Regulation of the lens transcriptome and chromatin architecture by FOXE3

FOXE3 对晶状体转录组和染色质结构的调节

基本信息

批准号：
10546497
负责人：
MICHAEL L ROBINSON
金额：
$ 18.06万
依托单位：
MIAMI UNIVERSITY OXFORD
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10546497
关键词：
ATAC-seq Affect Alleles Anterior Antibodies Architecture Binding Binding Sites Bioinformatics Biological Assay Biological Models Cataract Cell Compartmentation Cell Differentiation process Cell Reprogramming ChIP-seq Characteristics Chromatin Chromatin Structure Computer Analysis Computer Models Consensus Coupled Crystalline Lens Crystallins DNA DNA Binding Data Defect Development Differentiation and Growth Disease Distal Down-Regulation Ectoderm Enhancers Epigenetic Process Epithelial Cells Epithelium Eye FOXE3 gene Family Feasibility Studies Funding Mechanisms Gene Dosage Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Genetic Transcription Genetic study Genome Genomics Goals Heart Homozygote Individual Irido-corneo-trabecular dysgenesis Knowledge Lens Placodes Lens development Luciferases Maps Mediating Methodology Methods Mission Modeling Molecular Molecular Analysis Mus Mutate Mutation Neuroectoderm Organ Organogenesis PAX7 gene Pattern Phenotype Proliferating Proteins Public Health Regulation Research Retina Role Shapes Site Structure Surface Ectoderm Testing Tissue Differentiation Tissues Transcription Coactivator Transcription Repressor Transcriptional Activation Transcriptional Regulation United States National Institutes of Health Up-Regulation Vesicle Vision research comparative computer studies differential expression fiber cell forkhead protein gastrulation gene regulatory network gene repression genetic regulatory protein genome-wide human disease in vivo lens lens morphogenesis loss of function mouse model mutant neural novel programs promoter prospective protein complex regenerative regenerative therapy single-cell RNA sequencing transcription factor transcriptome transcriptome sequencing transcriptomics

项目摘要

Revelation of novel molecular mechanisms that regulate transcriptional networks controlling cellular differentiation provides essential information relevant both to understanding organogenesis and for reprogramming cells for regenerative therapies. The ocular lens provides a simple, self-contained tissue with characteristic patterns of differentiation-specific gene expression to model how transcription factors regulate chromatin landscapes to direct specific transcriptional networks through cooperative interactions with enhancers, promoters, and other regulatory protein complexes. The forkhead transcription factor, FOXE3, is an abundant transcription factor expressed in the early lens forming ectoderm, and maintained in the lens epithelium, downstream of PAX6 expression. In fact, mutations in FOXE3 mirror many of the ocular phenotypes (Peters anomaly, cataracts, reduced epithelia proliferation and fiber cell differentiation) resulting from deficiencies in PAX6 or AP-2, the two other abundantly expressed transcription factors in lens epithelium. However, little information exists concerning how FOXE3 regulates lens development. An RNA-seq analysis in lenses from a newly created Foxe3 allele identified numerous differentially regulated genes. These included downregulation of many classical lens identity genes (including Bfsp1, Bfsp2, Dnase2b and multiple crystallins) and upregulation of many genes associated with neural and or retina differentiation (including Nr2e1, Otx2, Ascl1, Tbx3, Rax, Vsx2, Lhx2 and Six6). This surprising shift in gene expression in FOXE3 deficient lenses, coupled with the structural similarity of FOXE3 to several pioneer transcription factors that can act as key drivers of cellular differentiation and reprogramming, led to the hypothesis that transcriptional regulation of gene expression in lens mediated by FOXE3 is determined by its dynamic interactions with chromatin resulting in its presence in both open and closed chromatin through cluster of adjacent cis-regulatory sites and trans- acting DNA-binding transcription factors in the promoters and enhancers. Two specific aims will test this hypothesis. 1) To determine how the loss of FOXE3 function affects chromatin landscape and gene control in the lens, combinations of ATAC-seq, scRNA-seq and bulk RNA-seq will be employed on Foxe3 null lenses. 2) To determine the cis-regulatory grammar of FOXE3-bound promoters and enhancers in lens, FOXE3 binding sites in lens chromatin will be discovered using CUT&RUN followed by bioinformatic analysis to identify the consensus FOXE3 binding motif and adjacent transcription factor binding motifs. This information will be integrated to discover direct FOXE3 transcriptional targets, which will be validated by RT-qPCR and luciferase assays. The fundamental information gained by these approaches will fuel broader and systematic analysis of molecular mechanisms of gene control by FOX transcription factors focused on their impact on chromatin structural dynamics for tissue differentiation and cellular reprogramming.

调节控制细胞的转录网络的新型分子机制的启示差异化提供了与理解器官发生和有关的重要信息重编程细胞用于再生疗法。眼镜提供了一个简单，独立的组织分化特异性基因表达的特征模式，以模拟转录因子如何调节染色质景观通过与增强剂的合作相互作用，指导特定的转录网络，启动子和其他调节蛋白复合物。叉子转录因子FOXE3是绝对的转录因子在形成外胚层的早期晶状体中表达，并保持在晶状体上皮中， PAX6表达的下游。实际上，FOXE3中的突变反映了许多眼类表型（Peters 异常，白内障，上皮增殖减少和纤维细胞分化），导致缺乏 PAX6或AP-2，另外两个绝对表达的转录因子在镜片上皮中。但是，很少有关FOXE3如何调节晶状体开发的信息。来自镜片的RNA-seq分析新创建的FOXE3等位基因确定了许多不同调节的基因。这些包括下调许多经典镜头身份基因（包括BFSP1，BFSP2，DNASE2B和多个结晶蛋白）和上调与神经和视网膜分化相关的许多基因（包括NR2E1，OTX2，ASCL1，TBX3，RAX，，RAX， VSX2，LHX2和SIX6）。 FOXE3缺乏透镜中基因表达的这种令人惊讶的转变，加上 FOXE3与几个可以充当细胞的关键驱动因素的结构相似性分化和重编程导致了基因表达的转录调节的假设在由FOXE3介导的镜头中，由其与染色质的动态相互作用确定，导致其通过相邻的顺式调节位点和反式的簇中的开放和闭合染色质存在启动子和增强子中的作用DNA结合转录因子。两个具体的目标将测试假设。 1）确定FOXE3功能的损失如何影响染色质景观和基因控制 ATAC-SEQ，SCRNA-SEQ和BOLK RNA-SEQ的镜头，将携带在FOXE3 NULL镜头上。 2）为了确定镜头中FOXE3结合启动子和增强子的顺式调节语法，FOXE3结合镜头染色质中的位点将使用切割和运行，然后进行生物信息学分析，以识别共识FOXE3结合基序和相邻的转录因子结合基序。这些信息将是集成以发现直接FOXE3转录目标，该目标将由RT-QPCR和荧光素酶验证测定。这些方法获得的基本信息将推动更广泛的系统分析 FOX转录因子对基因控制的分子机制，重点是其对染色质的影响组织分化和细胞重编程的结构动力学。