Co‐regulation of long non‐coding RNAs and protein‐coding genes during cell quiescence

Abstract

Proliferating cells can exit the cell cycle reversibly to enter a quiescent state. Better understanding of cell quiescence is important to gain deeper insights into stem cell renewal, tissue homeostasis, and cancer dormancy. Entry into quiescence involves global changes in protein-coding genes. How these changes are regulated is not understood. Long noncoding RNAs (lncRNAs) are >200 nucleotides long and regulate the expression of protein-coding genes in cis or in trans. Here, we investigated the role of lncRNAs in the regulation of protein-coding genes during quiescence using a quiescence cell culture model based on human dermal fibroblasts. Proliferating (P) cells were introduced into quiescence by 7-days of serum-starvation (SS) or contact inhibition (CI) and then restimulated to a proliferating state for 24 hours (SS-R or CI-R). Using data from stranded RNA-seq of fibroblasts in the five conditions, differential gene expression analysis was performed using four different algorithms to obtain a list of protein-coding genes and lncRNAs that changed significantly (adjusted p<0.05) between the conditions with all four algorithms. With this pipeline, 141 and 114 lncRNAs changed significantly in expression between P and CI and between P and SS, respectively, and 80 lncRNAs were common between the two groups. This suggests that different quiescent conditions adopt similar lncRNA expression profiles. Out of 80 lncRNAs, 66 were upregulated and 14 were downregulated with quiescence. Analysis of 80 lncRNA promoters showed an enrichment of motifs for a set of transcription factors that were also upregulated with quiescence. A partial reversal of the profile during quiescence was observed when the cells were restimulated to cycle for 24 hours. Co-expression analysis revealed that in most instances (57%), expression changes with quiescence for lncRNAs and neighboring protein-coding genes (within 10kb) were in opposite directions, suggesting the lncRNAs may repress expression of protein-coding genes either directly or via sequestration of transcriptional machinery. These protein-coding genes were enriched for biological processes related to quiescence such as DNA replication and repair, DNA replication checkpoints, MAP kinase signaling and transcriptional regulation. Our results, therefore, indicate a possible function for lncRNAs in regulation of cell cycle and transcription with quiescence.