉴定了大约200个活体头面部增强剂。利用在FaceBase 1期间建立的协议和合作，我们将继续产生急需的体内转基因分析，并伴随着光学投影断层扫描，以表征驻留在FaceBase 2研究人员和外部小组确定的新的头面部基因座上的增强子。这将包括与颅面畸形相关的增强子变体的测试。3)虽然增强剂有望对颅面形态产生重大影响，但缺乏功能研究。我们将生成一个由10个基因敲除的小鼠品系组成的小组，携带头面部发育或疾病相关基因座上个别增强子的缺失。将使用表情图谱和高分辨率形态测量法对小鼠进行表型鉴定。在初步研究中，我们显示了三种不同增强子缺失对颅面基因表达和形态的显著影响。通过我们的努力产生的所有数据集、载体、转基因胚胎和基因敲除小鼠系将作为资源提供给颅面研究社区。我们坚定地致力于我们与The Hub和其他分支项目的持续合作互动，为通过FaceBase计划实现的独特研究机会做出贡献并加以利用。

DESCRIPTION (provided by applicant): 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. 3) While enhancers are expected to have significant impact on craniofacial morphology, functional studies are lacking. We will generate a panel of 10 knockout mouse lines carrying deletions of individual enhancers at craniofacial development- or disease- associated loci. Mice will be phenotyped using expression profiling and high-resolution morphometry. In preliminary studies, we showed significant effects of three different enhancer deletions on craniofacial gene expression and morphology. All datasets, vectors, transgenic embryos, and knockout mouse lines 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.