Circadian analysis of the mouse retinal pigment epithelium transcriptome

Abstract

The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h 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 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions 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. 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, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes 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. 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.

Keywords: Circadian; Mouse; Retinal pigment epithelium; Transcriptome.