F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019

REVIEW

A period without PER: understanding 24-hour rhythms withoutclassic transcription and translation feedback loops [version 1;
peer review: 2 approved]
Arthur Millius

1,2, Koji L. Ode3, Hiroki R. Ueda1,3

1Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan
2Laboratory of Systems Immunology and Laboratory of Host Defense, Immunology Frontier Research Center, Osaka University, Suita, Osaka,

565-0871, Japan
3Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033,
Japan

published: 16 Apr 2019, 8(F1000 Faculty Rev):499 (v1 First
https://doi.org/10.12688/f1000research.18158.1)
Latest published: 16 Apr 2019, 8(F1000 Faculty Rev):499 (https://doi.org/10.12688/f1000research.18158.1)

Abstract
Since Ronald Konopka and Seymour Benzer’s discovery of the gene Period in
the 1970s, the circadian rhythm field has diligently investigated regulatory
mechanisms and intracellular transcriptional and translation feedback loops
involving Period, and these investigations culminated in a 2017 Nobel Prize inPhysiology or Medicine for Michael W. Young, Michael Rosbash, and Jeffrey C.
Hall. Although research on 24-hour behavior rhythms started with Period, aseries of discoveries in the past decade have shown us that post-transcriptional
regulation and protein modification, such as phosphorylation and oxidation, arealternatives ways to building a ticking clock.
Keywords
circadian rhythms, post-transcriptional oscillator, transcriptional-translation
feedback loop, red blood cells, dopaminergic ultradian oscillator, peroxiredoxin,
phosphorylation

Open Peer Review
Referee Status:
Invited Referees

12version 1
published
16 Apr 2019

F1000 Faculty Reviews are commissioned
from members of the prestigious F1000
Faculty. In order to make these reviews ascomprehensive and accessible as possible,
peer review takes place before publication; thereferees are listed below, but their reports are
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1 John O'Neill, MRC Laboratory of
Molecular Biology, UK
2 Carrie Partch, University of California
Santa Cruz, USA
Any comments on the article can be found at
the end of the article.

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F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019

Corresponding author: Hiroki R. Ueda (uedah-tky@umin.ac.jp)
Author roles: Millius A: Writing – Original Draft Preparation, Writing – Review & Editing; Ode KL: Writing – Original Draft Preparation; Ueda HR:
Conceptualization, Supervision, Writing – Review & Editing
Competing interests: No competing interests were disclosed.
Grant information: This work was supported by a grant from the Japan Agency for Medical Research and Development (AMED)-Core Research
for Evolution Science and Technology (CREST) (JP17gm0610006; AMED/Ministry of Education, Culture, Sports, Science and Technology[MEXT]; to HRU), CREST (Japan Science and Technology Agency/MEXT; to HRU), Brain Mapping by Integrated Neurotechnologies for Disease
Studies (Brain/MINDS) (AMED/MEXT; to HRU), Basic Science and Platform Technology Program for Innovative Biological Medicine(AMED/MEXT; to HRU), a Grant-in-Aid for Scientific Research (Japan Society for the Promotion of Science 18H05270 to HRU), a Grant-in-Aid for
Scientific Research on Innovative Areas (23115006 to HRU), the strategic programs for research and development (President’s Discretionary
Fund) of RIKEN (to HRU), an intramural Grant-in-Aid from the RIKEN Quantitative Biology Center (to HRU), a Grant-in-Aid for Young Scientists(18K14683 to KLO), the RIKEN Special Postdoctoral Research Program (to AM), and a Grant-in-Aid for Early-Career Scientists (18K14755 to AM).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright: © 2019 Millius A et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite this article: Millius A, Ode KL and Ueda HR. A period without PER: understanding 24-hour rhythms without classictranscription and translation feedback loops [version 1; peer review: 2 approved] F1000Research 2019, 8(F1000 Faculty Rev):499 (https://doi.org/10.12688/f1000research.18158.1)
First published: 16 Apr 2019, 8(F1000 Faculty Rev):499 (https://doi.org/10.12688/f1000research.18158.1)

