The Circadian Clocks of Mice and Men

Major strides have been made toward understanding the molecular mechanism of rodent clocks. But how far do those steps carry us toward understanding circadian clock function in humans? Practical advances in human genetics, not the least of which is the availability of a virtually complete genome sequence, now allow animal research to be complemented by genetic analysis of human families with sleep abnormalities. In a recent study led by Ptacek and colleagues, the first fruits of this approach have been harvested (Toh et al., 2001xToh, K.L, Jones, C.R, He, Y, Eide, E.J, Hinz, W.A, Virshup, D.M, Ptacek, L.J, and Fu, Y.-H. Science. 2001; 291: 1040–1043Crossref | PubMed | Scopus (824)See all ReferencesToh et al., 2001 and references therein).In 1999, familial advanced sleep-phase syndrome (FASPS) was documented in three families (Jones et al., 1999xJones, C.R, Campbell, S.S, Zone, S.E, Cooper, F, DeSano, A, Murphy, P.J, Jones, B, Czajkowski, L, and Ptacek, L.J. Nat. Med. 1999; 5: 1062–1065Crossref | PubMed | Scopus (335)See all References(Jones et al., 1999). Affected individuals experienced early evening sleepiness (around 7:30 p.m.) and early morning awakening (around 4:30 a.m.). Studying volunteers in a sleep lab revealed that individuals with FASPS had a circadian period about an hour shorter than normal. Thus, a sleep disorder in humans was associated with a genetic mutation affecting circadian clock function. Moreover, the Mendelian inheritance of the syndrome implied a single gene mutation within each family.Taking one of the FASPS families, Ptacek and coworkers used multiple sets of dense genomic markers to map the mutation (Toh et al., 2001xToh, K.L, Jones, C.R, He, Y, Eide, E.J, Hinz, W.A, Virshup, D.M, Ptacek, L.J, and Fu, Y.-H. Science. 2001; 291: 1040–1043Crossref | PubMed | Scopus (824)See all References(Toh et al., 2001). Remarkably, the mutant gene turned out to be hPer2, the human homolog of mPer2! Most research in rodents carries the assumption that the findings will approximate human biology, but it is enormously satisfying to see the proof. The hPer2 mutation changes serine 662 to a glycine (S662G), and this occurs in a region of hPER2 homologous to the casein kinase I epsilon (CKIϵ) binding region of mPER1 and mPER2 (Toh et al., 2001xToh, K.L, Jones, C.R, He, Y, Eide, E.J, Hinz, W.A, Virshup, D.M, Ptacek, L.J, and Fu, Y.-H. Science. 2001; 291: 1040–1043Crossref | PubMed | Scopus (824)See all References(Toh et al., 2001). Serine 662 is in fact part of a consensus CKIϵ phosphorylation site, and the S662G substitution renders the mutant protein less readily phosphorylated by CKIϵ than the wild-type hPER2 in vitro. Similar results were obtained for mPER2 and a mutant mPER2 equivalent to the human FASPS mutation.The phosphorylation of PER2 by CKIϵ appears important for circadian period in mammals. mPer2 is an essential clock component in mice (Zheng et al., 1999xZheng, B, Larkin, D.W, Albrecht, U, Sun, Z.S, Sage, M, Eichele, G, Lee, C.C, and Bradley, A. Nature. 1999; 400: 169–173Crossref | PubMed | Scopus (19)See all References(Zheng et al., 1999), and CKIϵ is an important clock component in Syrian hamsters, since a defect in the hamster CKIϵ gene corresponds to the short-period Tau mutation (Lowrey et al., 2000xLowrey, P.L, Shimomura, K, Antoch, M.P, Yamazaki, S, Zemenides, P.D, Ralph, M.R, Menaker, M, and Takahashi, J.S. Science. 2000; 288: 483–491Crossref | PubMed | Scopus (530)See all References(Lowrey et al., 2000). Together with cell culture experiments showing binding and phosphorylation of mPER1 and mPER2 by CKIϵ and research in Drosophila showing the importance of the CKIϵ homolog DOUBLE-TIME for destabilizing dPER, the genetic data in both rodents and humans strongly suggest that CKIϵ-dependent phosphorylation of PER proteins is an essential and ancient part of the circadian clock mechanism (references in Toh et al., 2001xToh, K.L, Jones, C.R, He, Y, Eide, E.J, Hinz, W.A, Virshup, D.M, Ptacek, L.J, and Fu, Y.-H. Science. 2001; 291: 1040–1043Crossref | PubMed | Scopus (824)See all ReferencesToh et al., 2001). Transcriptional feedback is not enough; posttranslational modifications are crucial for conveying time delays necessary for circadian clock function. It is unclear how phosphorylation contributes to these time delays in mammals, or how the mutation in hPER2 results in a short circadian period. In cell culture, CKIϵ-dependent phosphorylation of mammalian PER proteins affects their turnover rate and their nuclear translocation (references in Toh et al., 2001xToh, K.L, Jones, C.R, He, Y, Eide, E.J, Hinz, W.A, Virshup, D.M, Ptacek, L.J, and Fu, Y.-H. Science. 2001; 291: 1040–1043Crossref | PubMed | Scopus (824)See all ReferencesToh et al., 2001), but whether this is true in vivo, or whether phosphorylation affects some other activities of PER proteins, remains to be determined.Other families with FASPS do not possess mutations in the coding region of hPer2, raising the exciting possibility that genetic analysis of these families will identify other alleles of hPer2 and/or disease-linked alterations in additional genes. Perhaps identification of additional mutations will again affirm the homology among the clocks of humans, mice, and men. In addition to previously described clock gene homologs, new clock gene candidates have been unearthed recently by “data-mining” the human genome using sequence homology searches (Clayton et al., 2001xClayton, J.D, Kyriacou, C.P, and Reppert, S.M. Nature. 2001; 409: 829–831Crossref | PubMed | Scopus (67)See all References(Clayton et al., 2001). Genetic analysis of FASPS could verify whether these or previously discovered genes are important for human circadian rhythms. A genetic approach may also lead to the discovery of novel genes, providing new insights into mammalian clock mechanisms. New vistas opened by human genetic tools can then be more finely explored using laboratory animals, such as transgenic or “knockin” mice made to recapitulate the human mutations.Circadian clock research in mammals is now entering the “post-genomic” era. And it seems certain that circadian biologists, poised with microarrays in hand, will continue to have the time of their lives for some time to come.