Predicting Tissue Specific Gli3 Regulatory Activity Using Hand2

使用 Hand2 预测组织特异性 Gli3 调节活动

• 批准号: 10647737
• 负责人: Samantha A Brugmann
• 金额: $ 77.88万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-16 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647737
• 关键词: Activator Appliances; Address; Affect; Affinity; Alleles; Architecture; Automobile Driving; BHLH Protein; Binding; Binding Sites; Biochemical; Biological Assay; CRISPR/Cas technology; Cell Nucleus; Cells; Central Nervous System; ChIP-seq; Chromatin; Chromatin Remodeling Factor; Complement; Complex; Congenital Abnormality; Craniofacial Abnormalities; DNA Binding; Data; Development; Disease; Embryonic Development; Engineering; Enhancers; Epigenetic Process; Epitopes; Equation; Erinaceidae; Face; Family; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Health; Human; Individual; Joints; Knowledge; Length; Limb structure; Malignant Neoplasms; Mandible; Mandibular Prominence; Modality; Modeling; Mus; Mutagenesis; Output; Pathologic; Pathway interactions; Population; Positioning Attribute; Protein Isoforms; Proteins; Proteolytic Processing; Public Health; Reporter; Research; Role; Signal Pathway; Site; Specificity; Structural Congenital Anomalies; Syndrome; System; Technology; Testing; Therapeutic Intervention; Tissues; Transcription Coactivator; Transcription Repressor; Transcriptional Activation; Transcriptional Regulation; Work; cofactor; combinatorial; craniofacial; craniofacial complex; craniofacial development; design; experimental study; gene regulatory network; human disease; improved; in vivo; innovation; insight; multiple omics; mutant; novel; progenitor; programs; response; single cell analysis; single nucleus RNA-sequencing; skeletogenesis; smoothened signaling pathway; tandem mass spectrometry; tool; transcription factor

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.

项目概要 Hedgehog (Hh) 信号通路对于正常胚胎发育至关重要，当受到干扰时， 经常导致人类疾病，包括影响颅面复合体发育的疾病。这 Gli 转录因子是该通路的下游效应子，一直是许多研究的主题 因为它们与许多颅面综合征（例如格里格头多指并指症）有关，并且可以 作为 Hh 通路的转录激活子和阻遏子。 Gli3最稳定、含量最丰富 作为阻遏剂。尽管如此，我们最近的工作确定了 Gli3 激活剂 (Gli3A) 的特定且必要的作用 下颌骨正常发育过程中的功能，需要额外的调节输入来传达强大的功能 Gli3A 反应。对于 Gli3 作为双峰转录因子发挥作用所需的条件知之甚少 作为发育过程中的有效激活剂。解决全长 Gli3 的现有知识差距 转化为激活剂后，我们为 Gli3 设计了一组内源表位标记的等位基因。有了这些 我们建议使用新工具：（目标1）确定染色质可及性是否/如何调节 Gli3A 功能； （目标2） 研究辅助因子和调节语法在调节增强子输出中的作用；和（目标3）公正 鉴定细胞核内 Gli3A 的蛋白质相互作用因子。我们将重点关注颅面发育，特别是 考虑到 Gli 的要求，下颌骨的发育作为测试这些原理的相关模型 舌发生和下颌骨骼发生期间的活动。这个发展系统将使我们能够 确定染色质可及性的要求，详尽地询问监管语法，以及 鉴定 Gli3 激活剂复合物的成分。最近的技术进步将使假设成为可能 通过单细胞分析染色质可及性和预测突变体的转录谱进行测试 有开拓活动。为了验证我们的发现，我们将进行体内实验来测试增强子活性 并应用 CRISPR/Cas9 诱变来功能评估天然结合位点要求。总的来说，我们的 研究将揭示 Gli 引导的细胞程序的监管原则，当这些程序被破坏时 可能导致一系列人类疾病，从结构性出生缺陷到癌症。了解这些如何 在正常开发过程中部署和解释的计划有改善人类健康的潜力 通过扩大治疗干预措施，有助于减轻通路失调。