Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock

The role of mPer1 and mPer2 in regulating circadian rhythms was assessed by disrupting these genes. Mice homozygous for the targeted allele of either mPer1 or mPer2 had severely disrupted locomotor activity rhythms during extended exposure to constant darkness. Clock gene RNA rhythms were blunted in the suprachiasmatic nucleus of mPer2 mutant mice, but not of mPER1-deficient mice. Peak mPER and mCRY1 protein levels were reduced in both lines. Behavioral rhythms of mPer1/mPer3 and mPer2/mPer3 double-mutant mice resembled rhythms of mice with disruption of mPer1 or mPer2 alone, respectively, confirming the placement of mPer3 outside the core circadian clockwork. In contrast, mPer1/mPer2 double-mutant mice were immediately arrhythmic. Thus, mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.     

Direct association between mouse PERIOD and CKIepsilon is critical for a functioning circadian clock

The mPER1 and mPER2 proteins have important roles in the circadian clock mechanism, whereas mPER3 is expendable. Here we examine the posttranslational regulation of mPER3 in vivo in mouse liver and compare it to the other mPER proteins to define the salient features required for clock function. Like mPER1 and mPER2, mPER3 is phosphorylated, changes cellular location, and interacts with other clock proteins in a time-dependent manner. Consistent with behavioral data from mPer2/3 and mPer1/3 double-mutant mice, either mPER1 or mPER2 alone can sustain rhythmic posttranslational events. However, mPER3 is unable to sustain molecular rhythmicity in mPer1/2 double-mutant mice. Indeed, mPER3 is always cytoplasmic and is not phosphorylated in the livers of mPer1-deficient mice, suggesting that mPER3 is regulated by mPER1 at a posttranslational level. In vitro studies with chimeric proteins suggest that the inability of mPER3 to support circadian clock function results in part from lack of direct and stable interaction with casein kinase Iepsilon (CKIepsilon). We thus propose that the CKIepsilon-binding domain is critical not only for mPER phosphorylation but also for a functioning circadian clock.     

mPer2 antisense oligonucleotides inhibit mPER2 expression but not circadian rhythms of physiological activity in cultured suprachiasmatic nucleus neurons

Various day-night rhythms, observed at molecular, cellular, and behavioral levels, are governed by an endogenous circadian clock, predominantly functioning in the hypothalamic suprachiasmatic nucleus (SCN). A class of clock genes, mammalian Period (mPer), is known to be rhythmically expressed in SCN neurons, but the correlation between mPER protein levels and autonomous rhythmic activity in SCN neurons is not well understood. Therefore, we blocked mPer translation using antisense phosphothioate oligonucleotides (ODNs) for mPer1 and mPer2 mRNAs and examined the effects on the circadian rhythm of cytosolic Ca2+ concentration and action potentials in SCN slice cultures. Treatment with mPer2 ODNs (20microM for 3 days) but not randomized control ODNs significantly reduced mPER2 immunoreactivity (-63%) in the SCN. Nevertheless, mPer1/2 ODNs treatment inhibited neither action potential firing rhythms nor cytosolic Ca2+ rhythms. These suggest that circadian rhythms in mPER protein levels are not necessarily coupled to autonomous rhythmic activity in SCN neurons.     

Serine 714 might be implicated in the regulation of the phosphorylation in other areas of mPer1 protein

The phosphorylation of mPer proteins may play important roles in the mechanism of the circadian clock via changes in subcellular localization and degradation. However, the mechanism has remained unclear. Previously, we identified three putative casein kinase (CK)1epsilon phosphorylation motif clusters in mPer1. In this work, we examined the role of the phosphorylation of serine residue, Ser(S)714, in mPer1. mPer1 S[714-726]A mutant, in which potential phosphorylation serine residues replaced by alanine residues, is rapidly phosphorylated compared with wild-type mPer1 by CK1epsilon. Coexpression with S[714]G mutant of mPer1 advanced phase of circadian expression of mPer2-luc expression, which was monitored by in vitro bioluminescence system. This result showed that the mPER1 S[714]G mutation affects circadian core oscillator. Considering these, it seems that Ser 714 might be involved in the regulation of the phosphorylation of other sites in mPer1 by CK1epsilon.     

Light-induced phase shifts in mice lacking mPER1 or mPER2

Three homologs of the Drosophila Period gene have been identified in mammals. In mice, these three genes (mPer1, mPer2, and mPer3) have distinct roles in the circadian clockwork. While products of mPer1 and mPer2 play important roles in the maintenance of circadian rhythmicity, mPer3 gene products are dispensable for rhythmicity. Several studies also implicate mPER1 and mPER2 in transduction of photic information to the core circadian clockwork. The phase-shifting effects of light were examined in mPER1-deficient and mPER2-deficient mice using T cycle paradigms, in which mice received 1 h of light per day at an interval of T hours. To assess phase delays, repeated exposure to 1 h of light per day at T = 24 was used. To assess phase advances, exposure to 1-h light pulses at T = 22-h intervals was used. The degeneration of rhythmicity in the mutant mice prevented assessment of a response in most cases. Nevertheless, clear examples of phase delays and phase advances were observed in both mPer1 and mPer2 mutant mice. These results are not consistent with the hypothesis that mPER1 and mPER2 play necessary and nonoverlapping roles in mediating the effects of light on the circadian dock.