Cis -Combination of the Classic per S and per L Mutations Results in Arrhythmic Drosophila with Ectopic Accumulation of Hyperphosphorylated PERIOD Protein

Abstract

The 1st circadian “clock” gene identified was the X-linked period (per) gene in Drosophila melanogaster. In the pioneering initial report, Konopka and Benzer (1971) characterized 3 alleles of per that shortened (per ^S; ~19 h), lengthened (per ^L; ~29 h), or abolished (per ⁰) circadian behavioral rhythms. They also showed that transheterozygotes carrying the per ^S and per ^L mutations exhibit robust behavioral rhythms with nearly normal periods of ~23 h, highlighting the semidominant nature of many clock mutants. In this study, per ⁰ flies bearing a doubly mutated per transgene that carries both the per ^S and per ^L alleles (per ⁰; per ^S/L) were analyzed for behavioral and molecular rhythms. Unlike singly mutated versions, the per ⁰;per ^S/L transgenic flies are arrhythmic in constant dark conditions and exhibit little, if any, entrainment to daily light—dark cycles. In a wildtype per ⁺ background, expression of per ^S/L abolishes behavioral rhythms, indicating that it functions in a transdominant negative fashion. Biochemical analysis of head extracts revealed that only hyperphosphorylated isoforms of the PERS/L protein are detected throughout a daily cycle, and the levels remain constant. Intriguingly, little if any PERS/L is observed in key pacemaker neurons that control daily activity rhythms, consistent with the notion that hyperphosphorylated isoforms of PER are unstable. Nonetheless, PERS/L is detected in ectopic cells in the brain, in which it exhibits an unusual localization, mainly staining the periphery of the nucleus. These results suggest that posttranslational mechanisms play a key role in limiting the accumulation of PER to specific cells. On a broader scope, our results indicate that the semidominant effects of period-altering alleles observed in trans are not necessarily preserved in the cis-configuration and that novel phenotypes can emerge.

Keywords

Drosophila circadian rhythms period protein phosphorylation perS perL

References

Bae K. and Edery I. (2006) Regulating a circadian clock's period, phase and amplitude by phosphorylation: insights from Drosophila . J Biochem ( Tokyo ) 140: 609-617 .

Baylies MK , Bargiello TA , Jackson FR , and Young MW (1987) Changes in abundance or structure of the per gene product can alter periodicity of the Drosophila clock . Nature 326: 390-392 .

Baylies MK , Vosshall LB , Sehgal A. , and Young MW (1992) New short period mutations of the Drosophila clock gene per . Neuron 9: 575-581 .

Chang DC and Reppert SM (2003) A novel C-terminal domain of Drosophila PERIOD inhibits dCLOCK:CYCLE-mediated transcription . Curr Biol 13: 758-762 .

Curtin KD , Huang ZJ , and Rosbash M. (1995) Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock . Neuron 14: 365-372 .

Darlington TK , Wager-Smith K. , Ceriani MF , Staknis D. , Gekakis N. , Steeves TD , Weitz CJ , Takahashi JS , and Kay SA (1998) Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim . Science 280: 1599-1603 .

Edery I. , Rutila JE , and Rosbash M. (1994a) Phase shifting of the circadian clock by induction of the Drosophila period protein . Science 263: 237-240 .

Edery I. , Zwiebel LJ , Dembinska ME , and Rosbash M. (1994b) Temporal phosphorylation of the Drosophila period protein . Proc Natl Acad Sci U S A 91: 2260-2264 .

Gekakis N. , Saez L. , Delahaye-Brown AM , Myers MP , Sehgal A. , Young MW , and Weitz CJ (1995) Isolation of timeless by PER protein interaction: defective interaction between timeless protein and long-period mutant PERL . Science 270: 811-815 .

10.

Hall JC ( 2003) Genetics and molecular biology of rhythms in Drosophila and other insects . Adv Genet 48: 1-280 .

11.

Hamblen-Coyle MJ , Wheeler DA , Rutila JE , Rosbash M. , and Hall JC (1992) Behavior of period-altered circadian rhythm mutants of Drosophila in light:dark cycles (Diptera: Drosophilidae) . J Insect Beh 5: 417-446 .

12.

Hardin PE (2005) The circadian timekeeping system of Drosophila . Curr Biol 15: R714-R722 .

13.

Hardin PE , Hall JC , and Rosbash M. (1990) Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels . Nature 343: 536-540 .

14.

Helfrich-Forster C. (2005) Neurobiology of the fruit fly's circadian clock . Genes Brain Behav 4: 65-76 .

