Assessing Sox 10's effect on chromatin accessibility in enteric neuron lineage diversification

评估 Sox 10 对肠神经元谱系多样化中染色质可及性的影响

基本信息

批准号：
10749740
负责人：
Joseph T Benthal
金额：
$ 3.3万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10749740
关键词：
ATAC-seq Adult Affect Alleles Animal Model Animals Antibodies Architecture Binding Biological Biological Assay CRISPR interference Candidate Disease Gene Cell Fate Control Cell Lineage Cell Therapy Cells Chromatin Chromatin Remodeling Factor Chronic Colon Complex Congenital Megacolon DNA DNA Binding Data Data Set Defect Development Developmental Process Disease Distal Elements Enhancers Enteral Enteric Nervous System Epigenetic Process Equilibrium Excision Exhibits Fluorescence-Activated Cell Sorting Ganglia Gastroesophageal reflux disease Gastrointestinal Motility Gastrointestinal tract structure Gene Expression Genes Genomic Segment Genomics Genotype Human In Vitro Intestinal Motility Intestines Knowledge Learning Length Link Malignant Neoplasms Maps Mediating Modeling Molecular Mus Mutation Neural Crest Neural Crest Cell Neuroglia Neuronal Differentiation Neurons Normal Range Obstruction Patients Penetrance Phenotype Population Process Protein Isoforms Proteins Regulation Regulator Genes Regulatory Element Resolution Role Testing Tissue-Specific Gene Expression Tissues Variant Work chromatin modification chromatin remodeling developmental disease developmental genetics directed differentiation disease phenotype enteric neuropathy experience experimental study fetal gene regulatory network genetic architecture genome-wide genomic platform histone methylation histone modification migration motility disorder mouse model multiple omics mutant nerve stem cell nervous system development neurogenesis postnatal promoter single nucleus RNA-sequencing success transcription factor transcriptome sequencing

项目摘要

Project Description Normal development of the mammalian enteric nervous system requires migration of neural crest cells into and along the developing intestine. Perturbations of ENS development can result in changes in enteric ganglia that result in patient phenotypes such as gastroesophageal reflux, chronic pseudo-obstruction, and Hirschsprung’s disease (HSCR). HSCR is a complex genetic developmental disorder characterized by aganglionosis, the absence of enteric neurons in varying lengths, along the distal bowel. The genetic architecture of HSCR is not completely understood, with identified mutations documented in ~70% of patients. The transcription factor Sox10 is one gene that can be altered in HSCR. Our group has previously shown that the Sox10Dom mouse model of HSCR recapitulates the variable expressivity and penetrance of aganglionosis seen in patients. In addition, we have shown that postnatal Sox10Dom mice have altered ratios of enteric neuron types. This is an intriguing finding since Sox10 is not expressed in enteric neurons, although it is expressed in enteric neuronal progenitors (ENPs). This suggests that Sox10 could be regulating the differentiation process into the normal range of neuronal type proportions through an indirect mechanism. In other neural crest derived lineages, SOX10 interacts with chromatin remodeler proteins to control cell fate. Based on the lack of Sox10 expression in enteric neurons and SOX10’s participation in altering chromatin in other tissues, I hypothesize that Sox10 has a role in altering chromatin accessibility in the developing ENS. This hypothesis will be investigated through the following aims: Aim 1 will define effects of a Sox10 mutant allele on genome-wide chromatin accessibility and gene expression during mouse ENS neurogenesis. In this study, I will combine single nucleus RNA and ATAC-sequencing in ENPs. By comparing these data between wild-type and Sox10Dom ENPs, I will evaluate chromatin accessibility changes linked with gene expression downstream of a defective Sox10 isoform. These experiments will clarify Sox10’s role in the differentiation of ENPs towards neuronal fates. Aim 2 will define effects of altered Sox10 binding on chromatin modifications in mice. To determine how Sox10 mediates its indirect effect in the developing ENS, I will assay genome-wide SOX10 binding and histone modifications in wild type and Sox10Dom ENPs. These studies will link deficits in SOX10 binding to alternations in chromatin architecture, expanding the framework of genes in the developing ENP gene regulatory network. These studies will also reveal effects of defective SOX10 on histone modifications and will point towards candidate genomic elements to which SOX10 directly binds. Success of this project would identify genomic elements downstream of Sox10 that function during ENP differentiation into normal neuronal subtype proportions. Discernment of Sox10’s influence on regulatory genomic regions in ENPs will identify genes that are required for normal ENS development. This knowledge may be leveraged and applied to experiments in human ENS developmental mechanisms to direct differentiation towards neuronal fates, which may lead to cell therapies for patients with GI motility disorders such as HSCR.

项目描述哺乳动物肠神经系统的正常发育需要神经嵴细胞迁移到随着肠道的发育，ENS 发育的扰动会导致肠神经节的变化。导致患者出现胃食管反流、慢性假性梗阻和先天性巨结肠等表型疾病（HSCR）是一种复杂的遗传性发育障碍，其特征是神经节缺失。沿远端肠不存在不同长度的肠神经元 HSCR 的遗传结构并非如此。完全了解，约 70% 的患者记录了转录因子 Sox10 的突变。是一种可以在 HSCR 中改变的基因，我们的小组之前已经证明了 Sox10Dom 小鼠模型中的 HSCR 基因可以被改变。 HSCR 概括了患者中所见的神经节缺失的可变表达度和外显率。研究表明，出生后的 Sox10Dom 小鼠肠道神经元类型的比例发生了变化，这是一个有趣的发现。因为 Sox10 不在肠神经元中表达，尽管它在肠神经元祖细胞 (ENP) 中表达。这表明Sox10可能调节神经元类型向正常范围的分化过程在其他神经嵴衍生谱系中，SOX10 通过间接机制相互作用。基于肠神经元中缺乏 Sox10 表达的染色质重塑蛋白来控制细胞命运。 SOX10 参与改变其他组织中的染色质，我认为 Sox10 在改变发育中的 ENS 中的染色质可及性将通过以下目标进行研究：目标 1 将定义 Sox10 突变等位基因对全基因组染色质可及性和基因表达的影响在这项研究中，我将在小鼠 ENS 神经发生过程中结合单核 RNA 和 ATAC 测序。通过比较野生型和 Sox10Dom ENP 之间的这些数据，我将评估染色质可及性与有缺陷的 Sox10 同工型下游基因表达相关的变化这些实验将阐明。 Sox10 在 ENP 向神经元命运分化中的作用将定义改变的 Sox10 的影响。与小鼠染色质修饰的结合确定 Sox10 如何介导其对发育中的间接影响。 ENS，我将检测野生型和 Sox10Dom ENP 中的全基因组 SOX10 结合和组蛋白修饰。研究将 SOX10 结合缺陷与染色质结构的改变联系起来，从而扩展了 ENP 基因调控网络中正在发育的基因这些研究还将揭示 SOX10 缺陷的影响。组蛋白修饰，并将指向 SOX10 直接结合的候选基因组元件。该项目的成功将鉴定在 ENP 期间发挥作用的 Sox10 下游基因组元件分化为正常神经亚型比例辨别 Sox10 对调节的影响。 ENP 中的基因组区域将识别正常 ENS 发育所需的基因。可利用并应用于人类 ENS 发育机制的实验以指导分化神经元命运，这可能会导致针对 HSCR 等胃肠道运动障碍患者的细胞疗法。