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

• 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
• 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

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.

调控细胞转录网络的新分子机制的揭示 分化提供了重要的信息，相关的了解器官发生， 重新编程细胞用于再生疗法。眼用透镜提供了一种简单、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子的合作相互作用来指导特定的转录网络， 启动子和其它调节蛋白复合物。叉头转录因子FOXE 3是一种丰富的 在早期透镜形成外胚层中表达并维持在透镜上皮中的转录因子， PAX 6表达的下游。事实上，FOXE 3中的突变反映了许多眼部表型（Peters 异常、白内障、上皮细胞增殖和纤维细胞分化减少）。 PAX 6或AP-2是另外两种在透镜上皮中大量表达的转录因子。不过小 存在关于FOXE 3如何调节透镜发育的信息。一项来自一个人晶状体的RNA-seq分析 新创建的Foxe 3等位基因鉴定了许多差异调节基因。其中包括下调 许多经典的透镜身份基因（包括Bfsp 1、Bfsp 2、Dnase 2b和多种晶状体蛋白）和上调 许多与神经和/或视网膜分化相关的基因（包括Nr 2 e1，Otx 2，Ascl 1，Tbx 3，Rax， VSx 2、Lhx 2和Six 6）。FOXE 3缺陷晶状体中基因表达的这一令人惊讶的转变，加上 FOXE 3与几种先驱转录因子的结构相似性，这些转录因子可以作为细胞增殖的关键驱动因子。 分化和重编程，导致了基因表达的转录调控的假设， 由FOXE 3介导的透镜中的细胞凋亡是由其与染色质的动态相互作用决定的， 存在于开放和封闭染色质中，通过相邻的顺式调节位点簇和反式调节位点簇， 启动子和增强子中的作用DNA结合转录因子。两个具体目标将检验这一点 假说. 1)为了确定FOXE 3功能的丧失如何影响细胞中的染色质景观和基因控制， 透镜、ATAC-seq、scRNA-seq和bulkRNA-seq的组合将用于Foxe 3零透镜。（二） 为了确定透镜中FOXE 3结合启动子和增强子的顺式调节语法， 透镜染色质中的位点将使用CUT&RUN发现，然后进行生物信息学分析以鉴定 共有FOXE 3结合基序和相邻的转录因子结合基序。此信息将 整合以发现直接FOXE 3转录靶点，这将通过RT-qPCR和荧光素酶验证 测定。通过这些方法获得的基本信息将推动对以下问题进行更广泛和系统的分析： FOX转录因子调控基因的分子机制主要集中在对染色质的影响上 组织分化和细胞重编程的结构动力学。