Network features of the mammalian circadian clock

Abstract

The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. To assess the robustness of a human molecular clock, we systematically depleted known clock components and observed that circadian oscillations are maintained over a wide range of disruptions. We developed a novel strategy termed Gene Dosage Network Analysis (GDNA) in which small interfering RNA (siRNA)-induced dose-dependent changes in gene expression were used to build gene association networks consistent with known biochemical constraints. The use of multiple doses powered the analysis to uncover several novel network features of the circadian clock, including proportional responses and signal propagation through interacting genetic modules. We also observed several examples where a gene is up-regulated following knockdown of its paralog, suggesting the clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. We propose that these network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation.

Figure 1. Functional Effects on Oscillations Following Knockdown of Circadian Clock Components in U-2 OS Cells

U-2 OS cells were transfected with pools of four to five siRNAs for each gene, and oscillations in Bmal1-luciferase were measured for 5 d following synchronization with dexamethasone. Knockdown of each component was validated by quantitative RT-PCR from RNA isolated from a replicate sample. The relative amount of expression of each gene compared to the negative siRNA control sample is shown (insets). Results are representative of three independent biological replicates.

Figure 2. GDNA Following Genetic Perturbation of Bmal1, Clock, and Per1

(A, D, and G) Oscillations in Bmal1-luciferase were measured from cells transfected with increasing amounts of siRNAs targeting Bmal1, Clock, or Per1. (B, E, and H) RNA was isolated from replicate samples collected at time 0 (before synchronization) and expression of clock genes was determined by quantitative real time PCR. (C, F, and I) GDNAs were generated for siBmal1 (C), siClock (F), and siPer1(I) using expression data from each knockdown condition. Edges between genes were determined using nonparametric Pearson correlation (p-value < 0.10) and biochemical constraints, and gene expression changes are denoted as increases (red) and decreases (green). The gene being depleted is located at the top of network, with first order responses below as restricted by biochemistry, i.e. genes that decrease when Clock and Bmal1 activators are decreased (C and F) and genes that are increased with the Per1 repressor is decreased (I). Second-order responses are defined as those with correlated edges with the first-order responders. Black lines indicated published edges, and blue lines denote unpublished relationships.

Figure 3. Signal Propagation of Proportional Gene Expression Changes Is Observed upon Dose- Dependent Knockdown of Clock Components

(A–C) Examples of linear, proportional gene expression changes following knockdown of Bmal1 (A), Per1 (B), and Clock (C). (D and E) Disproportional responses are observed in Per2 gene expression following knockdown of Cry1 and Cry2 (D), and similar changes are observed in MEFs derived from Cry1/Cry2 double-knockout mice (E). (F) Signal propagation following knockdown of Per1 through a Repressor/Repressor module where knockdown of Per1 leads to increase of Rev-erbs, which in turn causes a decrease in Bmal1 expression. (G) An Activator/Repressor module relays the signal following knockdown of Bmal1, which leads to a decrease in Rev-erb alpha and a subsequent increase in Per2.

Figure 4. Unidirectional Paralog Compensation in the Circadian Repressors Cry1, Rev-erb beta, and Per1

(A, C, and E) U-2 OS cells were transfected with doses of siRNA as indicated, and gene expression was measured from samples collected 48 h after transfection. Knockdown of Cry1 (A), Rev-erb beta (C), and Per1 (E) leads to increased expression of the gene paralogs Cry2, Rev-erb alpha, and Per2 and Per3, respectively. (B, D, F, and G) The response is unidirectional, as a decrease of Cry2 (B), Rev-erb alpha (D), Per2 (F), or Per3 (G) does not result in an increase of respective gene paralogs. (H) Transcriptional repressors could directly regulate the expression of their gene paralog through response elements (RE) in upstream regulatory regions (H) (modified from [25]). In the case of the circadian clock network, we propose direct regulation of Cry1, Per1, and Rev-erb beta (Repressor 1) by Cry2, Per2/3, or Rev-erb alpha (Repressor 2), respectively.

Figure 5. Compensation in Nonparalogous Genes May Contribute to Oscillator Robustness

(A) U-2 OS cells transfected with siRNAs targeting Bmal1 or Cry1, individually or in combination as indicated, were synchronized, and luminescence levels were measured over 5 d. Data are representative of three independent biological replicates. Note that two examples are shown for the siCry1/siBmal1 combination knockdown. (B) Median and ranges of amplitude following single or combinatoric perturbation. The amplitude of the circadian signal was estimated using continuous wavelet decomposition and averaged across three replicates in three independent experiments. The median and range of the fold changes relative to the negative siRNA control are plotted. Both individual siRNA treatments are significantly down-regulated relative to the negative siRNA controls (p < 0.05, Mann-Whitney test), and the siCry1/siBmal1 double knockdown is also significantly down-regulated relative to siCry1 (p < 0.05, Mann-Whitney test).