Page 2 of 10

F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019

Introduction
The time-keeping mechanisms of circadian rhythms can be
regulated by multiple layers of different cellular networks,
including transcription-translation feedback loops (TTFLs) andpost-translation oscillators (PTOs)1. Circadian TTFLs generate oscillations in gene expression through delayed negative
feedback whereby expression of a transcription factor negatively regulates its own transcription2. The core of this genetic
network in mammals is the expression of a heterodimer of
BMAL1 (also called ARNTL) with either CLOCK or NPAS2,which binds at promoter cis-elements called E-boxes to drive
expression of genes encoding period (PER1-3), cryptochrome(CRY1-2), and nuclear receptor subfamily (NR1D1-2) proteins,which then repress Bmal1 expression by a series of separate
and interconnected feedback loops3,4. In contrast to behaviors
driven by cyclic differences in gene expression, PTOs generate
rhythms independent of transcription and translation through biochemical processes, such as phosphorylation, protein–protein
interactions, and other post-translational modifications. These
post-translational processes also alter TTFLs as well as posttranscriptional modification of transcripts involved in TTFLs5,6.
The most well-known PTO is the cyanobacteria KaiABC system, which consists of only three proteins and ATP7, but novel
PTOs may also exist in red blood cells (RBCs)8–14, which lack anucleus and the molecular machinery to drive TTFL rhythms.
In addition, a series of new and old observations of 24-hourrhythms in biological contexts where classic TTFLs are absent
or diminished (Figure 1)15–23 continue to puzzle researchers anddemonstrate that there are multiple ways to build a clock.
One of the first modern uses of the term “circadian” was todescribe 24-hour endogenous oscillators that alter Drosophila
fly behavior rhythms24, and the persistence of oscillations
at various temperatures was viewed as a defining feature ofcircadian rhythms25,26. The first genetic component of circadianrhythms was discovered in the 1970s when Ronald Konopka

in Seymour Benzer’s lab used chemical mutagenesis of
Drosophila to discover three alleles of the Period gene27. In the
1980s, rhythmicity of Period mutants was shown to be restored
by gene transfer28,29, confirming that Period both is necessary
and can restore rhythmic behaviors, such as eclosion andlocomotor activity, in flies. In 1990, Hardin et al. proposed that
PER protein altered the levels of Period mRNA in a negative
feedback loop30, but at the time it was unclear whether PER
directly suppressed Period transcription or whether the negative
feedback occurred through an indirect route. A few years later,
researchers discovered that this negative feedback was direct in
the bread mold Neurospora crassa model of circadian rhythms
because the frequency (FRQ) directly repressed its own
transcription31. In addition to Neurospora32 and Drosophila33,
TTFL models of circadian rhythms from plants34 to mammals35
have been elucidated and reviewed extensively.

Post-translational oscillators and post-translational
modifications: breaking the transcription-translation
feedback loop mold
The modern idea that TTFLs were necessary for 24-hourrhythms was shattered in 2005 when Nakajima et al. reconstituted
rhythmic 24-hour oscillations in protein phosphorylation withjust a small number of cyanobacterial proteins36. This seminal
moment in the circadian rhythm field spurred investigators to
examine other non-canonical rhythm-generating mechanisms andto unearth forgotten studies of PTOs. For example, in the 1960s,
it was shown that the unicellular alga Acetabularia undergoes
diurnal rhythms of photosynthesis, which persist even after the
nucleus has been artificially removed21.
There are a few more recent examples of organisms that havecircadian rhythms in the absence of TTFLs. In the unicellular
red alga Cyanidioschyzon merolae, circadian rhythms control
cell cycle progression in the absence of RNA translation22, andthe unicellular dinoflagellate Lingulodinium has daily rhythms

