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.