Circadian rhythms are 24-hour oscillations in behavior, physiology, and biochemistry that are generated by a cell-autonomous clock system found in all classes of living systems. To understand the molecular mechanism of the circadian clock system, our laboratory has used forward genetic approaches to discover genes that regulate circadian behavior in mice.
The Clock Gene
Using high-efficiency N-ethyl-N-nitrosourea mutagenesis, we isolated the first single-gene mutation that affects circadian rhythms in mice. The Clock mutation lengthens circadian period by 4 hours in homozygous mutants, followed by a complete loss of circadian rhythmicity in constant conditions. Using a combined approach of positional cloning and transgenic (functional) rescue of the Clock mutation in mice, we found that the Clock gene encodes a novel member of the basic helix-loop-helix (bHLH)-PAS family of transcription factors. The CLOCK protein acts as a heterodimeric transcription factor with a partner known as BMAL1 (also known as MOP3, ARNT-L, and CYCLE). The CLOCK-BMAL1 complex activates a number of target genes, including the mammalian homologs of the Drosophila period genes (mPer1, mPer2, mPer3), as well as the mammalian Cryptochrome genes (mCry1, mCry2).
Mammalian Cryptochrome Genes
Gene-targeting experiments have shown that the cryptochromes play an essential role in the control of circadian rhythms in mammals. Null mutations of mCry1 or mCry2 cause shortening and lengthening of the circadian period of mice, respectively, while Cry1/Cry2 double mutants completely lose circadian rhythms of behavior as well as molecular oscillations of mPer1 and mPer2 gene expression in the suprachiasmatic nucleus. Moreover, the CRY proteins are potent inhibitors of CLOCK/BMAL1-induced transcription, suggesting that the cryptochromes play an unexpected role as feedback elements of the circadian pacemaker itself.
Models of the Circadian Oscillator in Animals
From work in both Drosophila and mammals, it appears that a transcriptional autoregulatory feedback loop forms the basic mechanism of the circadian clock system. Although the core set of circadian genes is conserved in Drosophila and mice, there are fundamental differences in their regulation. There are at least six core genes that are thought to play a role in the circadian clock system of mice (Clock, Bmal1, mPer1, mPer2, mCry1, >mCry2). CLOCK and BMAL1 are positive elements that act upstream of the mPer and mCry genes to activate their transcription. As the levels of PER and CRY proteins accumulate, they interact among themselves and eventually translocate into the nucleus, where they inhibit the action of CLOCK/BMAL1 on their own transcription. With the decline of mPer and mCry transcription, the proteins then turn over and CLOCK and BMAL1 are released from negative feedback, leading to a new cycle of transcription. The mammalian transcriptional feedback loop is similar to that seen in Drosophila at the level of Per gene regulation; however, the cryptochromes play a completely different role as potent negative regulators. In addition, a role for the Drosophila timeless gene is absent in mammals, and the pathways for light input are divergent.
Casein Kinase Iε: A Conserved Circadian Pathway
Recently we used a positional syntenic cloning strategy to identify the circadian mutation tau in the hamster. The tau mutation was identified in 1988 as a spontaneous, semidominant autosomal mutation that shortens circadian period by 2 hours in heterozygotes and by 4 hours in homozygous mutants. We used genetically directed representational difference analysis (GDRDA) to isolate markers closely linked to the tau locus, since this species does not have genetic mapping resources. These GDRDA markers enabled us to identify a 15-cM region of conserved synteny between hamster and mouse chromosome 15. The comparative mouse and human maps allowed us to determine that the interval containing tau in hamster shared a region of conserved synteny with human chromosome 22. This helped us identify CKIε as a candidate gene, since the Drosophila circadian mutation double-time had recently been shown to encode a fly homolog of CKIε. The tau mutant enzyme has an arginine-to-cysteine amino acid substitution at a highly conserved residue, leading to a markedly reduced maximal enzyme velocity. Genetic and biochemical evidence shows that CKIε phosphorylates the mammalian PERIOD proteins, regulating their turnover. Because Per1 gene expression is altered in tau mutants, we propose that CKIε plays a significant role in delaying the negative-feedback signal within the transcription-translation–based autoregulatory loop that makes up the core of the circadian mechanism. Of the genes now identified with circadian function in mammals, CKIε is one of the first enzymes (as opposed to transcriptional regulators). As such, CKIε is a validated target for discovery of pharmaceutical compounds influencing circadian rhythms, sleep, and jet lag, as well as other physiological and metabolic processes under circadian regulation.
