The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse.

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 CLOCKregulated 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.cell cycle ͉ circadian rhythms ͉ Clock mutation ͉ gene expression ͉ protein-encoding transcriptome M any organisms have Ϸ24-h rhythms in metabolism, physiology, and behavior that are driven by cell autonomous circadian pacemakers (1). These circadian rhythms allow organisms to coordinate a myriad of physiological processes with the changing environment. In mammals, the circadian pacemaker is composed of interlocked transcription-translation feedback loops: the primary loop is composed of the basic helix-loophelix transcription factors CLOCK and BMAL1, which drive transcription of the Period (Per1, Per2) and Cryptochrome (Cry1, Cry2) genes (1, 2). PER and CRY proteins form the negative limb of the feedback loop by inhibiting their own CLOCK: BMAL1-induced transcription; turnover of PER and CRY allows the cycle to begin anew. The interlocked loop consists of REV-ERB-␣ and ROR␣, which repress and activate the Bmal1 gene, thereby modulating its function (3, 4). Mutation or deletion of Clock (5), Bmal1 (6), Per1/2 genes (7, 8), or Cry1/2 (9, 10) genes results in behavioral arrhythmicity and disruption of the autoregulatory loop, whereas disruption of components of the secondary loop results in short period-length phenotypes (3, 4).The molecular components of the circadian clock are present in the majority of neurons in the suprachiasmatic nucleus (SCN), a bilateral body in the anterior hypot...

We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.