15.

Houl JH , Yu W. , Dudek SM , and Hardin PE (2006) Drosophila CLOCK is constitutively expressed in circadian oscillator and non-oscillator cells . J Biol Rhythms 21: 93-103 .

16.

Huang ZJ , Edery I. , and Rosbash M. (1993) PAS is a dimerization domain common to Drosophila period and several transcription factors . Nature 364: 259-262 .

17.

Kaneko M. and Hall JC (2000) Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections . J Comp Neurol 422: 66-94 .

18.

Kim EY and Edery I. (2006) Balance between DBT/CKIepsilon kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein . Proc Natl Acad Sci U S A 103: 6178-6183 .

19.

Ko HW , Jiang J. , and Edery I. (2002) Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime . Nature 420: 673-678 .

20.

Konopka RJ and Benzer S. (1971) Clock mutants of Drosophila melanogaster . Proc Natl Acad Sci U S A 68: 2112-2116 .

21.

Konopka RJ , Pittendrigh C. , and Orr D. (1989) Reciprocal behaviour associated with altered homeostasis and photosensitivity of Drosophila clock mutants . J Neurogenet 6: 1-10 .

22.

Lee C. , Bae K. , and Edery I. (1998) The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation, and interactions with the PER-TIM complex . Neuron 21: 857-867 .

23.

Lyons LC , Darlington TK , Hao H. , Houl J. , Kay SA , and Hardin PE (2000) Specific sequences outside the E-box are required for proper per expression and behavioral rescue . J Biol Rhythms 15: 472-482 .

24.

Majercak J. , Chen WF , and Edery I. (2004) Splicing of the period gene 3′-terminal intron is regulated by light, circadian clock factors, and phospholipase C . Mol Cell Biol 24: 3359-3372 .

25.

Marrus SB , Zeng H. , and Rosbash M. (1996) Effect of constant light and circadian entrainment of perS flies: evidence for light-mediated delay of the negative feedback loop in Drosophila . Embo J 15: 6877-6886 .

26.

Myers MP , Wager-Smith K. , Rothenfluh-Hilfiker A. , and Young MW (1996) Light-induced degradation of TIMELESS and entrainment of the Drosophila circadian clock . Science 271: 1736-1740 .

27.

Rothenfluh A. , Abodeely M. , and Young MW (2000) Short-period mutations of per affect a double-time-dependent step in the Drosophila circadian clock . Curr Biol 10: 1399-1402 .

28.

Rutila JE , Edery I. , Hall JC , and Rosbash M. (1992) The analysis of new short-period circadian rhythm mutants suggests features of D. melanogaster period gene function . J Neurogenet 8: 101-113 .

29.

Sidote D. , Majercak J. , Parikh V. , and Edery I. (1998) Differential effects of light and heat on the Drosophila circadian clock proteins PER and TIM . Mol Cell Biol 18: 2004-2013.

30.

Tomioka K. , Sakamoto M. , Harui Y. , Matsumoto N. , and Matsumoto A. (1998) Light and temperature cooperate to regulate the circadian locomotor rhythm of wild type and period mutants of Drosophila melanogaster . J Insect Physiol 44: 587-596 .

31.

Vanselow K. , Vanselow JT , Westermark PO , Reischl S. , Maier B. , Korte T. , Herrmann A. , Herzel H. , Schlosser A. , and Kramer A. (2006) Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS) . Genes Dev 20: 2660-2672 .

32.

Wheeler DA , Hamblen-Coyle MJ , Dushay MS , and Hall JC (1993) Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both . J Biol Rhythms 8: 67-94 .

33.

Yildiz O. , Doi M. , Yujnovsky I. , Cardone L. , Berndt A. , Hennig S. , Schulze S. , Urbanke C. , Sassone-Corsi P. , and Wolf E. (2005) Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD . Mol Cell 17: 69-82 .

34.

Yu Q. , Jacquier AC , Citri Y. , Hamblen M. , Hall JC , and Rosbash M. (1987) Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster . Proc Natl Acad Sci U S A 84: 784-788 .

35.

Zeng H. , Qian Z. , Myers MP , and Rosbash M. (1996) A light-entrainment mechanism for the Drosophila circadian clock . Nature 380: 129-135 .

36.

Zerr DM , Hall JC , Rosbash M. , and Siwicki KK (1990) Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila . J Neurosci 10: 2749-2762 .

37.

Zordan M. , Osterwalder N. , Rosato E. , and Costa R. (2001) Extra ocular photic entrainment in Drosophila melanogaster . J Neurogenet 15: 97-116 .