Circadian analysis of the mouse retinal pigment epithelium transcriptome

Purpose: The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or two hours after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 hour before (CT23) and 1 hour after (CT1) subjective light onset) Methods: C57BL/6J male mice were maintained in DD for three days and euthanized under red light (< 1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. Results: 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥±2.0). Of the protein coding transcripts, a majority of them were classified as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included classes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Conclusions: Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation. Retinal pigment epithelium enriched samples isolated from whole eyes from 8-10 week old male C57BL/6J mice at 2 different circadian timepoints in triplicate研究背景与目的：已有多项研究报道，在多种昼行性与夜行性物种中，视网膜色素上皮（retinal pigment epithelium, RPE）会在光照起始后1至2小时出现感光细胞外节吞噬活性峰值。该吞噬活性峰值在恒亮或恒暗条件下仍可维持，表明其出现时序由昼夜节律钟驱动。本研究旨在探究两个不同昼夜时间点（昼夜时间点，CT；主观光照开始前1小时（CT23）与主观光照开始后1小时（CT1））下，RPE全转录组的变化情况。 实验方法：将C57BL/6J品系雄性小鼠置于黑暗环境（DD）中饲养3天，分别于CT23与CT1时点，在红光（<1勒克斯）环境下实施安乐死。从完整眼球中分离RPE组织，进行RNA文库构建，并依托Illumina HiSeq4000平台完成测序。 实验结果：CT23与CT1时点共检测到14083个小鼠RPE共有转录本，其中12005个为蛋白编码转录本，2078个为非蛋白编码转录本。与CT23相比，CT1时点有2421个蛋白编码转录本显著上调，仅3个显著下调；同时有12个非蛋白编码转录本显著上调，31个显著下调（p < 0.05，倍数变化≥±2.0）。在蛋白编码转录本中，绝大多数被归类为酶类、激酶类与转录调控因子，且其主要活性定位区域为细胞质、细胞核与细胞质膜。非蛋白编码转录本则涵盖长链非编码RNA与假基因等类别。基因本体分析与Ingenuity路径分析（Ingenuity Pathway Analysis, IPA）结果显示，差异表达转录本与整合素信号通路、氧化磷酸化、蛋白质磷酸化及肌动蛋白细胞骨架重塑密切相关，表明此前已确认的吞噬通路受昼夜节律钟调控。 研究结论：本研究分析发现了新的潜在参与吞噬活性昼夜调控的通路，例如通过增强氧化磷酸化提升线粒体呼吸功能。此外，本数据集提示所鉴定的非蛋白编码转录本可能在介导RPE吞噬的复杂生理功能中发挥调控作用。最后，与其他组织的已有研究结果一致，本研究结果表明约20%的RPE全转录组可能受昼夜节律钟调控。 本数据集样本为：从8-10周龄雄性C57BL/6J小鼠完整眼球中分离的RPE富集样本，涵盖两个昼夜时间点，每个时间点设置三次生物学重复。