Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation

Abstract

Circadian rhythms of cell and organismal physiology are controlled by an autoregulatory transcription-translation feedback loop that regulates the expression of rhythmic genes in a tissue-specific manner. Recent studies have suggested that components of the circadian pacemaker, such as the Clock and Per2 gene products, regulate a wide variety of processes, including obesity, sensitization to cocaine, cancer susceptibility, and morbidity to chemotherapeutic agents. To identify a more complete cohort of genes that are transcriptionally regulated by CLOCK and/or circadian rhythms, we used a DNA array interrogating the mouse protein-encoding transcriptome to measure gene expression in liver and skeletal muscle from WT and Clock mutant mice. In WT tissue, we found that a large percentage of expressed genes were transcription factors that were rhythmic in either muscle or liver, but not in both, suggesting that tissue-specific output of the pacemaker is regulated in part by a transcriptional cascade. In comparing tissues from WT and Clock mutant mice, we found that the Clock mutation affects the expression of many genes that are rhythmic in WT tissue, but also profoundly affects many nonrhythmic genes. In both liver and skeletal muscle, a significant number of CLOCK-regulated genes were associated with the cell cycle and cell proliferation. To determine whether the observed patterns in cell-cycle gene expression in Clock mutants resulted in functional dysregulation, we compared proliferation rates of fibroblasts derived from WT or Clock mutant embryos and found that the Clock mutation significantly inhibits cell growth and proliferation.

Fig. 1.

Rhythmic expressed genes in WT liver and skeletal muscle. (A and B) Liver and gastrocnemius muscle tissues were collected from WT mice over a 48-h period (30–74 h in DD), and mRNA expression was determined by using a custom Affymetrix whole mouse genome microarray. Expression data were subjected to COSOPT cosine analysis to identify transcripts that were expressed with an ≈24-h period. Rhythmic (MMCβ < 0.2) genes were plotted by peak phase for liver (A, 854 genes) and skeletal muscle (B, 383 genes). (C) These genes were then separated into phase clusters derived from COSOPT analysis. Rhythmic liver genes exhibited large-phase clusters at CT06 and CT18, whereas the largest skeletal muscle-phase cluster occurred at CT18.

Fig. 2.

The effect of the Clock mutation on WT circadian genes in mouse liver. (A) Pseudocolored graph depicting highly significant (MMCβ < 0.1) WT circadian genes (y axis) versus hours after DD (x axis) for WT or Clock mice. The graph is based on Z-score comparisons of each gene's individual time point intensity in relation to its average intensity for all times and genotypes, where red indicates higher expression and green indicates lower expression. Genes were ordered based on peak time in WT samples. The asterisk indicates genes that peak at CT10–14, which are in-phase with known CLOCK-BMAL1 target genes, such as Per2, Dbp, and Cry2. (B–D) Shown are graphs of fluorescence intensity (y axis) versus hours in DD (x axis) for examples of genes that are overall down-regulated (Dbp; B), up-regulated (Tmlhe; C), or peak-shifted (Casp6; D) in Clock (dashed line) versus WT (solid line) liver. Numbers in parentheses indicate how many genes fit the criteria for each grouping.

Fig. 3.

Effect of the Clock mutation on gene expression. Gene expression in liver and gastrocnemius muscle from Clock mice was compared with gene expression in WT mice. (A) Bar graphs represent the number of genes whose average expression over a 24-h period showed significant, >2-fold change in expression in Clock tissue. In both liver and muscle, the majority of CLOCK-controlled genes were down-regulated compared with WT expression. (B) Each graph shows 20 examples of genes whose average expression was up-regulated (Upper) or down-regulated (Lower) in Clock muscle. (C) Pie charts depict the most highly represented functional categories of genes changed in Clock mouse liver or muscle.

Fig. 4.

DNA synthesis and cell proliferation is diminished in Clock primary cells. (A) The average expression intensities from 24-h microarray data are shown for many genes that are involved in cell proliferation and exhibit significantly (P < 0.05; see SI Table 8) changed expression in Clock liver. (B and C) MEFs from WT and Clock embryos were cultured in 2% BCS for 48 h and then switched to media containing 10% FBS. The number of live cells (B) and quantity of DNA (C) were measured during low-serum and high-serum (+24 h, +36 h, +72 h) conditions. At least three wells per culture were measured at each time point, and all data were normalized to the average low-serum values for each culture. Asterisks represent the significant difference observed for both the relative cell number and relative DNA quantity of WT MEF cells versus Clock MEF cells at +36 h and +72 h, and the relative cell number of WT MEFs versus Clock MEFs at +24 h (∗, effect of genotype; two-way ANOVA). In addition, WT MEFs exhibited significantly higher relative cell number and DNA quantitation at +72h, compared with other time points (#, effect of time; two-way ANOVA). Error bars represent standard deviation from the means of normalized data from 12 independent WT and 10 independent Clock MEF cultures. (D) mRNA expression from MEFs grown in 10% FBS was determined by RT-PCR. Expression of the proproliferative genes Erk1, ERα, Egfr, and PI3K was significantly reduced in Clock MEFs (∗, P < 0.05; ∗∗,P < 0.01; Student's t test).