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。这些像元类型中的每一种都是必需的 以保证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表达 OTX2，一种同源结构域转录因子。OTX2在视网膜发育过程中通过前体细胞表达 产生五种类型的细胞，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OTX2的小鼠不能产生锥体或杆状感光细胞，也不能产生双极细胞 中间神经元。因此，视网膜中细胞命运的决定在很大程度上取决于OTX2表达的地点和时间。 为了了解OTX2的表达是如何调控的，我们搜索了它的增强子。增强剂是非 启动和稳定基因表达的DNA编码区。OTX2的三种潜在增强剂是 经鉴定由OTX2细胞表达。接下来，我们测试了这些增强剂是否必要 用于OTX2表达。CRISPR介导的增强子DHS4的缺失显示OTX2的减少 胚胎表达，但对出生后OTX2表达的影响不大。这表明其他人 OTX2增强剂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OTX2的另外两种增强剂DHS2和DHS15进行了实验。删除其中一个增强子显示 出生后OTX2的表达比DHS4的表达更强。有趣的是，删除这两个增强剂 同时在后来的时间点上对OTX2的还原没有相加的影响，这表明该复合体 OTX2增强剂的景观允许它们相互替代。我的观察使我得出了一个假设 在视网膜发育过程中，一个动态增强剂复合体启动并维持OTX2的表达。 我将在我的提案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，我将使用一个 高分辨染色体构象捕捉技术揭示OTX2增强子-启动子接触 视网膜发育的轨迹。此外，这项技术还将揭示OTX2的其他潜在增强剂。在……里面 我的第二个目标是，我将测试当增强剂被干扰时，这种增强剂复合体是如何被破坏的。为了做到这一点，我 将结合染色体构象捕捉和基于CRISPR的增强子扰动技术。这将是 请允许我了解增强器的动态，并确定增强器如何相互补充以确保 OTX2在视网膜发育过程中的表达这项建议的完成将提高我们对 视网膜发育，复杂的基因调控机制，为我提供了所需的经验 继续成功的职业生涯，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.