Published online: September 24, 2018

News & Views

Lost in clocks: non-canonical circadianoscillation discovered in Drosophila cells
Koji L Ode1 & Hiroki R Ueda1,2,3

In most organisms, cell-autonomous circadian clocks are driven by a transcription–translation negative feedback loop (TTFL).
Per was the first identified clock gene in
the fruit fly and is a core component of
the circadian TTFL. Surprisingly, in theirrecent study, Rey et al (2018) demonstrate
the presence of apparent circadian rhythmicity in a fly cell line that does not
express several core clock genes including
per (Rey et al, 2018). Quantitative multiomics measurements allowed the identification of unknown oscillating components
and revealed hundreds of transcripts,
proteins, and metabolites showing 24-hrhythmicity, suggesting that at least in
the fly, the circadian clock may be driven
by non-canonical (i.e., independent of perdriven TTFL) molecular mechanisms.
Mol Syst Biol. (2018) 14: e8567

See also: G Rey et al (August 2018)T

he fruit fly Drosophila melanogasterhas been one of the most importantmodel organisms for the analysis andunderstanding of circadian clocks. The identification of per as the gene responsible forbehavioral circadian rhythmicity led to thecanonical molecular model of circadianoscillator, where PER protein inhibits its
own transcription through the interaction
with other transcription regulators such as
TIM and CLK (Fig 1). This TTFL is further
controlled by post-translational regulationsuch as PER phosphorylation catalyzed by
the kinase DBT (Doubletime), a homolog of
mammalian Casein Kinase I (CKI). The overall structure of TTFL appears to beconserved in terms of the regulatory relationships between core circadian factors

among other organisms, and the TTFL
model has been considered as a canonicalcircadian oscillator.
However, several mechanisms driving
circadian clocks are independent of theTTFL. For example, the circadian clock incyanobacteria, driven by KaiA, KaiB, andKaiC, is an established example of a TTFLindependent mechanism. When KaiC is
incubated with KaiB, KaiC, and ATP in vitro,the phosphorylation status of KaiC oscillateswith a period of 24 h, indicating that a posttranslational mechanism that does not
involve transcription/translation is the corecircadian oscillator in this prokaryotic organism (Nakajima et al, 2005). The non-TTFL
model was extended to eukaryotic cells byfindings of circadian oscillations in theoxidation status of peroxiredoxin in enucleated mammalian red blood cells as well as
nucleated cells (O’Neill & Reddy, 2011).
Nonetheless, the extent to which the circadian clocks of non-cyanobacteria organisms
rely on canonical TTFL mechanisms, or onother non-TTFL mechanisms, is still underdiscussion, and the implications for cellularand organism-level circadian physiology
remain unclear.
The study by Reddy and colleagues
provides evidence indicating that circadianoscillations not driven by the canonical
TTFL can be observed for hundreds of transcripts, proteins, and metabolites (Rey et al,2018). This was demonstrated using Drosophila S2 cells, which do not express per, tim,and other core components of the canonicalcircadian TTFL. Up to now, circadian oscillations had not been detected using the classic
circadian reporters that rely on the canonical
TTFL model (e.g., the expression dynamics
of per). To identify molecules showingcircadian rhythmicity, Rey et al used quantitative omics approaches. They collected
time-course samples of S2 cells for 57 h at
3 h intervals under carefully controlled
conditions and used these samples toperform RNA-seq, mass spectrometry-based
proteomics and metabolomics analyses.
Genes and proteins involved in metabolic
processes were found to be enriched among
the rhythmic components. In agreementwith this finding, the abundance of several
metabolites showed circadian rhythmicity.
Interestingly, the rhythmic metabolites
include ATP and NADP, which often regulate enzymatic activities as a substrate orcofactor. Such metabolic rhythmicity may
not simply be the readout of protein oscillations—it is also possible that the metabolic
oscillations can in turn regulate protein
activities. Considering that the concentrationof ATP changes ~2-fold between peak andtrough in their analysis, one can readily
imagine that this non-canonical circadianoscillation may impact many aspects ofcellular physiology as well.
Rey et al might have discovered a newcircadian clock that has remained unknown
so far due to the focus on the canonicalmechanisms regulating the circadian clock.
Does a de novo autonomous oscillator exist
behind the observed rhythmicity in theabsence of a canonical circadian clock? If
the non-canonical oscillator has intrinsic
24 h periodicity, then S2 cells will be preferentially synchronized close to the 24 htemperature cycle, as used in this study, but
not well synchronized by a cyclic signal atdifferent frequencies. This can be tested infuture studies. In addition, if the noncanonical circadian rhythmicity is driven byan actual “circadian” oscillator, the period