Figure 1. Post-translation oscillators without transcription-translation feedback loops. Examples of post-translation oscillators
in enucleated cells such as Acetabularia and red blood cells, in unicellular alga lacking RNA rhythms, and in mice in which the classictranscription-translation feedback loop module is disrupted genetically or anatomically. KO, knockout; SCN, suprachiasmatic nucleus.
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F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019in bioluminescence and photosynthesis without a detectablechange in RNA transcript abundance and in the presence oftranscription inhibitors23. These studies suggest that protein
activities and post-translational modifications can serve as
24-hour oscillators. Research has centered on phosphorylation
as the period-determining post-translational modification37–40,
but other post-translational modifications, including methylation, acetylation, sumoylation, and ubiquitination, also alter
clock function41–44.
Importantly, circadian rhythms are insensitive to temperatureand this property of temperature compensation was identified
in biological time-keeping systems, such as those of bees, flies,
and marine organisms, as early as the 1950s and 1960s25,26,45,46.
Transcription and translation are temperature-dependent
reactions47–50, which suggests that post-translational activities
are important for temperature compensation. For example,
Isojima et al. revealed that phosphorylation by casein kinase I(CKI) is a temperature-insensitive period-determining process,and the degradation rate of PER2, which is regulated by CKI
phosphorylation, was found to be insensitive to temperature38.
Importantly, the phosphorylation of PER2-derived peptide by
CKI is insensitive to temperature in vitro. In 2015, the degradation of PER2 was found to occur in a more complex mode
composed of three distinct stages, and the duration of the second
stage depended on circadian time, which led to the identification of temperature-sensitive and -insensitive PER2 phosphorylation sites51. Thus, differences in the temperature sensitivity ofphosphorylation sites on the repressor, which alter degradation
rates at different temperatures, are responsible for temperaturecompensation, PER2 stability, and ultimately the length of thecircadian period. In 2017, Shinohara et al. identified a shortsequence region around residue K224 in CKI, which was
responsible for temperature compensation and converted a temperature-sensitive kinase into a temperature-insensitive one
in vitro52. Mutation of K224 shortens circadian behavioral
rhythms and alters the temperature dependency of the circadianclock in the sub-hypothalamic region of the brain52, called
the suprachiasmatic nucleus (SCN), which controls circadian
response to light. It is though noteworthy that K224 is part of theconsensus KRQK monopartite nuclear localization signal in CKI,
which makes it difficult to disentangle the effects of temperature dependence from that of localization in vivo. These studies
provide evidence for how post-translational activities modify
TTFL rhythms, but a series of new and old studies haverevealed that PTOs can drive rhythms even in the absence of
TTFL clocks.

Blood: a novel source of post-translational oscillator
rhythms
Mammals have a natural supply of enucleated cells in RBCs,
and researchers have plumbed this cell type for non-TTFL
rhythms. In the 1970s, circadian rhythms in ATPase activity andperiodic rhythms in enzymes—such as acetylcholinesterase,
glyceraldehyde-3-phosphate dehydrogenase, and glucose-6phosphate dehydrogenase—in RBCs were found (Table 1), but

