Biomedical subjects

Steven M Reppert

Publications and source records attributed to Steven M Reppert.

11 recordsLinked to original sources

PubMed2004

A rhythmic Ror.

The circadian clock mechanism in mammals involves two interlocking transcriptional feedback loops. Rev-erb alpha, through its role as a transcriptional repressor, was thought to be the primary determinant of the feedback loop that regulates Bmal1 transcription. Results reported by Sato et al. in this issue of Neuron now show that the transactivator Rora acts coordinately with Rev-erb alpha and that their competing activities on the same promoter element drive the rhythm in Bmal1 transcription. This finding defines the second feedback loop in mammals.

Animals↗

PubMed2004

Polarized light helps monarch butterflies navigate.

During their spectacular migratory journey in the fall, North American monarch butterflies (Danaus plexippus) use a time-compensated sun compass to help them navigate to their overwintering sites in central Mexico. One feature of the sun compass mechanism not fully explored in monarchs is the sunlight-dependent parameters used to navigate. We now provide data suggesting that the angle of polarized skylight (the e-vector) is a relevant orientation parameter. By placing butterflies in a flight simulator outdoors and using a linear polarizing filter, we show that manipulating the e-vector alters predictably the direction of oriented flight. Butterflies studied in either the morning or afternoon showed similar responses to filter rotation. Monarch butterflies possess the anatomical structure needed for polarized skylight detection, as rhabdoms in the dorsalmost row of photoreceptor cells in monarch eye show the organization characteristic of polarized-light receptors. The existence of polarized-light detection could allow migrants to accurately navigate under a variety of atmospheric conditions and reveals a critical input pathway into the sun compass mechanism.

Animal Migration↗

PubMed2004

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.

Animals↗

PubMed2003

Constructing a feedback loop with circadian clock molecules from the silkmoth, Antheraea pernyi.

Circadian clocks are important regulators of behavior and physiology. The circadian clock of Drosophila depends on an autoinhibitory feedback loop involving dCLOCK, CYCLE (also called dBMAL, for Drosophila brain and muscle ARNT-like protein), dPERIOD, and dTIMELESS. Recent studies suggest that the clock mechanism in other insect species may differ strikingly from that of Drosophila. We cloned Clock, Bmal, and Timeless homologs (apClock, apBmal, and apTimeless) from the silkmoth Antheraea pernyi, from which a Period homolog (apPeriod) has already been cloned. In Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL activates transcription through an E-box enhancer element found in the 5' region of the apPeriod gene. Furthermore, apPERIOD can robustly inhibit apCLOCK: apBMAL-mediated transactivation, and apTIMELESS can augment this inhibition. Thus, a complete feedback loop, resembling that found in Drosophila, can be constructed from silkmoth CLOCK, BMAL, PERIOD, and TIMELESS. Our results suggest that the circadian autoinhibitory feedback loop discovered in Drosophila is likely to be widespread among insects. However, whereas the transactivation domain in Drosophila lies in the C terminus of dCLOCK, in A. pernyi, it lies in the C terminus of apBMAL, which is highly conserved with the C termini of BMALs in other insects (except Drosophila) and in vertebrates. Our analysis sheds light on the molecular function and evolution of clock genes in the animal kingdom.

ARNTL Transcription Factors↗

PubMed2003

Illuminating the circadian clock in monarch butterfly migration.

Migratory monarch butterflies use a time-compensated Sun compass to navigate to their overwintering grounds in Mexico. Here, we report that constant light, which disrupts circadian clock function at both the behavioral and molecular levels in monarchs, also disrupts the time-compensated component of flight navigation. We further show that ultraviolet light is important for flight navigation but is not required for photic entrainment of circadian rhythms. Tracing these distinct light-input pathways into the brain should aid our understanding of the clock-compass mechanisms necessary for successful migration.

Animal Migration↗

PubMed2003

A novel C-terminal domain of drosophila PERIOD inhibits dCLOCK:CYCLE-mediated transcription.

The essence of the Drosophila circadian clock involves an autoregulatory feedback loop in which PERIOD (PER) and TIMELESS (TIM) inhibit their own transcription by association with the transcriptional activators dCLOCK (dCLK) and CYCLE (CYC). Because PER, dCLK, and CYC each contain a PAS domain, it has been assumed that these interaction domains are important for negative feedback. However, a critical role for PAS-PAS interactions in Drosophila clock function has not been shown. Nuclear transport of PER is also believed to be an essential regulatory step for negative feedback, but this has not been directly tested, and the relevant nuclear localization sequence (NLS) has not been functionally mapped. We evaluated these critical aspects of PER-mediated transcriptional inhibition in Drosophila Schneider 2 (S2) cells. We mapped the dCLK:CYC inhibition domain (CCID) of PER and discovered that it lies in the C terminus, downstream of the PAS domain. Using deletion mutants and site-directed mutagenesis, we identified a novel NLS in the CCID of PER that is a potent regulator of PER's nuclear transport in S2 cells. We further found that nuclear transport, primarily through this novel NLS, is essential for the inhibitory activity of PER. The data indicate that nuclear PER inhibits dCLK:CYC-mediated transcription through a novel domain that additionally contains a potent NLS.

