- Circadian oscillator
Circadian oscillators are components of the biological clocks that regulate the activities of organisms in relation to environmental cycles and provide an internal temporal framework. All circadian clocks, regardless of phylogenetic origin, consist of three major components:
- A central oscillator with a period of about 24 hours that keeps time
- A series of input pathways to this central oscillator to allow entrainment of the clock
- A series of output pathways tied to distinct phases of the oscillator that regulate overt rhythms in biochemistry, physiology, and behavior throughout the organism
The clock is reset as the environment changes through an organism's ability to sense external time cues of which the primary one is light. Circadian oscillators are ubiquitous in tissues of the body where they are synchronized by both endogenous and external signals to regulate transcriptional activity throughout the day in a tissue-specific manner. The basic molecular mechanisms of the biological clock have been defined in vertebrate species, Drosophila melanogaster, plants, fungi, and bacteria.
Recent studies employing genomic approaches have further elucidated understanding of the circadian oscillator mechanism by providing a large-scale view into an organism's network and genetic architecture.
Transcriptional and translational control
Evidence for a genetic basis of circadian rhythms in higher eukaryotes began with the discovery of the period (per) locus in Drosophila melanogaster from forward genetic screens completed by Ronald Konopka and Semour Benzer in 1971. Through the analysis of per circadian mutants and additional mutations on Drosophila clock genes, it was demonstrated that there is an underlying generative molecular mechanism of the circadian clock that consists of a set of core clock genes and their protein products, which together participate in positive and negative autoregulatory feedback loops of transcription and translation. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Similar mechanisms have been demonstrated in mammals and other organisms.
Several mammalian clock genes have been identified and characterized through experiments on animals harboring naturally occurring, chemically induced, and targeted knockout mutations, and various comparative genomic approaches. The majority of identified clock components are transcriptional activators or repressors that modulate protein stability and nuclear translocation, and create two interlocking feedback loops. In the primary feedback loop, members of the basic helix-loop-helix (bHLH)-PAS (Period-Arnt-Single-minded) transcription factor family, CLOCK and BMAL1, heterodimerize in the cytoplasm to form a complex that, following translocation to the nucleus, initiates transcription of target genes such as the core clock genes Period genes (PER1, PER2, and PER3) and two Cryptochrome genes (CRY1 and CRY2). Negative feedback is achieved by PER:CRY heterodimers that translocate back to the nucleus to repress their own transcription by inhibiting the activity of the CLOCK:BMAL1 complexes. Another regulatory loop is induced when CLOCK:BMAL1 heterodimers activate the transcription of Rev-ErbA and Rora, two retinoic acid-related orphan nuclear receptors. REV-ERBa and RORa subsequently compete to bind retinoic acid-related orphan receptor response elements (ROREs) present in Bmal1 promoter. Through the subsequent binding of ROREs, members of ROR and REV-ERB are able to regulate Bmal1. While RORs activate transcription of Bmal1, REV-ERBs repress the same transcription process. Hence, the circadian oscillation of Bmal1 is both positively and negatively regulated by RORs and REV-ERBs.
In D. melanogaster, the gene cycle (CYC) is the orthologue of BMAL1 in mammals. Thus, CLOCK–CYC dimers activate the transcription of circadian genes. The gene timeless (TIM) is the orthologue for mammalian CRYs as the inhibitor; D. melanogaster CRY functions as a photoreceptor instead. In flies, CLK–CYC binds to the promoters of circadian-regulated genes only at the time of transcription. A stabilizing loop also exists where the gene vrille (VRI) inhibits whereas PAR-domain protein-1 (PDP1) activates Clock transcription. In N. crassa, the clock mechanism is analogous, but non-orthologous, to that of mammals and flies.
The autoregulatory feedback loops in clocks take about 24 hour to complete a cycle and constitute a circadian molecular clock. This generation of the ~24-hour molecular clock is governed by post-translational modifications such as phosphorylation, sumolyation, histone acetylation and methylation, and ubiquitination. Reversible phosphorylation regulates important processes such as nuclear entry, formation of protein complexes and protein degradation. Each of these processes significantly contributes to the delay that keeps the period at ~24 hours and lends the precision of a circadian clock by affecting the stability of aforementioned core clock proteins. Thus, while transcriptional regulation generates rhythmic RNA levels, regulated posttranslational modifications control protein abundance, subcellular localization, and repressor activity of PER and CRY.
Proteins responsible for post-translational modification of clock genes include casein kinase I family members (CSNK1D and CSNK1E) and the F-box and leucine-rich repeat protein 3 (FBXL3). In mammals, Casein kinase 1 epsilon and Casein kinase 1 delta are critical factors that regulate the core circadian protein turnover. Experimental manipulation on either of these proteins results in dramatic effects on circadian periods, such as altered kinase activities and cause shorter circadian periods, and further demonstrates the importance of the post-translational regulation within the core mechanism of the circadian clock. These mutations have become of particular interest in humans as they are implicated in familial advanced sleep phase syndrome. A small ubiquitin-related modifier protein modification of BMAL1 has also been proposed as another level of post-translational regulation.
