Cis-regulatory analysis of Onecut1 in fate-restricted retinal progenitor cells. Cis-regulatory analysis of Onecut1 in fate-restricted retinal progenitor cells

Background: The vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction. Method: We identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests.Results: Two regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression. Conclusions: This work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs. Overall design: 2 samples, one replicate each. ThrbCRM1(+) and ThrbCRM1(-) were sorted and collected through FACS after electroporation with ThrbCRM1::GFP and UbiqC::TdT. ThrbCRM1(-) served as a comparison to ThrbCRM1(+)

背景：脊椎动物视网膜包含六大类神经元细胞。发育过程中，这些细胞均由表达Vsx2基因的多能视网膜祖细胞（multipotent retinal progenitor cells, RPCs）池分化产生。近期研究已鉴定出命运受限型视网膜祖细胞（fate-restricted RPCs），与多能RPCs相比，这类细胞的有丝分裂潜能与细胞命运选择范围均更为有限。一类以调控元件ThrbCRM1活性为标记的命运受限型RPCs，可同时分化为视锥感光细胞与水平细胞。这类细胞不表达Vsx2，但共表达可结合ThrbCRM1的转录因子（transcription factors, TFs）Onecut1与Otx2。目前尚不明确调控多能RPCs向命运受限型细胞转变的基因调控网络（gene regulatory networks, GRNs）组成。本研究旨在鉴定并评估Onecut1基因邻近的顺式调控元件，以明确参与RPC命运受限过程的GRNs。
方法：本研究通过转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）与序列保守性分析鉴定调控元件，随后基于时空标准通过报告基因实验筛选具有活性的元件。针对目标调控元件开展缺失与突变分析以鉴定其功能序列，并通过定量流式细胞术实验进行验证。最后，本研究将增强子-报告基因实验与候选TF过表达实验相结合，以评估TFs、增强子与脊椎动物视网膜早期发育之间的关联。统计检验方法包括方差分析（ANOVA）、Kruskal-Wallis检验与非配对t检验。
结果：本研究鉴定出两个可在ThrbCRM1(+)命运受限型RPCs中发挥活性的调控元件ECR9与ECR65。两类元件的关键序列均被鉴定为候选碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）结合位点。候选bHLH TF的过表达实验揭示了特异性的增强子-bHLH相互作用。Nhlh1过表达可将ECR65的活性范围扩展至Vsx2(+) RPC群体，而NeuroD1/NeuroG2/NeuroD4的过表达对ECR9具有类似效应。此外，可激活异位ECR9报告基因的bHLH因子，能够诱导内源性Otx2的表达。
结论：本研究通过大规模筛选，鉴定出Onecut1基因位点附近具有时空特异性的调控元件。利用这些元件，可区分发育中视网膜内的不同细胞群体。此外，命运受限型调控元件对bHLH因子的响应具有特异性，这表明视网膜bHLH因子在多能RPCs向命运受限型RPCs转变的过程中，可作为Otx2与Onecut1基因的上游调控因子发挥作用。
实验整体设计：共设置2个样本，每个样本设置1次生物学重复。将ThrbCRM1::GFP与UbiqC::TdT通过电穿孔法转染细胞后，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分选出ThrbCRM1(+)与ThrbCRM1(-)细胞群体，其中ThrbCRM1(-)细胞作为ThrbCRM1(+)细胞的对照样本。