Available online at www.sciencedirect.com

Challenges in synthetically designing mammalian circadianclocksEtsuo A Susaki1,3, Jörg Stelling4,5 and Hiroki R Ueda1,2,6,7
Synthetic biology, in which complex, dynamic biologicalsystems are designed or reconstructed from basic biologicalcomponents, can help elucidate the design principles of such
systems. However, this engineering approach has only been
applied to a few simple biological systems. The circadian clock
is appropriate for this approach, since it is a dynamic systemwith complex transcriptional and post-transcriptional circuitsthat have been comprehensively described. Rational synthesisof the properties of the suprachiasmatic nucleus, the central
clock tissue of the circadian system that controls many
dynamic behaviors, will be important for understanding theneural-circuit systems that control physiological behaviors.
These approaches will provide a deeper understanding of thebiological clock, and of clinical problems associated with it,such as sleep disorders.
Addresses
1
Laboratory for Systems Biology, RIKEN Center for Developmental
Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 6500047, Japan
2
Functional Genomics Unit, RIKEN Center for Developmental Biology,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
3
Research Fellow of the Japan Society for the Promotion of Science, 1-8,
Chiyoda-ku, Tokyo 102-8472, Japan
4
Department of Biosystems Science and Engineering, ETH Zurich,
Mattenstrasse 26, 4058 Basel, Switzerland
5
Swiss Institute of Bioinformatics, ETH Zurich, 8092 Zurich, Switzerland
6
Graduate School of Science, Osaka University, 1-1 Machikaneyama,
Toyonaka, Osaka 560-0043, Japan
7
Department of Mathematics, Graduate School of Science, Kyoto
University, Kyoto 606-8502, Japan
Corresponding author: Ueda, Hiroki R (uedah-tky@umin.ac.jp)

Current Opinion in Biotechnology 2010, 21:556–565
This review comes from a themed issue on
Systems biology
Edited by Vitor Martins dos Santos and Jiri Damborsky
Available online 12th August 20100958-1669/$ – see front matter# 2010 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2010.07.011

The recent demands for integrating large-scale datasets toreach a deeper understanding of living systems has set thestage for the advent of systems and synthetic biology[1,2]. Complex and dynamic biological phenomena,such as the circadian clock, are suitable subjects for these
newly emerging approaches [2], in which developing
an understanding of a system is a four-step process:Current Opinion in Biotechnology 2010, 21:556–565

identification, analysis, control, and design of the system
of interest. The components and their networks sufficientto the function of the system are confirmed in the final
step, design, in which the original system is reconstructedfrom scratch, using rationally synthesized biological components, pathways, and networks. Here, we use themammalian circadian system as an example to discuss
how this synthetic approach can be applied to gain acomprehensive understanding of the systems. In engineering and therapeutic scenarios, this approach can help
manufacture precise and timely interventions of theintracellular or intercellular regulatory mechanisms andrational reprogramming of cell/tissue phenotypes of thecircadian clock, which will be of growing importanceparticularly in medical applications.

Overview of the mammalian circadian clock
and application of synthetic biology
The circadian clock is an evolutionarily conserved molecular biological timing system. Its underlying mechanisms consist of intracellular auto-regulatory feedback
loops in which specific proteins called clock proteins rhythmically activate or repress each other [2,3,4,5,6].
Circadian clocks in multi-cellular organisms are organized
as a hierarchy of circadian oscillators. Peripheral circadianclock cells are widely distributed in a variety of tissues
throughout the body [2,3,4,5,6,7]. One central
clock tissue located in the suprachiasmatic nucleus (SCN)of the anterior hypothalamus regulates the circadianrhythms of these peripheral clock cells in mammals. Morespecifically, the SCN orchestrates these circadianrhythms according to external cues, including light[3,4,6]. The circadian clocks in central and peripheral tissues are intimately involved in the regulation of
metabolic and physiologic processes. Impairment of thecircadian clock is associated with numerous diseases,
including sleep disorders, depression, cancer, and dementia [2,3].
Synthetic approaches to understanding mammalian circadian clocks can be divided into synthesis of a molecular
clock, and synthesis of the central clock. The first involves
the rational design of minimal artificial transcriptional
and post-transcriptional networks, often these are (simplified) replicas of the original structure and function thatis shared by central and peripheral clock cells. The
second application involves a rational design of theproperties of the SCN, the central clock tissue. Thisrequires implementing the signal transduction network,
electrophysiological network, and intercellular circuits of
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Challenges in designing mammalian circadian clocks Susaki, Stelling and Ueda 557

the SCN (e.g., gating, electrophysiological oscillations,and coupling) in non-SCN cells.

