Regulation of the lens transcriptome and chromatin architecture by FOXE3

调控控制细胞的转录网络的新分子机制的揭示 区分提供了与理解器官发生和 对细胞进行重新编程以进行再生治疗。人工晶状体提供了简单、自给自足的组织， 分化特异性基因表达的特征模式以模拟转录因子如何调控 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， 启动子和其他调节蛋白复合体。叉头转录因子FOXE3是一种丰富的 转录因子在晶状体形成外胚层的早期表达，并在晶状体上皮细胞中维持。 PAX6表达下游。事实上，FOXE3的突变反映了许多眼部表型(彼得斯 畸形、白内障、上皮细胞增殖减少和纤维细胞分化) 另外两种转录因子Pax6或AP-2在晶状体上皮细胞中大量表达。然而，几乎没有 关于FOXE3如何调节晶状体发育的信息已经存在。一种来自中国人晶状体中的RNA-SEQ分析 新创建的Foxe3等位基因确定了许多差异调控基因。这些措施包括下调对 许多经典的晶状体识别基因(包括Bfsp1、Bfsp2、Dnase2b和多晶体蛋白)和上调 在许多与神经和/或视网膜分化相关的基因中(包括Nr2e1，OTX2，Ascl1，Tbx3，Rax， Vsx2、LHx2和Six6)。FOXE3缺陷晶状体基因表达的这种令人惊讶的变化，加上 FOXE3与几种可作为细胞关键驱动因子的先驱转录因子的结构相似性 分化和重新编程，导致了基因表达的转录调节的假说 在FOXE3介导的晶状体中是由其与染色质的动态相互作用决定的，从而导致其 开放染色质和闭合染色质通过相邻的顺式调控位点簇和反式- 在启动子和增强子中起作用的DNA结合转录因子。两个具体的目标将检验这一点 假设。1)确定FOXE3功能的丧失如何影响染色质景观和基因控制 该透镜是ATAC-seq、scRNA-seq和Bulk RNA-seq的组合，将用于Foxe3零透镜。2) 确定晶状体中FOXE3结合的启动子和增强子的顺式调控语法，FOXE3结合 将使用Cut&Run发现晶状体染色质中的位置，然后进行生物信息学分析以确定 一致的FOXE3结合基序和相邻的转录因子结合基序。这一信息将是 整合以发现FOXE3的直接转录靶点，这将通过RT-qPCR和荧光素酶进行验证 化验。通过这些方法获得的基本信息将有助于更广泛和系统地分析 FOX转录因子调控基因的分子机制及其对染色质的影响 组织分化和细胞重编程的结构动力学。

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.