1 Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. E-mail: uedah-tky@umin.ac.jp
2 Laboratory for Synthetic Biology, Center for Biosystems Dynamics Research, RIKEN, Osaka, Japan
3 International Research Center for Neurointelligence (WPI-IRCN), UTIAS, The University of Tokyo, Tokyo, Japan
DOI 10.15252/msb.20188567ª 2018 The Authors. Published under the terms of the CC BY 4.0 license

Molecular Systems Biology 14: e8567 | 2018

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Published online: September 24, 2018

Molecular Systems Biology

Lost in clocks

Canonical circadian clock model

TIM

Koji L Ode & Hiroki R Ueda

Drosophila S2 cell line

TIM

PER

PER

PER

PER
TRANSLATION

TRANSLATION

Feedbacktranscriptional
repression
TimeTime
TRANSCRIPTION

TRANSCRIPTION

per gene

CYC

per gene

CLKCYC

CLK

Output genes

Transcriptome
profileTranscriptome
profileTimeTime

Proteome
profileProteome
profileNon canonicalcircadian oscillator

Metabolome
profile© EMBOMetabolome
profile?

Figure 1. Circadian rhythmicity observed in the absence of canonical clock gene expression.
CYC and CLK form a heterodimer that binds to a promoter element and activates the expression of downstream genes. per gene expression is activated by CYC/CLK. PER
protein then binds to TIM and represses the activity of CYC/CLK. The overall mechanism creates a negative feedback loop. This negative feedback loop is considered as thecore circadian oscillator in Drosophila, generating circadian oscillation in many transcripts, proteins, and metabolites. Drosophila S2 cells do not express CLK, PER, and TIM;
thus, the core feedback mechanism does not operate in this cell line. Surprisingly, Rey et al (2018) found hundreds of transcripts, proteins, and metabolites that show
apparent circadian rhythmicity under constant culture conditions, suggesting that this rhythmicity is generated through non-canonical circadian mechanisms.

of oscillation is expected to be around 24 hregardless of the period of the temperature
cycle used for the synchronization. In orderto perform rigorous analyses of the oscillator’s properties, it is important to find arobust reporter that represents the non-canonical circadian rhythmicity. The multi-omics
dataset presented in this study is a useful
resource for finding such a reporter, which
will then allow genetic and chemical screens
to discover components regulating 24-h

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Molecular Systems Biology 14: e8567 | 2018

period length. This strategy has been previously employed to find core components
involved in the canonical TTFL.
A potential candidate regulator could be
the kinase DBT, since DBT and its mammalian homolog CKI are not only critical for
period determination of the canonical TTFLbased clock but also control the peroxiredoxin cycle period (O’Neill et al, 2011).
Notably, a recent study demonstrated that
this kinase also controls the period of (non-circadian) metabolic oscillations in yeast(Causton et al, 2015). Future analyses in thisdirection may uncover the components
driving this newly reported circadian rhythmicity and resolve whether the oscillation
is founded on a TTFL, post-translational
mechanisms, metabolic networks, or theircombination. Another important feature
characterizing the circadian oscillation istemperature compensation. Circadian clocks
do not conform to the usual biochemicalª 2018 The Authors