it was unclear whether the rhythms were robust or persistentbeyond 24 hours10.
In 2011, an anti-oxidant enzyme called peroxiredoxin (PRX)
in cultured human RBCs was found to have temperatureindependent circadian cycles of hyperoxidation for up to
76 hours11. Because RBCs lack a nucleus and the rhythms
persisted in the presence of transcription and translation
inhibitors, a novel non-transcriptional-based circadian oscillator in mammals was proposed. Analysis of the PRX rhythms
relied solely on PRX1, PRX2, and PRX-SO2/3 (hyperoxidized
PRX form) antibodies. In particular, the PRX-SO2/3 antibody
recognizes multiple hyperoxidized forms of PRX53, results in
up to eight different bands on non-reducing sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)11, andproduces multiple non-specific bands that can confound interpretation of the hyperoxidized signal54,55, which make determination of the correct PRX isoform technically difficult and in-gel
controls essential. Nevertheless, the same researchers discoveredthat hyperoxidized PRX-SO2/3 rhythms were conserved in a wide
range of species55,56.
In mice, blocking hemoglobin oxygen transport by incubation with carbon monoxide eliminates PRX2 hyperoxidized
rhythms57. Hemoglobin auto-oxidation in RBCs generates
superoxide, which is converted to H2O2 by superoxide dismutase
1 (SOD1)58,59, and H2O2 is subsequently reduced by catalase,
glutathione peroxidase, and PRXs54,58,60, which results in theoxidation of these proteins61. Oxidation of PRX2 is reversed bysulfiredoxin (SRX)62–64, but rhythms in PRX2 oxidation in mice
are not mediated by the rhythmic reduction of hyperoxidized
PRX2 by SRX but rather through rhythmic degradation by
20S proteasomes, and only about 1% of the total PRX pool is
modified in a circadian manner57. Mitochondria-specific PRX(PRX3) is also reversibly inactivated by hyperoxidation, reduced,
and reactivated by SRX, and hyperoxidized PRX3 and SRX
undergo anti-phasic circadian oscillations in the mitochondria
in various mice tissues, which links mitochondria function
to circadian rhythms65. Another group revealed about three peaks
in hyperoxidized PRX-SO2/3 rhythms in mice over a 48-hour
period (instead of two as would be expected for a circadianrhythm) and showed that rhythms were impaired in SOD1mutant mice66. There is still uncertainty regarding the origin
of PRX hyperoxidation rhythms, but the data suggest that
PRX-SO2/3 oscillations are more an output of rhythm-generatingmachinery involving the 20S proteasome rather than a dailyoxidation-reduction cycle.
Deconstructing PRX rhythms biochemically and using nonantibody methods, such as mass spectrometry, to directly
detect the hyperoxidized cysteine residue or redox-sensitive
fluorescent proteins may bolster understanding of this novel
PTO. However, biochemical reconstruction is difficult because
RBC lysis causes gradual loss of PRX-SO2/3 signal over a 48-hour
period9. However, these types of approaches have revealed thatpotassium-containing media enhances PRX2-SO2/3 rhythms9,

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F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019

Table 1. Oscillatory phenomena observed in human red blood cells.
Molecule

Year

PeriodImpactReference

Glucose-6-phosphate
dehydrogenase1975 ~12 hours Observed two peaks in enzyme activity over a 24-hour period in threedifferent individuals

13

Glutamate oxaloacetatetransaminase

1975 ~12 hours Observed two peaks in enzyme activity over a 24-hour period in two
different individuals

13

Acid phosphatase1975 ~24 hours Observed one peak in enzyme activity over a 24-hour period in one
individual in plasma-free human red blood cell suspensions

13

Acetylcholinesterase

1975 ~24 hours Observed one peak in enzyme activity over a 24-hour period in two
individuals

13

Glucose-6-phosphate
dehydrogenase

1976 ~12 hours Observed two peaks in activity over a 24-hour period with one patternpeaking at 4 p.m. and midnight and the other peaking at midnight and8 p.m. in six and five individuals, respectively

146-phophogluconate
dehydrogenase

1976 ~12 hours Observed two peaks in activity over a 24-hour period peaking at
4 a.m. and 4 p.m. in 11 individuals

14

Lactic dehydrogenase

1976 ~12 hours Observed two peaks in activity over a 24-hour period with one patternpeaking at noon and midnight and the other peaking at 4 a.m. and4 p.m. in four and seven individuals, respectively

14

Aspirate aminotransferase

1976 ~12 hours Observed two peaks in activity over a 24-hour period peaking at 4 a.m.
and 4 p.m. in 11 individuals

14

Hexokinase

1976 ~24 hours Observed one peak in activity over a 24-hour period peaking at 4 p.m.
in 11 individuals

14

Potassium efflux

1976 NS

Membrane potential

1976 ~24 hours Observed two peaks in membrane potential by DiOC5(3) over a 48-hour
period in an unknown number of individuals (averaged data reported)

12

Mg-dependent ATPase1976 ~24 hours Observed one peak in activity from human blood bank bags incubatedat 37 °C for 27 hours (average of eight samples)