Transcriptional networks of mammalian
circadian rhythms

TTA[T/C]GTAA), and night-time (RevErbA/ROR binding element or RRE [A/T]A[A/T]NT[A/G]GGTCA)
elements [2,3]. Many molecules controlling the circadian transcription program via the three CCEs have beenreported, as described in Figure 1.

The circadian system in multi-cellular organisms has welldefined dynamic properties, including: first, endogenous
oscillations with an approximately 24-h period; second,
entrainment to external environmental changes (temperature and light cycles) and third, temperature compensation
over a wide range of temperatures. In mammalianclocks, circadian transcriptional oscillations are governed,
at least in part, by transcriptional programs that rely on
at least three clock-controlled cis-elements (CCEs): morning (E-box/E0 -box, CACGT[G/T]), day-time (D-box,

The E-box-mediated transcriptional program is critical in
the core auto-regulatory loop of the mammalian circadianclock [2,3,5,7]. According to a current clock model,
bHLH-PAS transcription activators such as BMAL1 and
CLOCK form heterodimers that bind to E-box/E0 -box ciselements present in the promoter regions of their target
genes, which include the Per and Cry1 genes. In turn, theCRYs and PERs induced by the BMAL1/CLOCK heterodimers form repressor complexes; these physicallyFigure 1

Overview of the transcriptional network of the mammalian circadian clock. Genes, CCEs, transcriptional/translational expression, activation, andrepression are depicted as ovals, rectangles, gray lines, green lines, and orange lines, respectively. The E-box-mediated transcription program is
directly or indirectly controlled by at least 11 transcription factors. These include four basic helix–loop–helix (bHLH)-PAS transcription activators,
Clock, Npas2, Bmal1 (also known as Arntl or Mop3), and Bmal2; three Period genes, Per1, Per2, and Per3; two Cryptochrome transcription repressors,
Cry1 and Cry2; and two other bHLH transcription factors, Bhlhb2 and Bhlhb3 (also known as Dec1 and Dec2). At least four bZIP-family genes, Dbp,
Hlf, Tef and E4bp4 (also known as Nfil3), and five orphan nuclear hormone receptors, Nr1d1, Nr1d2 (also known as RevErbAa, RevErbAb), Rora, Rorb
and Rorc, control the D-box- and RRE-mediated transcription programs, respectively.
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558 Systems biology

Figure 2

Synthetic clock outputs with a natural wiring mechanism. (a) Ukai-Tadenuma and colleagues developed an artificial in vitro transcriptional cycling
assay system in mouse NIH3T3 cells to determine whether cis-elementary transcriptional regulations via the three main CCEs (the E/E0 -box
(morning), the D-box (day-time), and the RRE (night-time)) are sufficient to create clock outputs. This system was composed of an artificial activator(destabilized GAL4 fused to VP16 transcriptional activator; dGAL4-VP16) and an artificial repressor (destabilized GAL4; dGAL4), which wereexpressed under the control of either of CCEs (3  CCE) and regulate the expression of destabilized luciferase (dLuc) reporter gene via competitive
binding to its upstream activator sequences (four tandem repeats of the GAL4-binding sequence; 4  UAS) fused with a minimal CMV promoter(CMV mini). Using the assay system, the investigators reconstructed a natural circadian output, (b), day-time in this example, using a morning
activator and night-time repressor as suggested from previous information. They also successfully designed an unnatural, artificial circadian output
with various combinations of these CCEs with the transcriptional regulators, (c), early day-time in this example, using a morning activator and daytime repressor.

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Challenges in designing mammalian circadian clocks Susaki, Stelling and Ueda 559

associate with the BMAL1/CLOCK heterodimers to
inhibit E-box-mediated transcription [2,3,5,7].
However, the mechanisms behind the circuit’s dynamic
properties remain largely elusive.

