Identification and characterization of early photoreceptor cis-regulatory elements and their relation to Onecut1.
Identification and characterization of early photoreceptor cis-regulatory elements and their relation to Onecut1.
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DOI：
10.1186/s13064-018-0121-x
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发表时间：
2018-11-22
影响因子：
3.6
通讯作者：
Emerson MM
中科院分区：
文献类型：
--
作者：
Jean-Charles N;Buenaventura DF;Emerson MM
视锥和视杆光感受器是人类视网膜疾病中受影响的两种主要细胞类型。对抗这些疾病的潜在策略是使用基因疗法来拯救受损的光感受器或产生新的功能性光感受器以取代患病视网膜中丢失的光感受器。视锥细胞、视杆细胞或这两种类型光感受器的顺式调节元件是成功实施这两种策略的关键组成部分。本研究的目的是确定和表征细胞类型的特异性和活性的顺式调节元件在开发光感受器。通过电穿孔将顺式调控元件导入发育中的鸡和小鼠视网膜。使用共聚焦显微镜测定与细胞类型标志物相关的报告活性的表征。此外，开发了两种高通量流式细胞术测定来评估这些元件是否在感光细胞特化网络中Onecut1的下游。大多数顺式调控元件在视锥细胞和视杆细胞中都是活跃的，并且在很大程度上不受Onecut1显性负性结构的影响。与Thrb，Nr2e3和视紫红质基因相关的元素在视锥细胞或视杆细胞中表现出高度富集的活性，并受到Onecut1信号干扰的影响。视紫红质启动子活性受Onecut1活性的影响最大，其诱导可由Maf家族转录因子L-Maf调节。观察到Nr2e3元件在视锥光感受器中具有活性，并且Nr2e3蛋白在发育中的视锥光感受器中表达，这表明该优势杆基因在视锥光感受器发育中的作用。这里提出的分析提供了一个实验框架，以确定在特定的遗传网络中的感光元件的特异性和强度在发展过程中。Onecut1转录因子就是这样一个因子，它影响视锥细胞和视杆细胞特有的基因调控网络，但不影响两者共有的基因调控网络。本文的在线版本（10.1186/s13064 - 018 - 0121-x）包含补充材料，可供授权用户使用。
Cone and rod photoreceptors are two of the primary cell types affected in human retinal disease. Potential strategies to combat these diseases are the use of gene therapy to rescue compromised photoreceptors or to generate new functional photoreceptors to replace those lost in the diseased retina. Cis-regulatory elements specific to cones, rods, or both types of photoreceptors are critical components of successful implementation of these two strategies. The purpose of this study was to identify and characterize the cell type specificity and activity of cis-regulatory elements active in developing photoreceptors. Cis-regulatory elements were introduced into the developing chicken and mouse retina by electroporation. Characterization of reporter activity in relation with cell type markers was determined using confocal microscopy. In addition, two high-throughput flow cytometry assay were developed to assess whether these elements were downstream of Onecut1 in the photoreceptor specification network. The majority of cis-regulatory elements were active in both cone and rod photoreceptors and were largely uninfluenced by a Onecut1 dominant-negative construct. Elements associated with the Thrb, Nr2e3, and Rhodopsin genes showed highly enriched activity in cones or rods, and were affected by interference in Onecut1 signaling. Rhodopsin promoter activity was the most highly influenced by Onecut1 activity and its induction could be modulated by the Maf family transcription factor L-Maf. Nr2e3 elements were observed to have activity in cone photoreceptors and Nr2e3 protein was expressed in developing cone photoreceptors, suggesting a role for this predominant rod gene in cone photoreceptor development. The analysis presented here provides an experimental framework to determine the specificity and strength of photoreceptor elements within specific genetic networks during development. The Onecut1 transcription factor is one such factor that influences the gene regulatory networks specific to cones and rods, but not those that are common to both. The online version of this article (10.1186/s13064-018-0121-x) contains supplementary material, which is available to authorized users.
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影响因子：
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