8

Acetylcholinesterase

1978 NS

10

Peroxiredoxin

2011 ~24 hours Observed three peaks of peroxiredoxin dimer oxidation and PRX-SO2/3
abundance over a 60-hour period in three individuals

11

NADH

2011 ~24 hours Observed three peaks in NADH abundance over a 60-hour period inthree individuals

11

NADPH

2011 ~24 hours Observed three peaks in NADPH abundance over a 60-hour period inthree individuals

11

Membrane potential

2017 ~24 hours Observed two peaks in membrane potential by dielectrophoresis,
DiOC5(3), and mass spectrometry over a 48-hour period in fourbiological replicates

9

Membrane conductance andcytoplasm conductivity

2017 ~24 hours Observed two or three peaks in membrane conductance and cytoplasm 9
conductivity over a 48-hour period in four individuals

Intracellular potassium

2017 ~24 hours Observed two peaks in intracellular potassium concentrations over a
48-hour period in four biological replicates

Observed a steady increase in potassium efflux over a 48-hour period in 12
an unknown number of individuals (averaged data reported)

Observed variations in acetylcholinesterase activity over a 24-hour
period in four individuals, but variations had lower amplitude thanreference13 and were not circadian9NS, not significant.

and chemical perturbation with Conoidin A, a PRX2 inhibitor67,
shortens PER2:LUCIFERASE rhythms in immortalized mouse
fibroblasts68.
The exact mechanism of rhythmic PRX oxidation is still unclear,but researchers have begun to examine other general rhythmic
behaviors in RBCs. Although no one has followed up on thecircadian, ultradian, and irregular rhythms of various enzyme
activities in the 1970s, in 2011 researchers reported circadianchanges in NADH and NADPH levels11, and in 2017 researchers reproduced circadian changes in RBC membrane potential9
observed in an article published in 197612. Paradoxically, theresearchers reported circadian changes in potassium concentration in 2017, whereas no rhythms in potassium were observedin 1976; instead, a gradual and steady increase in potassium
efflux occurred over the 48-hour observation period. Whether
these differences arise from measuring slightly different
potassium populations (intracellular versus extracellular), a small
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F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019

sample size, technical differences in methods and individuals, oran actual biological phenomenon remains to be determined.

Post-translation control of circadian period intranscription-translation feedback loop modelorganisms
There are shared design principles between the perioddetermination processes of PTO-based and TTFL-based
oscillators. A PTO generates rhythmic changes in proteinstates without changing the amount of protein itself. On theother hand, rhythmic protein synthesis and degradation are essential for TTFL-based oscillations, and mechanisms that control
protein abundance are critical for controlling the circadian period.
This idea is widely accepted for circadian TTFL oscillators
because there is a significant correlation between the half-lifeof transcription repressor mutants, such as in Drosophila PER
and Neurospora FRQ, and circadian period69,70. The correlationsuggests that faster degradation of circadian repressors accelerates clock speed. In mammalian circadian clocks, F-box
proteins recruit E3 ubiquitin ligase complexes that license
PER and CRY degradation, which modulates period length71.
Although the circadian TTFL-based oscillators involve posttranslational regulation as period-determination mechanisms,
modification of transcription repressors regulates period length
by changing repressor stability. For example, a mutation
in CKIε that destabilizes mammalian PER results in period
shortening72,73, and mutation of a phosphorylation site on PERthat destabilizes PER also results in period shortening74,75.
Other kinases, such as AMPK and DNA-PK, control period
length by altering CRY stability through phosphorylation76,77.
In addition, stabilization of CRY by small molecules lengthens
the period78, and destabilization of CRY by degron tagging of
CRY shortens the period79, strongly suggesting the causal
relationship between CRY stability and period length.
However, a recent study of the Neurospora circadian clock
challenged this protein stability–period length paradigm of
period determination in a TTFL-based oscillator80. Researchers
used an FWD-1–deleted strain, which is an F-box protein thatcauses proteolysis of phosphorylated FRQ. The Δfrd-1 strain
results in a markedly increased FRQ half-life, and new FRQ is
produced even in the presence of hyperphosphorylated FRQ.
Nonetheless, circadian oscillation of FRQ-promoter activities
persists with modest change in period length, and several
short-period mutations of FRQ still have a short period in aΔfrd-1 background in which the stability of FRQ is significantly
increased. Because mutation of phosphorylation sites in FRQ
still alters the period and because generic inhibition of kinase
activity lengthens the period even in the absence of FWD-1,
these data suggest that a protein-state not a protein-abundance
attribute, namely phosphorylation, controls period length.
A similar uncoupling of protein stability and circadian period
may occur even in the TTFL clock in mammals. A recent study of
CRY1 mutations in phosphorylation sites by Ode et al. revealed
that multiple phosphorylation sites near the co-factor binding
pocket of CRY1 markedly changes period length while having
only a modest effect on CRY1 half-life79. Mutagenesis of CRY1