Circadian Control of Global Gene Expression
In addition to the core circadian clock mechanism, a number of "clock-controlled" genes are driven as output pathways by the circadian system. To begin to identify such output pathways systematically, we have used high-density oligonucleotide arrays to discover cycling genes in the mouse. This analysis revealed approximately 650 cycling transcripts in the suprachiasmatic nucleus (SCN), which contains the master circadian pacemaker in mammals, and in the liver, which represents a major target tissue for circadian regulation. In each tissue examined, approximately 10 percent of all expressed transcripts were under circadian control. In comparing the two tissues, however, the majority of cycling transcripts were unique to each tissue. Subsequent work has borne out these results: about 8–10 percent of all expressed genes in any tissue are under circadian regulation, and only about 10 percent of cycling genes in any pair of tissues are the same. To determine whether these cycling transcripts are under control of the Clock gene, we analyzed Clock mutant mice and sequences of candidate promoter regions of cycling genes. These analyses suggested that a relatively small number of output genes are directly regulated by the core circadian mechanism.
In the SCN, genes involved in protein biosynthesis and trafficking, including ribosomal synthesis, translation initiation, folding, targeting, post-translational modification, and transport, are under coordinated circadian control. In addition, genes involved in energy metabolism, the redox state of the cell, and cell signaling also show circadian variation in their steady-state message levels. In the liver, basic cellular pathways, such as glycolysis, fatty acid metabolism, cholesterol biosynthesis, and xenobiotic and intermediate metabolism, are under circadian regulation. In addition, synthesis of many plasma proteins involved in immune function and coagulation is under circadian control. Rate-limiting steps in these various pathways are key sites of circadian control, highlighting the fundamental role that circadian clocks play in cellular and organismal physiology. These global views of circadian gene expression reveal the fundamental role that circadian clocks play at the cellular level and suggest that they are deeply embedded in the metabolism of living systems.
Chimera Analysis of the Clock Mutation in Mice
Although substantial progress has been made in our understanding of the molecular mechanism of circadian rhythms, the regulation of circadian behavior is not yet understood in cellular or molecular terms. As described above, the Clock mutation lengthens the periodicity and reduces the amplitude of circadian rhythms in mice. The cell-intrinsic effects of Clock can be observed at the level of single neurons in the suprachiasmatic nucleus. To address how cells of contrasting genotype interact in vivo to control circadian behavior, we have analyzed a series of Clock mutant mouse aggregation chimeras. Circadian behavior in Clock/Clock <—> wild-type chimeric individuals is determined by the proportion of mutant versus normal cells. A majority of either wild-type or mutant cells is required to dominate behavior in vivo. Significantly, a number of intermediate phenotypes, including Clock/+ phenocopies and novel combinations of the parental behavioral characteristics, were seen in balanced chimeras, revealing a variety of modes of oscillator interaction. Our results demonstrate that cell-cell interaction is crucial for the generation and expression of coherent circadian rhythms at the organismal level.
The last five years have witnessed a remarkable set of discoveries concerning the circadian clock mechanism in mammals. An important goal for our future work is to test the current models of the circadian mechanism and to determine whether the proposed circadian pathways are valid in the intact organism. It is also important to define how environmental inputs converge upon the core circadian mechanism and to discover how this mechanism regulates its various outputs.
