Transcriptional regulatory mechanisms of vertebrate regeneration

脊椎动物再生的转录调控机制

基本信息

批准号：
10208975
负责人：
Andrea Elizabeth Wills
金额：
$ 33.42万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-15 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10208975
关键词：
ATAC-seq Adult Amputation Animal Model Animals Binding Sites Biological Biology CRISPR/Cas technology Cell Count Cells Chromatin Competence Coupled Dendrites Development Embryo Enhancers Event FOXO1A gene Failure Gene Activation Gene Expression Gene Expression Regulation Genes Genetic Transcription Genomic Segment Goals Hour Human Injury Intrinsic factor Knock-out Lead Mediating Molecular Molecular Conformation Morphogenesis Motor Mutagenesis Natural regeneration Neuraxis Neurons Nucleic Acid Regulatory Sequences Outcome Pathway interactions Patients Population Rana Recovery of Function Regenerative research Regenerative response Regulation Regulator Genes Regulatory Element Role Sensory Signal Transduction Spinal Cord Spinal cord injury Spinal cord injury patients System Systems Analysis Tadpoles Tail Testing Tissues Up-Regulation Vertebrates Work Xenopus axon regeneration base central nervous system injury chromatin remodeling extracellular functional genomics genome-wide improved insight loss of function mature animal mutant nerve stem cell neurogenesis neuronal growth regenerative relating to nervous system repaired response severe injury spinal cord regeneration targeted treatment therapeutically effective tissue regeneration tool transcription factor wound

项目摘要

Why do humans fail to regenerate injured central nervous system tissues, when other vertebrates do so readily? In this proposal we take aim at this fundamental question by defining the cell-intrinsic mechanisms that enable spinal cord regeneration in the frog Xenopus tropicalis. Tadpoles of this species are able to regenerate spinal cord tissues and motor function following injury, while adult animals cannot. We will exploit this temporal competence to regenerate in order to understand how regeneration normally proceeds as well as why it might fail. This distinctive biology coupled with the deep set of available tools for functional and genomic analysis makes X. tropicalis a uniquely powerful system for analysis of regeneration. A central goal of spinal cord regeneration research is to identify the cell-intrinsic factors that enable neurogenesis and axon regeneration. Our preliminary analyses in this system have uncovered new insights into these factors and the gene regulatory mechanisms that may form the basis for regenerative competence. First, we have found that tens of thousands of genomic regions shift rapidly to an accessible chromatin conformation, and then unexpectedly to an inaccessible conformation, within the first few hours of regeneration. These rearrangements take place in regions that are heavily enriched for binding sites of FoxO1 and Ascl1, factors that have pioneer activity and critical roles in neural progenitor function. Second, genes specific to differentiated neurons are expressed within hours of amputation, and are surprisingly independent of neural induction and neurogenesis gene activation. Based on these observations, we hypothesize that regenerative competence relies on three features: 1) a robust neural progenitor population, 2) a rapid burst of chromatin remodeling in neural progenitor cells carried out by Ascl1, FoxO1, and other pioneer factors, and 3) activation of neuronal specific genes that allow axonogenesis and neuronal growth in existing differentiated neurons. In this proposal, we will test these predictions by identifying the transcription factors that mediate chromatin remodeling in isolated neural progenitors. We will functionally test the role of Ascl1 and FoxO1 in regeneration using loss-of-function mutants for these factors. We will then identify whether upregulation of axonogenesis genes in regenerating tadpoles represents neuronal repair or neurogenesis, and interrogate whether these genes are upregulated using embryonic gene regulatory elements or regeneration-specific regulatory elements. Finally, we will identify whether regeneration in adult frogs fails due to lack of neural progenitors, failure to initiate chromatin remodeling, or failure to upregulate neuronal morphogenesis genes. By systematically characterizing the events that define regeneration competence in Xenopus, we expect to identify molecular mechanisms that can be targeted for more effective therapeutics in human spinal cord injury patients.

当其他脊椎动物这样做时，为什么人类无法再生受伤的中枢神经系统组织容易？在此提案中，我们通过定义细胞内部机制来实现这个基本问题在青蛙爪蟾tropicalis中启用脊髓再生。该物种的t能够再生受伤后脊髓组织和运动功能，而成年动物不能。我们将利用这个时间为了了解再生通常如何进行以及为什么可能会导致再生的能力失败。这种独特的生物学以及用于功能和基因组分析的深层可用工具使X. Tropicalis成为分析再生的独特功能。脊髓的主要目标再生研究是为了确定能够神经发生和轴突再生的细胞中性因素。我们在该系统中的初步分析发现了对这些因素和基因的新见解可能构成再生能力基础的监管机制。首先，我们发现数十个成千上万的基因组区域迅速转向可访问的染色质构象，然后出乎意料地转向在再生的前几个小时内，无法接近的构象。这些重排发生在FOXO1和ASCL1的结合位点大量丰富的区域，具有先驱活动和在神经祖细胞功能中的关键作用。其次，表达针对分化神经元的基因截肢后的几个小时内，与神经诱导和神经发生基因无关激活。基于这些观察结果，我们假设再生能力取决于三个特征：1）强大的神经祖细胞，2）神经祖细胞中的染色质重塑快速爆发通过ASCL1，FOXO1和其他先锋因素进行的细胞，以及3）激活神经元特异性基因允许现有分化神经元中的轴突生成和神经元生长。在此提案中，我们将测试这些通过识别介导隔离神经中染色质重塑的转录因子来预测祖先。我们将使用功能丧失的突变体在功能上测试ASCL1和FOXO1在再生中的作用对于这些因素。然后，我们将确定轴突生成基因在再生t中是否上调代表神经元修复或神经发生，并询问这些基因是否被上调胚胎基因调节元件或再生特异性调节元件。最后，我们将确定成年青蛙的再生是否由于缺乏神经祖细胞而失败，无法启动染色质重塑或未能上调神经元形态发生基因。通过系统地表征定义爪蟾中再生能力的事件，我们期望确定可以分子机制针对人脊髓损伤患者更有效的治疗剂。