Functional analysis of Vsx2 super-enhancer reveals an autoregulatory network governing Vsx2 expression during retinogenesis [RNA-seq_mouse]

The roles of retinal cis-regulatory landscape in controlling the expression of gene regulatory networks important for retinogenesis remain poorly understood. Vsx2 is a transcription factor essential for retinal proliferation and bipolar cell differentiation but the molecular mechanisms underlying its developmental roles are unclear. Here, we profiled VSX2 genomic occupancy during mouse retinogenesis, revealing extensive retinal gene regulatory networks associated with Vsx2 during development. We defined an autoregulatory loop in which VSX2 binds and transactivates its own enhancer in association with the transcription factor PAX6 . The Vsx2 regulatory landscape contains elements that are required for Vsx2 expression, retinal proliferation and proper cell type differentiation. We further show that retinae in which the Vsx2 enhancer landscape has been largely deleted suffer a bias toward photoreceptor production. Genomic data indicate that VSX2 occupies cis-regulatory elements nearby genes associated with photoreceptor differentiation and homeostasis in mouse and human retinae, including a conserved region nearby the rod-specifying factor Prdm1. We provide evidence that VSX2 associates with OTX2 and can act to suppress OTX2-dependent enhancer transactivation of Prdm1 enhancer. Taken together, our analyses illuminate important mechanistic insights on how VSX2 is engaged with gene regulatory networks that are essential for retinal proliferation and cell fate acquisition. RNA-seq on mouse VSX2 SE WT at E14.5 (three replicates), VSX2 SE KO at E14.5 (three replicates), VSX2 EN1 WT at E14.5 (four replicates) and VSX2 EN1 KO at E14.5 (four replicates).目前学界对视网膜顺式调控景观（cis-regulatory landscape）调控视网膜发生过程中关键基因调控网络表达的作用机制仍知之甚少。Vsx2是一种对视网膜增殖与双极细胞分化至关重要的转录因子，但其发挥发育调控作用的分子机制尚未明确。本研究对小鼠视网膜发生过程中VSX2的基因组结合位点进行了全谱分析，揭示了发育阶段与Vsx2紧密关联的庞大视网膜基因调控网络。我们鉴定出一条自调控环路：VSX2可与转录因子PAX6协同结合，并反式激活自身的增强子。Vsx2的调控景观包含了其自身表达、视网膜增殖以及正常细胞类型分化所必需的调控元件。我们进一步证实，当Vsx2增强子景观被大规模缺失时，视网膜会出现感光细胞生成偏倚的表型。基因组数据显示，在小鼠与人类视网膜中，VSX2可结合于与感光细胞分化及稳态相关基因附近的顺式调控元件，其中包括视杆细胞特异性因子Prdm1附近的一段保守区域。本研究证实，VSX2可与OTX2发生相互作用，并能够抑制OTX2依赖的Prdm1增强子反式激活过程。综上，本研究阐明了VSX2如何与视网膜增殖及细胞命运获取所必需的基因调控网络相互作用的重要机制。本研究对E14.5天小鼠的VSX2 SE野生型（3次生物学重复）、VSX2 SE敲除型（3次生物学重复）、VSX2 EN1野生型（4次生物学重复）以及VSX2 EN1敲除型（4次生物学重复）样本开展了RNA测序。