Gene regulation of retinal cell differentiation

Abstract Retinal progenitor cells possess remarkable multipotency since they can differentiate into all retinal neurons and Muller glia in the retina. This multipotency is controlled by a sparse number of homeodomain and Sox family transcription factors (TFs), such as Six3, Six6, Sox2, and Pax6. Mutations in these genes cause human congenital eye abnormalities, such as microphthalmia, anophthalmia, and coloboma. Loss-of- function studies in mice demonstrate that these multipotency TFs are essential for the competence of retinal progenitor cells. Despite these advances, direct target genes of these multipotency TFs, mechanisms of target regulation in chromatin, and molecular underpinnings of the multipotency remain elusive. To address these critical knowledge gaps, we set out to profile chromatin landscapes of multipotency TFs in retinal development using CUT&RUN. In Six3-occupied peaks, the most enriched de novo motif was often neighbored by a Sox2-like motif, suggesting cooperative DNA binding between Six3 and Sox2. We performed single-cell ATAC sequencing and found that Six3 and Six6 jointly regulate chromatin accessibility. Additionally, we identified components in SIX3, SIX6, and SOX2 protein complexes isolated from retinal progenitor cells. Subunits of BAF and/or PBAF complexes (mammalian SWI/SNF complexes), KDMs, and HDACs were highly enriched in Six3 complexes and to a lesser extent in Six6 and Sox2 complexes. Additionally, SIX3 protein complexes contained variable amounts of PAX6, SOX2, and SIX6 proteins. The goal of this program is to elucidate molecular underpinnings of the multipotency and lineage differentiation of retinal progenitor cells. We hypothesize that multipotency TFs Six3, Six6, Sox2, and Pax6 have both overlapping and distinct chromatin occupancies; these TFs cooperatively bind to cis-acting DNA elements and then recruit chromatin remodeling proteins to regulate chromatin states in a context- dependent manner for target regulation; their target genes collectively execute the control of the multipotency and lineage differentiation of retinal progenitor cells. This hypothesis will be tested in two Aims. Aim 1 is to elucidate how multipotency TFs of the retina regulate target genes in chromatin by assessing chromatin occupancy, DNA-binding cooperativity, and chromatin remodeler recruitment. Aim 2 is to determine how Six3, Six6, and Sox2 regulate chromatin accessibility at a cellular resolution. Elucidation of the functions of multipotency TFs will provide pivotal insight into congenital eye abnormalities. Chromatin occupancies of TFs, chromatin remodelers, and histone post-translational modifications in retinas will be valuable resources for functional annotations of non-coding retinal disease SNPs in humans.