ARNTL Transcription Factors↗

PubMed2003

Targeted disruption of the mouse Mel(1b) melatonin receptor.

Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel(1a) melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel(1b) melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel(1b) receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel(1b) receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel(1a) receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel(1a) receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel(1a)-Mel(1b) double-mutant mice, indicating that the Mel(1b) receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

Amino Acid Sequence↗

PubMed2002

Rhythmic histone acetylation underlies transcription in the mammalian circadian clock.

In the mouse circadian clock, a transcriptional feedback loop is at the centre of the clockwork mechanism. Clock and Bmal1 are essential transcription factors that drive the expression of three period genes (Per1-3) and two cryptochrome genes (Cry1 and Cry2). The Cry proteins feedback to inhibit Clock/Bmal1-mediated transcription by a mechanism that does not alter Clock/Bmal1 binding to DNA. Here we show that transcriptional regulation of the core clock mechanism in mouse liver is accompanied by rhythms in H3 histone acetylation, and that H3 acetylation is a potential target of the inhibitory action of Cry. The promoter regions of the Per1, Per2 and Cry1 genes exhibit circadian rhythms in H3 acetylation and RNA polymerase II binding that are synchronous with the corresponding steady-state messenger RNA rhythms. The histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner. Moreover, the Cry proteins inhibit a p300-induced increase in Clock/Bmal1-mediated transcription. The delayed timing of the Cry1 mRNA rhythm, relative to the Per rhythms, is due to the coordinated activities of Rev-Erbalpha and Clock/Bmal1, and defines a new mechanism for circadian phase control.

Acetylation↗

PubMed2002

Coordination of circadian timing in mammals.

Time in the biological sense is measured by cycles that range from milliseconds to years. Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous and well-studied timing systems. At the core of this timing mechanism is an intricate molecular mechanism that ticks away in many different tissues throughout the body. However, these independent rhythms are tamed by a master clock in the brain, which coordinates tissue-specific rhythms according to light input it receives from the outside world.

Active Transport, Cell Nucleus↗

PubMed2002

Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK/BMAL1 activity.

Circadian rhythmicity in mammals is under the control of a molecular pacemaker constituted of clock gene products organized in transcriptional autoregulatory loops. Phase resetting of the clock in response to light involves dynamic changes in the expression of several clock genes. The molecular pathways used by light to influence pacemaker-driven oscillation of clock genes remain poorly understood. We explored the functional integration of both light- and clock-responsive transcriptional regulation at the promoter level of the Period (Per) genes. Three Per genes exist in the mouse. Whereas mPer1 and mPer2 are light-inducible in clock neurons of the hypothalamic suprachiasmatic nucleus, mPer3 is not. We have studied the promoter structure of the three mPer genes and compared their regulation. All three mPer promoters contain E-boxes and respond to the CLOCK/brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) heterodimer. On the other hand, only mPer1 and mPer2 promoters contain bona fide cAMP-responsive elements (CREs) that bind CRE-binding protein (CREB) from suprachiasmatic nucleus protein extracts. The mPer1 promoter is responsive to synergistic activation of the cAMP and mitogen-activated protein kinase pathways, a physiological response that requires integrity of the CRE. In contrast, activation of mPer promoters by CLOCK/BMAL1 occurs regardless of an intact CRE. Altogether, these results constitute strong evidence that CREB acts as a pivotal endpoint of signaling pathways for the regulation of mPer genes. Our results reveal that signaling-dependent activation of mPer genes is distinct from the CLOCK/BMAL1-driven transcription required within the clock feedback loop.

ARNTL Transcription Factors↗

PubMed2002

Redox potential: differential roles in dCRY and mCRY1 functions.

Cryptochromes (CRYs) are flavoproteins important for the molecular clocks of animals. The Drosophila cryptochrome (dCRY) is a circadian photoreceptor, whereas mouse cryptochromes (mCRY1 and mCRY2) are essential negative elements of circadian clock transcriptional feedback loops. It has been proposed that reduction/oxidation (redox) reactions are important for dCRY light responsiveness and mCRY1 transcriptional inhibition. We therefore evaluated the role of redox in light-dependent activation of dCRY and in mCRY1 transcriptional inhibition in Drosophila Schneider 2 cells. Using site-directed mutagenesis, three of the four conserved flavin binding residues in dCRY were found to be essential for light responses, whereas three of the four corresponding residues in mCRY1 did not abolish transcriptional responses. Two tryptophan residues in dCRY are critical for its function and are likely involved in an intramolecular redox reaction. The corresponding tryptophan residues do not play a redox-mediated role in mCRY1 function. The data provide a multistep redox model for the light-dependent activities of dCRY and suggest that such a model does not apply to mCRY1 transcriptional responses.

Animals↗