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

In The Loop：研究视网膜发育过程中 OTX2 增强剂介导的调节

• 批准号: 10752407
• 负责人: Ian J Purvis
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752407
• 关键词: Ablation; Adopted; Adult; Amacrine Cells; Animals; Biological Assay; Bite; Brain; CRISPR interference; CRISPR/Cas technology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Compensation; Complex; DNA; DNA Sequence; Development; Electroporation; Embryo; Enhancers; Ensure; Frequencies; Gene Expression; Gene Silencing; Interneurons; Knowledge; Light; Liver; Mediating; Modeling; Mus; Occupations; Pattern; Phenotype; Photoreceptors; Plasmids; Play; Production; Regulation; Regulator Genes; Resolution; Retina; Retinal Cone; Role; Techniques; Testing; Tissues; Untranslated RNA; Vertebrate Photoreceptors; Vision; career; cell type; chromosome conformation capture; experience; experimental study; homeodomain; horizontal cell; improved; loss of function; novel; postmitotic; postnatal; precursor cell; prevent; promoter; retinal rods; transcription factor; visual information

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.

项目摘要 视网膜是一种由七种主要细胞类型组成的复杂组织。这些细胞类型都需要 对于正常的视网膜功能，因此视力。哺乳动物视网膜中的所有细胞均在发育过程中形成 并且必须持续动物的生命。三种视网膜细胞类型的产生取决于表达 OTX2（同源域转录因子）的OTX2。 OTX2在视网膜发育过程中由前体表示 引起五种细胞类型，但仅由光感受器和双极细胞保持成熟。丧失 功能研究表明，缺乏OTX2的小鼠无法产生锥形或杆感光体，也无法产生双极细胞 中间神经元。因此，视网膜中的细胞命运决策在很大程度上取决于表达OTX2的何时何时。 为了了解如何调节OTX2的表达，我们搜索了其增强子。增强剂是非 - 启动和稳定基因表达的DNA的编码区域。 OTX2的三个潜在增强子是 鉴定并证明由OTX2+细胞表达。接下来，我们测试了这些增强器是否需要 用于OTX2表达。 CRISPR介导的一个增强器DHS4的删除显示OTX2降低了 在胚胎上表达，但对产后OTX2表达的影响适度。这表明其他 OTX2增强子用于产后视网膜发育。为了调查这一点，我进行了CRISPR删除 OTX2的其他两个增强子，称为DHS2和DHS15。删除两个增强器显示 与DHS4相比，otx2表达的降低更强。有趣的是，删除两个增强器 同时在以后的时间点上对otx2的减少没有添加作用，这表明该复合物 OTX2增强剂的景观使它们可以互相替代。我的观察使我假设 动态增强子复合物在视网膜发育过程中启动并保持OTX2表达。 我将通过完成两个具体目标来检验我的提案中的假设。在我的第一个目标中，我将采用 高分辨率染色体构象捕获技术，以揭示OTX2的增强剂启动器触点 跨视网膜发育的基因座。此外，该技术将揭示OTX2的其他潜在增强子。在 我的第二个目标是，当增强剂受到干扰时，我将测试该增强子络合物如何破坏。为此，我 将结合染色体构象捕获与基于CRISPR的增强剂扰动技术。这会 允许我辨别增强器动态，并确定增强剂如何相互补偿以确保 视网膜发育过程中的OTX2表达。该提案的完成将提高我们对 视网膜发展，复杂的基因调节机制，并为我提供所需的经验 继续领导我自己的学术实验室的成功职业。