Department of Genetics, INSERM, U968, Paris, France.
PLoS One. 2010 Oct 7;5(10):e13075. doi: 10.1371/journal.pone.0013075.
RdCVF and RdCVF2, encoded by the nucleoredoxin-like genes NXNL1 and NXNL2, are trophic factors with therapeutic potential that are involved in cone photoreceptor survival. Studying how their expression is regulated in the retina has implications for understanding both their activity and the mechanisms determining cell-type specificity within the retina.
METHODOLOGY/PRINCIPAL FINDINGS: In order to define and characterize their promoters, a series of luciferase/GFP reporter constructs that contain various fragments of the 5'-upstream region of each gene, both murine and human, were tested in photoreceptor-like and non-photoreceptor cell lines and also in a biologically more relevant mouse retinal explant system. For NXNL1, 5'-deletion analysis identified the human -205/+57 bp and murine -351/+51 bp regions as having promoter activity. Moreover, in the retinal explants these constructs drove expression specifically to photoreceptor cells. For NXNL2, the human -393/+27 bp and murine -195/+70 bp regions were found to be sufficient for promoter activity. However, despite the fact that endogenous NXNL2 expression is photoreceptor-specific within the retina, neither of these DNA sequences nor larger upstream regions demonstrated photoreceptor-specific expression. Further analysis showed that a 79 bp NXNL2 positive regulatory sequence (-393 to 315 bp) combined with a 134 bp inactive minimal NXNL1 promoter fragment (-77 to +57 bp) was able to drive photoreceptor-specific expression, suggesting that the minimal NXNL1 fragment contains latent elements that encode cell-type specificity. Finally, based on bioinformatic analysis that suggested the importance of a CRX binding site within the minimal NXNL1 fragment, we found by mutation analysis that, depending on the context, the CRX site can play a dual role.
CONCLUSIONS/SIGNIFICANCE: The regulation of the Nucleoredoxin-like genes involves a CRX responsive element that can act as both as a positive regulator of promoter activity and as a modulator of cell-type specificity.
RdCVF 和 RdCVF2 是由核还原酶样基因 NXNL1 和 NXNL2 编码的营养因子，具有治疗潜力，参与视锥细胞感光器的存活。研究它们在视网膜中的表达调控，对于理解它们的活性以及决定视网膜中细胞类型特异性的机制都具有重要意义。
方法/主要发现：为了定义和描述它们的启动子，我们构建了一系列包含每个基因（包括鼠和人）5' 上游区域不同片段的荧光素酶/GFP 报告基因构建体，在光感受器样和非光感受器细胞系中以及在更具生物学相关性的鼠视网膜外植体系统中进行了检测。对于 NXNL1，5' 缺失分析确定了人类 -205/+57 bp 和鼠 -351/+51 bp 区域具有启动子活性。此外，在视网膜外植体中，这些构建体特异性地驱动感光器细胞的表达。对于 NXNL2，人类 -393/+27 bp 和鼠 -195/+70 bp 区域被发现足以发挥启动子活性。然而，尽管内源性 NXNL2 在视网膜中是感光器特异性表达的，但这些 DNA 序列或更大的上游区域都没有表现出感光器特异性表达。进一步的分析表明，一个 79 bp 的 NXNL2 阳性调控序列（-393 到 315 bp）与一个 134 bp 的非活性最小 NXNL1 启动子片段（-77 到 +57 bp）相结合，能够驱动感光器特异性表达，这表明最小的 NXNL1 片段包含编码细胞类型特异性的潜在元件。最后，基于生物信息学分析表明最小 NXNL1 片段内的 CRX 结合位点的重要性，我们通过突变分析发现，视黄醛结合蛋白（CRX）位点可以根据上下文发挥双重作用。
结论/意义：核还原酶样基因的调控涉及一个 CRX 反应元件，该元件既能作为启动子活性的正调控因子，又能作为细胞类型特异性的调节剂。