Published online: September 24, 2018

Koji L Ode & Hiroki R Ueda

principle that would mean that high temperatures shorten the period of the clock andlow temperatures extend it. Instead, whentemperature is kept constantly low or high,period remains invariant. Accordingly, Rey
et al examined whether the period of S2 cellrhythmicity is affected by changing the incubation temperature. Although their results
supported that period is not affected bytemperature, the fact that S2 cells do nottolerate temperatures higher than 28°C
limits the range of temperature change that
could be tested experimentally. Further evaluation of temperature compensation over awider range of physiological temperatures
would need to be undertaken in futurework.
Although the study by Rey et al focusedon a particular Drosophila cell line, it isreasonable to speculate that the non-canonical circadian oscillator operates in other
Drosophila cells, in which the per-driven
canonical circadian oscillator exists. It
would be interesting to examine whether thenon-canonical rhythmicity can be observedin cells in which the “canonical” clock genes
have been knocked out. It is also possiblethat similar non-canonical circadian rhythmicity could exist in other organisms.
Interestingly, multi-omics datasets frommammals share some features found in themulti-omics profiles of the Drosophila cellswith non-canonical circadian rhythmicity.
For example, a comparison between RNAseq and proteomics profiles from mouseª 2018 The Authors

Molecular Systems Biology

Lost in clocks

liver highlighted the uncoupling of rhythmictranscripts and rhythmic proteins and thelarge time delay between the peak phase oftranscripts and corresponding proteins(Mauvoisin et al, 2014; Robles et al, 2014).
According to the canonical TTFL-basedmodel, such complex behavior is generated
through the downstream modification of
canonical circadian oscillator output, but it
is possible that non-canonical circadianmechanisms are also responsible forcomplex rhythmic behavior.
Identification of the underlying molecularmechanisms and evaluation of its physiological significance are important future directions that will provide more conclusive
support for the presence of a non-canonicaland de novo circadian clock responsible forthe apparent circadian rhythmicity observedin the study by Rey et al. If cells areequipped with multiple coupled circadianoscillators, this will change the way wethink of the overall organization of circadianrhythmic output within the cell.

-independent rhythmic proteomes implement
distinct diurnal functions in mouse liver.
Proc Natl Acad Sci USA 111:
167 – 172
Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama
Y, Iwasaki H, Oyama T, Kondo T (2005)
Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylation in vitro.
Science 308: 414 – 415
O’Neill JS, Reddy AB (2011) Circadian clocks inhuman red blood cells. Nature 469: 498 – 503
O’Neill JS, van Ooijen G, Dixon LE, Troein C,Corellou F, Bouget FY, Reddy AB, Millar AJ(2011) Circadian rhythms persist withouttranscription in a eukaryote. Nature 469:
554 – 558
Rey G, Milev NB, Valekunja UK, Ch R, Ray S, Silva
Dos Santos M, Nagy AD, Antrobus R, MacRae JI,Reddy AB (2018) Metabolic oscillations on thecircadian time scale in Drosophila cells lacking
clock genes. Mol Syst Biol 14: e8376
Robles MS, Cox J, Mann M (2014) In-vivo
quantitative proteomics reveals a keycontribution of post-transcriptional
mechanisms to the circadian regulation

Referencesof liver metabolism. PLoS Genet 10:
e1004047

Causton HC, Feeney KA, Ziegler CA, O’Neill JS(2015) Metabolic cycles in yeast share featuresconserved among circadian rhythms. Curr Biol

License: This is an open access article under the25: 1056 – 1062

terms of the Creative Commons Attribution 4.0

Mauvoisin D, Wang J, Jouffe C, Martin E, Atger F,

License, which permits use, distribution and repro-Waridel P, Quadroni M, Gachon F, Naef F

duction in any medium, provided the original work

(2014) Circadian clock-dependent andis properly cited.

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