Circadian oscillator
Encyclopedia
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:
  1. A central oscillator with a period of about 24 hours that keeps time
  2. A series of input pathways to this central oscillator to allow entrainment of the clock
  3. 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
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...

, plants, fungi, and bacteria
Bacterial circadian rhythms
Bacterial circadian rhythms, like other circadian rhythms, are endogenous "biological clocks" that have the following three characteristics: in constant conditions Bacterial circadian rhythms, like other circadian rhythms, are endogenous "biological clocks" that have the following three...

.

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 rhythm
Circadian rhythm
A circadian rhythm, popularly referred to as body clock, is an endogenously driven , roughly 24-hour cycle in biochemical, physiological, or behavioural processes. Circadian rhythms have been widely observed in plants, animals, fungi and cyanobacteria...

s in higher eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...

s began with the discovery of the period
Period (gene)
Period is a gene located on the X chromosome of Drosophila melanogaster. Oscillations in levels of both per transcript and its corresponding protein PER have a period of approximately 24 hours and together play a central role in the molecular mechanism of the Drosophila biological clock driving...

(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
Feedback
Feedback describes the situation when output from an event or phenomenon in the past will influence an occurrence or occurrences of the same Feedback describes the situation when output from (or information about the result of) an event or phenomenon in the past will influence an occurrence or...

 of transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...

 and translation
Translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of...

. 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.

Mammalian clocks

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
Feedback
Feedback describes the situation when output from an event or phenomenon in the past will influence an occurrence or occurrences of the same Feedback describes the situation when output from (or information about the result of) an event or phenomenon in the past will influence an occurrence or...

 loops. In the primary feedback loop, members of the basic helix-loop-helix (bHLH)-PAS (Period-Arnt-Single-minded) transcription factor family, CLOCK
CLOCK
Clock is a gene encoding a basic-helix-loop-helix-PAS transcription factor that affects both the persistence and period of circadian rhythms...

 and BMAL1
ARNTL
Aryl hydrocarbon receptor nuclear translocator-like, also known as ARNTL, Bmal1, or Mop3, is a gene which is associated with susceptibility to hypertension and type 2 diabetes.-Function:...

, 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
PER1
Period circadian protein homolog 1 is a protein that in humans is encoded by the PER1 gene....

, PER2
PER2
Period circadian protein homolog 2 is a protein that in humans is encoded by the PER2 gene.PER2 contains glucocorticoid response elements and a GRE within the core clock gene Per2 is continuously occupied during rhythmic expression and essential for glucocorticoid regulation of PER2 in vivo...

, and PER3
PER3
Period circadian protein homolog 3 is a protein that in humans is encoded by the PER3 gene....

) 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
Rev-ErbA
The Rev-ErbA proteins are members of the nuclear receptor family of intracellular transcription factors. There are two forms of the receptor, alpha and beta, each encoded by a separate gene ....

 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.

Other organisms

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.

Post-translational modification

The autoregulatory feedback
Feedback
Feedback describes the situation when output from an event or phenomenon in the past will influence an occurrence or occurrences of the same Feedback describes the situation when output from (or information about the result of) an event or phenomenon in the past will influence an occurrence or...

 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
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....

, sumolyation, histone acetylation and methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...

, 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
CSNK1D
Casein kinase I isoform delta is an enzyme that in humans is encoded by the CSNK1D gene.-Interactions:CSNK1D has been shown to interact with Gap junction protein, alpha 1.-Further reading:...

 and CSNK1E
CSNK1E
Casein kinase I isoform epsilon is an enzyme that in humans is encoded by the CSNK1E gene.-Interactions:CSNK1E has been shown to interact with PER1 and AXIN1.-References:...

) and the F-box and leucine-rich repeat protein 3 (FBXL3
FBXL3
F-box/LRR-repeat protein 3 is a protein that in humans is encoded by the FBXL3 gene.-Further reading:...

). In mammals, Casein kinase 1 epsilon
CSNK1E
Casein kinase I isoform epsilon is an enzyme that in humans is encoded by the CSNK1E gene.-Interactions:CSNK1E has been shown to interact with PER1 and AXIN1.-References:...

 and Casein kinase 1 delta
CSNK1D
Casein kinase I isoform delta is an enzyme that in humans is encoded by the CSNK1D gene.-Interactions:CSNK1D has been shown to interact with Gap junction protein, alpha 1.-Further reading:...

 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

Modern experimental approaches using systems biology
Systems biology
Systems biology is a term used to describe a number of trends in bioscience research, and a movement which draws on those trends. Proponents describe systems biology as a biology-based inter-disciplinary study field that focuses on complex interactions in biological systems, claiming that it uses...

 have identified many novel components in biological clocks that suggest an integrative view on how organisms maintain circadian oscillation.

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
Small interfering RNA
Small interfering RNA , sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference pathway, where it...

 (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
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...

 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
Small interfering RNA
Small interfering RNA , sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference pathway, where it...

 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
Rev-ErbA alpha
Rev-ErbA alpha also known as NR1D1 , is a protein that in humans is encoded by the NR1D1 gene.Rev-erbα is member of the Rev-ErbA family of nuclear receptors and is a transcriptional repressor...

 and Rev-ErbA beta
Rev-ErbA beta
Rev-ErbA beta also known as NR1D2 is a protein that in humans is encoded by the NR1D2 gene.Rev-erbβ is a member of the Rev-ErbA family of transcription factors...

 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
PER1
Period circadian protein homolog 1 is a protein that in humans is encoded by the PER1 gene....

 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
Genomics
Genomics is a discipline in genetics concerning the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis,...

 to explore network features in the circadian oscillator.

Another study conducted by Zhang et al. also employed a genome-wide small interfering RNA
Small interfering RNA
Small interfering RNA , sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference pathway, where it...

 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
Periodic function
In mathematics, a periodic function is a function that repeats its values in regular intervals or periods. The most important examples are the trigonometric functions, which repeat over intervals of length 2π radians. Periodic functions are used throughout science to describe oscillations,...

 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
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....

 and hedgehog signaling pathway
Hedgehog signaling pathway
In a growing embryo, cells develop differently in the head or tail end of the embryo, the left or right, and other positions. They also form segments which develop into different body parts. The hedgehog signaling pathway gives cells information that they need to make the embryo develop properly....

, the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...

, 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 systems biology
Systems biology
Systems biology is a term used to describe a number of trends in bioscience research, and a movement which draws on those trends. Proponents describe systems biology as a biology-based inter-disciplinary study field that focuses on complex interactions in biological systems, claiming that it uses...

 approach may relate circadian rhythms to cellular phenomena that were not originally considered regulators of circadian oscillation.

External links

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