timrit Lengthens circadian period in a temperature-dependent manner through suppression of PERIOD protein cycling and nuclear localization.
A fundamental feature of circadian clocks is temperature compensation of period. The free-running period of ritsu (timrit) (a novel allele of timeless [tim]) mutants is drastically lengthened in a temperature-dependent manner. PER and TIM protein levels become lower in timrit mutants as temperature becomes higher. This mutation reduces per mRNA but not tim mRNA abundance. PER constitutively driven by the rhodopsin1 promoter is lowered in rit mutants, indicating that timrit mainly affects the per feedback loop at a posttranscriptional level. An excess of per+ gene dosage can ameliorate all rit phenotypes, including the weak nuclear localization of PER, suggesting that timrit affects circadian rhythms by reducing PER abundance and its subsequent transportation into nuclei as temperature increases. (+info)
Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism.
Recent discoveries have identified a framework for the core circadian clock mechanism in mammals. Development of this framework has been based entirely on the expression patterns of so-called "clock genes" in the suprachiasmatic nuclei (SCN), the principal clock of mammals. We now provide data concerning the protein expression patterns of two of these genes, mPer1 and mTim. Our studies show that mPER1 and mTIM are nuclear antigens expressed in the SCN and extensively throughout the forebrain. Expression of mPER1 in the SCN was rhythmic under entrained conditions and with clear circadian cycling under free-running conditions. Expression of mPER1 elsewhere in the mouse forebrain was not rhythmic. In contrast to mPER1, mTIM expression in the SCN did not vary with time in mice housed in either a light/dark cycle or in constant dim red light. The phase relationship between mPer1 RNA and mPER1 cycles in the SCN is consistent with a negative feedback model of the mammalian clock. The invariant nature of nuclear mTIM in the SCN suggests that its participation in negative feedback occurs only after mPER1 has entered the nucleus, and that the abundance of mTIM is not regulated by the circadian clock or the light/dark cycle. (+info)
Oscillation and light induction of timeless mRNA in the mammalian circadian clock.
Circadian rhythms in Drosophila melanogaster depend on a molecular feedback loop generated by oscillating products of the period (per) and timeless (tim) genes. In mammals, three per homologs are cyclically expressed in the suprachiasmatic nucleus (SCN), site of the circadian clock, and two of these, mPer1 and mPer2, are induced in response to light. Although this light response distinguishes the mammalian clock from its Drosophila counterpart, overall regulation, including homologous transcriptional activators, appears to be similar. Thus, the basic mechanisms used to generate circadian timing have been conserved. However, contrary to expectations, the recently isolated mammalian tim homolog was reported not to cycle. In this study, we examined mRNA levels of the same tim homolog using a different probe. We observed a significant (approximately threefold) diurnal variation in mTim expression within mouse SCN using two independent methods. Peak levels were evident at the day-to-night transition in light-entrained animals, and the oscillation persisted on the second day in constant conditions. Furthermore, light pulses known to induce phase delays caused significant elevation in mTim mRNA. In contrast, phase-advancing light pulses did not affect mTim levels. The mTim expression profile and the response to nocturnal light are similar to mPer2 and are delayed compared with mPer1. We conclude that temporal ordering of mTim and mPer2 parallels that of their fly homologs. We predict that mTIM may be the preferred functional partner for mPER2 and that expression of mTim and mPer2 may, in fact, be driven by mPER1. (+info)
PER and TIM inhibit the DNA binding activity of a Drosophila CLOCK-CYC/dBMAL1 heterodimer without disrupting formation of the heterodimer: a basis for circadian transcription.
The Drosophila CLOCK (dCLOCK) and CYCLE (CYC) (also referred to as dBMAL1) proteins are members of the basic helix-loop-helix PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of the circadian clock genes period (per) and timeless (tim). Several lines of evidence indicate that PER, TIM, or a PER-TIM heterodimer somehow inhibit the transcriptional activity of a putative dCLOCK-CYC complex, generating a negative-feedback loop that is a core element of the Drosophila circadian oscillator. In this report we show that PER and/or TIM inhibits the binding of a dCLOCK-CYC heterodimer to an E-box-containing DNA fragment that is present in the 5' nontranscribed region of per and acts as a circadian enhancer element. Surprisingly, inhibition of this DNA binding activity by PER, TIM, or both is not accompanied by disruption of the association between dCLOCK and CYC. The results suggest that the interaction of PER, TIM, or both with the dCLOCK-CYC heterodimer induces a conformational change or masks protein regions in the heterodimer, leading to a reduction in DNA binding activity. Together with other findings, our results strongly suggest that daily cycles in the association of PER and TIM with the dCLOCK-CYC complex probably contribute to rhythmic expression of per and tim. (+info)
Light-dependent sequestration of TIMELESS by CRYPTOCHROME.
Most organisms have circadian clocks consisting of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In this study, CRYPTOCHROME (CRY), a protein involved in circadian photoperception in Drosophila, is shown to block the function of PERIOD/TIMELESS (PER/TIM) heterodimeric complexes in a light-dependent fashion. TIM degradation does not occur under these conditions; thus, TIM degradation is uncoupled from abrogation of its function by light. CRY and TIM are part of the same complex and directly interact in yeast in a light-dependent fashion. PER/TIM and CRY influence the subcellular distribution of these protein complexes, which reside primarily in the nucleus after the perception of a light signal. Thus, CRY acts as a circadian photoreceptor by directly interacting with core components of the circadian clock. (+info)
mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.
We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially. (+info)
Requirement of circadian genes for cocaine sensitization in Drosophila.
The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies lacking the gene timeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase. (+info)
Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2.
Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. A previous study showed that mice lacking the Cry2 gene had reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and had an intrinsic period 1 hr longer than normal. In this study, Cry1(-/-) and Cry1(-/-)Cry2(-/-) mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1(-/-) mice had a circadian period 1 hr shorter than wild type and the Cry1(-/-)Cry2(-/-) mice were arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN was blunted in Cry1(-/-) and abolished in Cry1(-/-)Cry2(-/-) mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1(-/-) and Cry1(-/-)Cry2(-/-) animals. Importantly, in double mutants, mPer1 expression was constitutively elevated and no rhythmicity was detected in either 12-hr light/12-hr dark or DD, whereas mPer2 expression appeared rhythmic in 12-hr light/12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals. (+info)