Predicting Tissue Specific Gli3 Regulatory Activity Using Hand2

项目摘要 Hedgehog（Hh）信号通路对于正常胚胎发育是必不可少的，当受到干扰时， 经常导致人类疾病，包括那些影响颅面复合体发育的疾病。的 Gli转录因子是该通路的下游效应子，已成为许多研究的主题 因为它们与许多颅面综合征（例如，格里格头多并指畸形）和可以 作为Hh途径的转录激活因子和抑制因子发挥作用。Gli 3是最稳定和丰富的 作为一种抑制剂。尽管如此，我们最近的工作确定了Gli 3激活剂（Gli 3A）的特定和必要作用。 在下颌骨的正常发育过程中，需要额外的调节输入来传达稳健的 Gli 3A反应。Gli 3作为一种双峰转录因子，其功能需要什么还知之甚少 在发育过程中作为一种有效的激活剂。为了解决全长Gli 3是如何在 转化为激活剂，我们设计了一组Gli 3的内源性表位标记等位基因。与这些 我们提出了新的工具：（Aim 1）确定染色质可及性是否/如何调节Gli 3A功能;（Aim 2） 研究辅助因子和调节语法在调节增强子输出中的作用;以及（Aim 3）无偏地 识别细胞核内Gli 3A的蛋白质相互作用物。我们将着重于颅面发育，特别是 下颌骨的发育，作为测试这些原则的相关模型，考虑到Gli的要求 舌发生和下颌骨骨发生过程中的活动。这个发展系统将使我们能够 确定染色质可及性的要求，详尽地询问调控语法，以及 鉴定Gli 3激活剂复合物的成分。最近的技术进步将使假设 通过单细胞分析预测的突变体的染色质可及性和转录谱进行测试， 具有开拓性的活动。为了验证我们的发现，我们将进行体内实验来测试增强子的活性 并应用CRISPR/Cas9诱变来功能性地评估天然结合位点需求。总体而言，我们 这些研究将揭示Gli指导的细胞程序的调控原则， 可能导致从结构性出生缺陷到癌症的一系列人类疾病。了解这些 在正常发育期间部署和解释的程序有可能改善人类健康 通过扩大治疗干预，可以帮助减轻途径失调。

Project Summary The Hedgehog (Hh) signaling pathway is essential for normal embryonic development and when perturbed, frequently results in human disease, including those that impact development of the craniofacial complex. The Gli transcription factors are the downstream effectors of the pathway and have been the subject of much research as they are associated with a number of craniofacial syndromes (e.g., Grieg cephalopolysyndactyly) and can function as both transcriptional activators and repressors of the Hh pathway. Gli3 is most stable and abundant as a repressor. Despite this, our recent work identified a specific and necessary role for Gli3 activator (Gli3A) function during normal development of the mandible that requires additional regulatory inputs to convey a robust Gli3A response. Little is known regarding what is required for Gli3, as a bimodal transcription factor, to function as a potent activator during development. To address existing knowledge gaps in how full-length Gli3 is converted into an activator, we engineered a set of endogenously epitope tagged alleles for Gli3. With these novel tools we propose to: (Aim1) determine if/how chromatin accessibility modulates Gli3A function; (Aim2) investigate the role of co-factors and regulatory grammar in regulating enhancer output; and (Aim3) unbiasedly identify protein interactors of Gli3A within the nucleus. We will focus on craniofacial development, specifically development of the mandible, as a relevant model for testing these principles given the requirement for Gli activity during glossogenesis and mandibular skeletogenesis. This developmental system will allow us to determine the requirement for chromatin accessibility, exhaustively interrogate the regulatory grammar, and identify the constituents of the Gli3 activator complex. Recent technological advances will enable hypothesis testing through single-cell analysis of chromatin accessibility and transcription profiles from mutants predicted to have pioneering activity. To validate our findings, we will perform in vivo experiments to test enhancer activity and apply CRISPR/Cas9 mutagenesis to functionally assess native binding site requirements. Collectively, our studies will shed light on the regulatory principles governing Gli-directed cellular programs that when disrupted can result in range of human disorders ranging from structural birth defects to cancer. Understanding how these programs are deployed and interpreted during normal development has the potential to improve human health through the expansion of therapeutic interventions that can help mitigate pathway dysregulation.