Predicting Tissue Specific Gli3 Regulatory Activity Using Hand2

项目摘要 Hedgehog(HH)信号通路对于正常的胚胎发育是必不可少的，当受到干扰时， 经常导致人类疾病，包括那些影响头面部复合体发育的疾病。这个 Gli转录因子是该途径的下游效应因子，已成为许多研究的主题 因为它们与许多颅面综合征(例如格里格头多发性指病)有关，并且可以 既是HH途径的转录激活因子，又是转录抑制因子。Gli3是最稳定和最丰富的 作为一个压迫者。尽管如此，我们最近的工作确定了Gli3激活剂(Gli3A)的一个特定和必要的角色 在下颌骨正常发育期间的功能，需要额外的调节输入来传达一个健壮的 Gli3A回应。关于Gli3作为一个双峰转录因子需要什么才能发挥作用，目前还知之甚少 在发展过程中作为一个强有力的激活剂。为了解决Gli3全长的现有知识差距 转化为激活剂后，我们为Gli3设计了一组内源性表位标记的等位基因。有了这些 我们提出了新的工具来：(AIM1)确定染色质的可及性是否/如何调节Gli3A功能；(AIM2) 研究协同因子和调控语法在调节增强子输出中的作用；和(Aim3)公正地 确定Gli3A在细胞核内的蛋白质相互作用。我们将专注于颅面发育，特别是 下颌骨的开发，作为测试这些原理的相关模型，考虑到Gli的要求 舌骨发育和下颌骨发育过程中的活动性。这一发展系统将使我们能够 确定染色质可及性的要求，详尽地询问调节语法，以及 鉴定Gli3激活剂复合体的成分。最近的技术进步将使假说成为可能 通过单细胞分析对预测到的突变体的染色质可及性和转录图谱进行测试 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剂的活性 并应用CRISPR/Cas9突变法从功能上评估天然结合位点的要求。总的来说，我们的 研究将阐明管理Gli定向细胞程序的监管原则，这些原则在被破坏时 会导致一系列人类疾病，从结构性出生缺陷到癌症。了解这些功能是如何 在正常发展过程中部署和解释方案有可能改善人类健康 通过扩大治疗干预措施，帮助缓解途径失调。

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.