and CRY2 revealed mutations in a secondary co-factor binding
pocket which shorten the period without reducing CRY1
stability81. Furthermore, an exon-skipping mutation in CRY1
found from a human family with delayed sleep phase syndrome lengthens the period without affecting CRY1 stability82.
Therefore, mammalian CRY may also control the circadianperiod independently of its abundance.
If protein abundance control does not explain all aspects ofperiod determination, what is the nature of state control of
TTFL-based oscillator proteins such as multisite phosphorylation of FRQ, PER, and CRY? One of the shared properties of
period-determining repressor proteins is structural flexibility.
Most FRQ and PER regions modified by multisite phosphorylation are intrinsically disordered, highly flexible, and variable83,84.
The multisite phosphorylation region of CRY1 critical for
period control also occurs on a flexible loop region. Theseflexible regions may undergo a relatively large conformation
change that may underlie slow dynamics (that is, 24 hours) ofprotein activity change. The intrinsically disordered C-terminal
domain of BMAL1 controls the period through a slow conformation change with a high energy barrier85. Conformation
changes may lead to a slow and coherent re-organization of
the macromolecular repressor complex86, which is consistent
with the dynamics of the cyanobacteria PTO87,88 in which theslow dynamics of the intrinsic conformational change of KaiC89
couple to the re-organization of the KaiABC complex90. An
atomic-scale understanding of the repressor complex in a
TTFL-based oscillator may reveal subtle differences in molecular mechanisms of 24-hour period determination between PTOand TTFL-based oscillators.

Oscillations without classic transcription-translation
feedback loop oscillators
Several classic models of circadian rhythms have persistent
24-hour rhythms even when the circadian TTFL machinery isabsent or disrupted. In S2 cells, which are generally regarded
as non-rhythmic, a multi-omics approach recently revealed
hundreds of genes, proteins, and metabolites with 24-hourrhythms20. Although this approach seems to suggest thepresence of a novel non-canonical oscillator with 24-hour
periodicity, it does not preclude possible cell cycle effects
from the roughly 24-hour doubling time of S2 cells or the
possibility of classic circadian clock components operating
below the experimental limits of detection. For example, largescale proteomics studies of circadian variation frequently failto detect circadian proteins91,92 because there may be only a few
hundred to a thousand protein copies per cell93. Thus, genetic
knockout (KO) of canonical clock genes is needed to definitively
determine whether rhythms derive from a novel oscillator.
In mammals, genetic and anatomical ablation of the circadian machinery normally disrupts 24-hour behavioral rhythms,
but rhythms persist under specialized situations. For example,
SCN-lesioned rats administered methamphetamine in the drinking water retain circadian behaviors of activity in constantlight conditions17. This so-called methamphetamine-sensitive
oscillator also does not depend on classic circadian genes,
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F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019such as Per1-2, Cry1-2, Bmal1, Npas2, and Clock18,94. Recent
data suggest that the methamphetamine-sensitive oscillator isa long-period manifestation of a tunable dopamine ultradianoscillator95,96. KO of a dopamine transporter in SCN-lesioned
or Bmal1 KO mice, which prevents dopamine reuptake indopaminergic neurons, increases the period of the ultradianrhythms. Similarly, administration of methamphetamine, which
increases extracellular dopamine concentrations, lengthens
ultradian rhythms in a dose-dependent manner from 4 hours
to an astonishing 48 hours. In contrast, the anti-psychotic
drug haloperidol, which selectively blocks the dopamine D2
receptor, shortens long-period rhythms induced by methamphetamine in wild-type and Bmal1 KO mice95. These datasuggest that dopamine neurons are a second independent
rhythm-generating mechanism in the brain, and future studies
using chemical and genetic approaches to perturb dopamine
pathways coupled with recently developed brain-clearing
techniques97–100 may enable a more complete understanding of
the neural architecture of this dopamine ultradian oscillator.

