Genomic, Transgenic and Knockout Resources for Craniofacial Enhancer Studies

用于颅面增强剂研究的基因组、转基因和基因敲除资源

• 批准号: 8850845
• 负责人: Axel Visel
• 金额: $ 63.89万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-20 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8850845
• 关键词: 8q24; Affect; Biological Assay; ChIP-seq; Code; Collaborations; Communities; Complement; Complex; Computational Biology; Computer Analysis; Conceptions; Congenital Abnormality; Craniofacial Abnormalities; DNA; Data; Data Set; Development; Disease; Distal; Distant; E1A-associated p300 protein; Embryo; Enhancers; Face; FaceBase; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Variation; Genetic study; Genome; Genomics; Health; Histones; Human; Human Genetics; Human Genome; Individual; Knock-out; Knockout Mice; Location; Maps; Modeling; Molecular Profiling; Morphology; Mus; Optics; Phenotype; Play; Postdoctoral Fellow; Predisposition; Process; Proteins; Protocols documentation; Regulator Genes; Reporter; Research; Research Personnel; Resolution; Resources; Role; Secondary Palate; Staging; Structure; Testing; Time; Tissues; Transgenic Mice; Transgenic Organisms; Untranslated RNA; Variant; Work; base; clinical care; comparative; craniofacial; craniofacial development; genome-wide; histone modification; human tissue; improved; in vivo; insight; member; morphometry; mouse genome; mouse model; new technology; novel strategies; programs; repository; spatiotemporal; tomography; trait; transcriptome sequencing; vector

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.

描述（由申请人提供）：遗传学研究表明，嵌入人类基因组大量非编码部分的远距离作用调节序列（增强子）在颅面发育和颅面出生缺陷易感性中发挥重要作用。这些远距离作用增强子的机制探索仍然是困难的，因为大多数颅面增强子的基因组位置和体内功能仍然未知。作为FaceBase 1的成员，我们为控制颅面发育的远端增强子生成了第一组注释和功能数据。这些资源被证明是颅面研究界的重要价值。然而，这些努力只捕获了一小部分在颅面发育过程中有活性的增强子。在这里，我们建议更全面地使用新的和互补的方法来表征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定位预测的增强子。在初步研究中，我们使用ChIP-seq与增强子相关蛋白p300来鉴定数千个增强子，这些增强子预计在小鼠面部e11.5和发育后期的次级腭中具有活性。使用ChIP-seq进行组蛋白修饰（H3 K4 me 1，H3 K27 ac，H3 K27 me 3），这将需要更少的组织，并将增强子发现的灵敏度提高一个数量级，我们将在三个发育阶段（e11.5，e13.5，e15.5）从发育小鼠面部的所有子区域获得更高分辨率的数据。我们将在人类胚胎面部组织上使用ChIP-seq来补充这种基于小鼠的努力，以确定在小鼠中功能上不保守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约200种颅面增强子。利用FaceBase 1期间建立的协议和合作，我们将继续生成急需的体内转基因检测，并伴有光学投影断层扫描，以表征FaceBase 2研究人员和外部团体确定的新颅面位点中的增强子。这将包括测试与颅面畸形相关的增强子变体。3)虽然增强剂有望对颅面形态产生显著影响，但缺乏功能研究。我们将产生一组10个敲除小鼠品系，其在颅面发育或疾病相关基因座处携带单个增强子的缺失。将使用表达谱分析和高分辨率形态测定法对小鼠进行表型分析。在初步研究中，我们发现三种不同的增强子缺失对颅面基因表达和形态有显著影响。通过我们的努力产生的所有数据集，载体，转基因胚胎和敲除小鼠品系将作为颅面研究社区的资源。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计划提供独特的研究机会并利用这些机会。