Transcriptional Dynamics of Photoreceptor Gene Regulation.

Project Summary/Abstract The homeodomain transcription factor Crx is expressed at the final mitosis of photoreceptor precursors, is involved in epigenetic remodeling, binds to promoters to activate photoreceptor- specific genes and silence other genes. In this proposal, we will investigate target search dynamics in live photoreceptors – how it transits the complex nuclear environment to find regulatory DNA target elements within the mass of non-specific DNA in chromatin. We will develop quantitative single-particle tracking super-resolution imaging (sptPALM) to characterize Crx dynamics in a new model. We have created a knock-in mouse line in which the coding region of a next-generation photoswitchable fluorescent protein, mEos4b, was inserted into the Crx gene. Since mEos4b is in the endogenous Crx locus, expression levels will be similar to native concentrations throughout development. We will answer the following three fundamental questions: 1) What mobility modes are employed by Crx in photoreceptor nuclei? 2) How long is Crx bound to chromatin? 3) Are Crx dynamics different in rods, cones, photoreceptor precursors, or bipolar cells? Our model will give us unprecedented access to nuclear dynamics of Crx throughout the lifespan of photoreceptors.