In The Loop: Investigating Enhancer-Mediated Regulation of OTX2 During Retinal Development

项目摘要 视网膜是由七种主要细胞类型组成的复杂组织。每种细胞类型都需要 正常的视网膜功能和视力。哺乳动物视网膜中的所有细胞都是在发育过程中形成的 并且必须持续动物的一生。三种视网膜细胞类型的产生依赖于 Otx 2是一种同源结构域转录因子。OTX 2在视网膜发育过程中通过前体表达， 产生五种细胞类型，但它只由光感受器和双极细胞维持到成熟。损失- 功能研究表明，缺乏Otx 2的小鼠不能产生锥或杆光感受器，也不能产生双极细胞 中间神经元因此，视网膜中的细胞命运决定在很大程度上取决于Otx 2表达的位置和时间。 为了了解Otx 2的表达是如何调节的，我们寻找了它的增强子。增强剂是非- 启动和稳定基因表达的DNA编码区。三种可能的Otx 2增强子是 鉴定并显示由OTX 2+细胞表达。我们接下来测试这些增强子是否是必需的 Otx 2表达。CRISPR介导的一个增强子DHS 4的缺失揭示了OTX 2的减少。 胚胎中的表达，但对出生后的OTX 2表达的影响是适度的。这表明，其他 otx 2增强子用于出生后视网膜发育。为了调查这一点，我进行了CRISPR删除， 对Otx 2的另外两种增强子（称为DHS 2和DHS 15）的实验。缺失任一增强子显示 出生后OTX 2表达的降低比DHS 4更强。有趣的是，删除两个增强子 同时在随后的时间点对OTX 2减少没有累加效应，表明复合物 Otx 2增强子的景观允许它们相互替代。我的观察让我假设 动态增强子复合物在视网膜发育期间启动并维持Otx 2表达。 我将在我的建议中通过完成两个具体目标来检验这一假设。在我的第一个目标中，我将使用 高分辨率染色体构象捕获技术，以揭示Otx 2处的增强子-启动子接触 在视网膜发育过程中的位置此外，这项技术将揭示Otx 2的其他潜在增强子。在 我的第二个目标是，当增强子受到干扰时，我将测试这种增强子复合体是如何被破坏的。这么做我 将联合收割机染色体构象捕获与基于CRISPR的增强子扰动技术相结合。这将 让我辨别增强动力学，并确定增强剂如何相互补偿，以确保 otx 2在视网膜发育中的表达完成这一建议将增进我们对以下问题的了解： 视网膜发育，复杂的基因调控机制，并为我提供所需的经验， 继续领导我自己的学术实验室

Project Summary The retina is a complex tissue composed of seven major cell types. Each of these cell types is needed for normal retinal function and therefore vision. All cells in the mammalian retina are formed during development and must last the lifetime of the animal. The production of three retinal cell types is dependent on the expression of Otx2, a homeodomain transcription factor. Otx2 is expressed during retinal development by precursors that give rise to five cell types, but it is only maintained by photoreceptors and bipolar cells into maturity. Loss-of- function studies show that mice lacking Otx2 cannot produce cone or rod photoreceptors, nor bipolar cell interneurons. Thus, cell fate decisions in the retina depend heavily on where and when Otx2 is expressed. To understand how Otx2 expression is regulated, we searched for its enhancers. Enhancers are non- coding regions of DNA that initiate and stabilize gene expression. Three potential enhancers of Otx2 were identified and shown to be expressed by OTX2+ cells. We next tested whether these enhancers were necessary for Otx2 expression. CRISPR-mediated deletion of one enhancer, DHS4, revealed a reduction in OTX2 expression embryonically yet the effect on postnatal OTX2 expression was modest. This suggested that other Otx2 enhancers are utilized in postnatal retinal development. To investigate this, I conducted CRISPR deletion experiments on the other two enhancers of Otx2, termed DHS2 and DHS15. Deletion of either enhancer showed a stronger reduction in OTX2 expression postnatally than DHS4. Interestingly, deleting both enhancers simultaneously did not have an additive effect on OTX2 reduction at later timepoints, suggesting that the complex landscape of Otx2 enhancers allows them to substitute for each other. My observations led me to hypothesize that a dynamic enhancer complex initiates and maintains Otx2 expression during retinal development. I will test this hypothesis in my proposal by completing two specific aims. In my first aim, I will employ a high-resolution chromosome conformation capture technique to reveal enhancer-promoter contacts at the Otx2 locus across retinal development. Additionally, this technique will reveal other potential enhancers of Otx2. In my second aim, I will test how this enhancer complex is disrupted when enhancers are perturbed. To do this, I will combine chromosome conformation capture with CRISPR-based enhancer perturbation techniques. This will allow me to discern enhancer dynamics and determine how enhancers compensate for each other to ensure Otx2 expression during retinal development. The completion of this proposal will improve our understanding of retinal development, complex gene regulatory mechanisms and provide me with the experience needed to continue onto a successful career leading my own academic lab.