Systems biology approaches to elucidate oscillating mechanisms
Recently, Baggs et al. developed a novel strategy termed "Gene Dosage Network Analysis" (GDNA) to describe network features in the human circadian clock that contribute to an organism's robustness against genetic perturbations. In their study, the authors used small interfering RNA (siRNA) to induce dose-dependent changes in gene expression of clock components within immortalized human osteosarcoma U2OS cells in order to build gene association networks consistent with known biochemical constraints in the mammalian circadian clock. Employing multiple doses of siRNA powered their quantitative RT-PCR analysis to uncover several network features of the circadian clock, including proportional responses of gene expression, signal propagation through interacting modules, and compensation through gene expression changes.
Proportional responses in downstream gene expression following siRNA-induced perturbation revealed levels of expression that were actively altered with respect to the gene being knocked down. For example, when Bmal1 was knocked down in a dose-dependent manner, Rev-ErbA alpha and Rev-ErbA beta mRNA levels were shown to decrease in a linear, proportional manner. This supported previous findings that Bma1 directly activates Rev-erb genes and further suggests Bma1 as a strong contributor to Rev-erb expression.
In addition, the GDNA method provided a framework to study biological relay mechanisms in circadian networks through which modules communicate changes in gene expression. The authors observed signal propagation through interactions between activators and repressors, and uncovered unidirectional paralog compensation among several clock gene repressors—for example, when PER1 is depleted, there is an increase in Rev-erbs, which in turns propagates a signal to decrease expression in BMAL1, the target of the Rev-erb repressors.
By examining knockdown of several transcriptional repressors, GDNA also revealed paralog compensation where gene paralogs were upregulated through an active mechanism by which gene function is replaced following knockdown in a nonredunant manner—that is, one component is sufficient to sustain function. These results further suggested that a clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. In essence, the authors proposed that the observed network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation. Following this logic, we may use genomics to explore network features in the circadian oscillator.
Another study conducted by Zhang et al. also employed a genome-wide small interfering RNA screen in U2OS cell line to identify additional clock genes and modifiers using luciferase reporter gene expression. Knockdown of nearly 1000 genes reduced rhythm amplitude. The authors found and confirmed hundreds of potent effects on period length or increased amplitude in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling pathway, the cell cycle, and folate metabolism. Coupled with data demonstrating that many of these pathways are clock-regulated, Zhang et al. postulated that the clock is interconnected with many aspects of cellular function.
- ^ a b Ueda, H.R., et al. 2005. System-level identification of transcriptional circuits underlying mammalian circadian clocks. Nature Genet. 37: 187–192.
- ^ Harmer, S.L., et al. 2001. Molecular bases of circadian rhythms. Annu. Rev. Cell Dev. Biol. 17: 215–53
- ^ a b Lowrey, P.L. and J.S. Takahashi. 2004. Mammalian Circadian Biology: Elucidating Genome-Wide Levels of Temporal Organization. Annu. Rev. Genomics Hum. Genet. 5: 407–441.
- ^ a b c Zhang, E.E., et al. 2009. A Genome-wide RNAi Screen for Modifiers of the Circadian Clock in Human Cells. Cell 139: 199–210
- ^ a b c d e Baggs J.E., et al. 2009. Network Features of the Mammalian Circadian Clock. PLoS Biol 7(3): e1000052. doi:10.1371/journal.pbio.1000052
- ^ Konopka R.J., Benzer S. 1971, Clock mutants of Drosophila melanogaster. Proc. Natl. Acad. Sci. 68: 2112–2116
- ^ Bagiello T.A., Jackson, F.R., Young M.W. 1984. Restoration of circadian behavioural rhythms by gene transfer in Drosophila. Nature 312: 752–754.
- ^ Shearman L.P., Sriram S., Weaver D.R., Maywood E.S., Chaves I., et al. 2000. Interacting molecular loops in the mammalian circadian clock. Science 288: 1013–19
- ^ a b c d e f Ko, C. H.; Takahashi, J. S. (2006). "Molecular components of the mammalian circadian clock". Human Molecular Genetics 15: R271–R277. doi:10.1093/hmg/ddl207. PMID 16987893.
- ^ a b c d Gallego, M., and Virshup, D.M. 2007. Post-translational modifications regulate the ticking of the circadian clock. Nature Reviews. 8: 139–148
- ^ Brunner, M. and Schafmeier, T. 2006. Transcriptional and posttranscriptional regulation of the circadian clocks of cyanhobacteria and Neurospora. Genes Dev. 20: 723–733
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