microRNAs regulate cell-to-cell variability of endogenous target gene expression in developing mouse thymocytes

The development and homeostasis of multicellular organisms relies on gene regulation within individual constituent cells. Gene regulatory circuits that increase the robustness of gene expression frequently incorporate microRNAs as post-transcriptional regulators. Computational approaches, synthetic gene circuits and observations in model organisms predict that the co-regulation of microRNAs and their target mRNAs can reduce cell-to-cell variability in the expression of target genes. However, whether microRNAs directly regulate variability of endogenous gene expression remains to be tested in mammalian cells. Here we use quantitative flow cytometry to show that microRNAs impact on cell-to-cell variability of protein expression in developing mouse thymocytes. We find two distinct mechanisms that control variation in the activation-induced expression of the microRNA target CD69. First, the expression of miR-17 and miR-20a, two members of the miR-17-92 cluster, is co-regulated with the target mRNA Cd69 to form an activation-induced incoherent feed-forward loop. Another microRNA, miR-181a, acts at least in part upstream of the target mRNA Cd69 to modulate cellular responses to activation. The ability of microRNAs to render gene expression more uniform across mammalian cell populations may be important for normal development and for disease.
Author Summary microRNAs are integral to many developmental processes and may 'canalise' development by reducing cell-to-cell variation in gene expression. This idea is supported by computational studies that have modeled the impact of microRNAs on the expression of their targets and the construction of artificial incoherent feedforward loops using synthetic biology tools. Here we show that this interesting principle of microRNA regulation actually occurs in a mammalian developmental system. We examine cell-to-cell variation of protein expression in developing mouse thymocytes by quantitative flow cytometry and find that the absence of microRNAs results in increased cell-to-cell variation in the expression of the microRNA target Cd69. Mechanistically, T cell receptor signaling induces both Cd69 and miR-17 and miR-20a, two microRNAs that target Cd69. Co-regulation of microRNAs and their target mRNA dampens the expression of Cd69 and forms an incoherent feedforward loop that reduces cell-to-cell variation on CD69 expression. In addition, miR-181, which also targets Cd69 and is a known modulator of T cell receptor signaling, also affects cell-to-cell variation of CD69 expression. The ability of microRNAs to control the uniformity of gene expression across mammalian cell populations may be important for normal development and for disease.