Conclusions
From blood to brain, these studies suggest that non-canonical
PTOs have an impact on circadian rhythms beyond the classic
PER negative feedback loop. However, recent studies of PER
itself, including temperature-sensitive phosphorylation sites51,
three prime untranslated region (3′-UTR) regulation101, and theseparation of Period2 rhythms from Bmal1 rhythms in the
SCN102, indicate that even a gene as well studied as Period canstill teach us new tricks about the period-determining mechanismsof circadian rhythms.

Abbreviations
AMPK, AMP-activated protein kinase; BMAL1, brain andmuscle Arnt-like protein 1; CKI, casein kinase I; CLOCK,

circadian locomotor output cycles kaput; CRY1-2, cryptochrome1-2; FRQ, frequency; FWD-1, F-box/WD-40-repeatcontaining protein 1; KO, knockout; NPAS2, neuronal PAS
domain-containing protein 2; NR1D1-2; nuclear receptor
subfamily 1 group D member 1-2; PER; period; PRX, peroxiredoxin; PTO, post-translational oscillator; RBC, red blood
cell; S2, Schneider 2; SCN, suprachiasmatic nucleus; SOD1,
superoxide dismutase 1; SRX, sulfiredoxin; TTFL, transcriptiontranslation feedback loop.

Grant information
This work was supported by a grant from the Japan Agency
for Medical Research and Development (AMED)-Core Research
for Evolution Science and Technology (CREST) (JP17gm0610006;
AMED/Ministry of Education, Culture, Sports, Science andTechnology [MEXT]; to HRU), CREST (Japan Science andTechnology Agency/MEXT; to HRU), Brain Mapping byIntegrated Neurotechnologies for Disease Studies (Brain/
MINDS) (AMED/MEXT; to HRU), Basic Science and Platform
Technology Program for Innovative Biological Medicine(AMED/MEXT; to HRU), a Grant-in-Aid for Scientific Research(Japan Society for the Promotion of Science 18H05270 to HRU),
a Grant-in-Aid for Scientific Research on Innovative Areas(23115006 to HRU), the strategic programs for research anddevelopment (President’s Discretionary Fund) of RIKEN (toHRU), an intramural Grant-in-Aid from the RIKEN Quantitative
Biology Center (to HRU), a Grant-in-Aid for Young Scientists(18K14683 to KLO), the RIKEN Special Postdoctoral Research
Program (to AM), and a Grant-in-Aid for Early-Career Scientists(18K14755 to AM).
The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

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F1000Research 2019, 8(F1000 Faculty Rev):499 Last updated: 16 APR 2019

Open Peer Review
Current Referee Status:
Editorial Note on the Review Process
F1000 Faculty Reviews are commissioned from members of the prestigious F1000 Faculty and are edited as aservice to readers. In order to make these reviews as comprehensive and accessible as possible, the referees
provide input before publication and only the final, revised version is published. The referees who approved thefinal version are listed with their names and affiliations but without their reports on earlier versions (any comments
will already have been addressed in the published version).

The referees who approved this article are:Version 1
Carrie Partch Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz,
CA, USA
Competing Interests: No competing interests were disclosed.
2 John O'Neill MRC Laboratory of Molecular Biology, Cambridge, UK
Competing Interests: No competing interests were disclosed.
1

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