Reconstruction and design of circadiantranscriptional networks
Simple regulatory modules, such as positive and negative
feedback loops, can generate complex dynamic behaviors, alone or when they are embedded in larger network
structures. Theoretically, a specific type of gene regulatory network can display a particular dynamic behavior,such as toggle switching, logic gating, or oscillations [8].
Recent work in synthetic biology focuses on forward
engineering gene regulatory networks similar to thoseobserved in known biological networks, and proving thenatural circuits’ design principles by synthesis (‘proof-bysynthesis’). The first step in designing a network structurewith specific dynamic behavior is often in silico modeling;
this is followed by an experimental implementation of thedesigned molecular network in cellulo.
Several studies have attempted to design and implement
purely artificial networks of de novo wiring to generate
systems exhibiting oscillatory behavior, a characteristic
dynamic behavior in the circadian system, by using a
repressilator transcriptional network [9], coupled positive
and negative feedback transcriptional loops [10–12,1314], or a transcriptional- and metabolic-integrated
network loop [15]. A study by Elowitz and colleagues was
one of the earliest synthetic approach in which they
designed and implemented the by now ‘classical’ repressilator gene circuit into Escherichia coli [9]. Later, morerobust — and tunable — oscillation systems were achievedin mammalian cells [12] as well as in E. coli [11,14].
A challenge of the reconstruction or design of a circadianoscillator with a relatively long period (nearly 24 h) andstable amplitudes has not yet been accomplished.
Recently, Tigges and colleagues successfully generated
an artificial transcriptional network with an oscillatory
period of about 26 h, but with fragile oscillations [13].
Another attempt is to use the natural components of thecircadian clock, which was performed by Chilov andcolleagues [16]. They tried to reconstruct a feedback
loop of the natural circadian oscillatory network, composed by inducibly expressed Bmal1/CROCK gene plus
E/E0 -box-connected Per/Cry gene and/or a reporter gene.
The system performed at least a single cycle of a clocklike oscillation, but sustained oscillatory expression of thereporter gene was not observed. Thus, it appears difficult
to artificially reproduce periodic dynamic behavior with asustainability and relatively long period (nearly 24 h). An
alternative approach to build a stable circadian oscillator is
to utilize wiring information in a natural circadian transcriptional network [17]. They succeeded to reconstruct, at least in part, sub-network of mammalianwww.sciencedirect.com

circadian clocks, and found that the phases of the transcriptional activator(s) and repressor(s) of the circadianclock can determine the downstream transcriptional
output phase, by using an in vitro cycling assay systemcomposed of an artificial activator and repressor, of which
expression timings were controlled via either of CCEs,
and an output reporter gene regulated by the activatorand repressor (Figure 2a). The artificial transcriptional
circuits successfully reproduced the natural circadian
output and they generated other unnatural phases by
various combinations of these CCEs with the transcriptional regulators (Figure 2b,c). However, they could notregenerate the morning phase. Taken together, the challenges of synthesizing a ‘perfect’ natural circadian clock
remain to be solved, as we work toward the complete
reconstruction of the transcriptional circuits underlying
the mammalian circadian clock.
We also note that synthetic-biological approaches can beapplied to other dynamic properties of circadian clocks
such as circadian output’s amplitude, temperature compensation and time delay. For example, artificial CCEs
were synthesized followed by their implementation into
cells [18]. Among the synthetically designed CCE
sequences, there were cis-elements with very high- or
low-amplitude circadian transcriptional activity. Highamplitude oscillations required an appropriate affinity
balance between the activators and the repressors. Thus,
novel design principles and underlying mechanisms werediscovered by the synthetic approach. In addition,
temperature compensation of the molecular clock isanother target of the synthetic-biological study, although
it seems necessary to design temperature-insensitive
enzyme [19]. Other synthetic approaches tried to createtime-delay circuits with feed-forward loops or multistep
reactions [20–22]. Such time-delay circuits can be an
important control motif in both circadian clock and othersignaling pathways in nature. Overall, these proof-bysynthesis approaches with natural circadian components
serve a dual purpose; they can investigate sufficiency ofidentified natural components and/or their interactions,and also reveal requirement of previously unidentified
components or interactions.

Design and implementation of dynamic
properties of the central clock tissue
The circadian clock adjusts an organism’s metabolic andphysiological activities to the environmental day–nightcycle of the earth. In mammals, organism-level circadianrhythmicity is critically regulated by one central pacemaker, located in the SCN of the hypothalamus[3,4,6]. The central clock tissue is of particular interest, because it regulates many cyclic metabolic andphysiologic processes, such as the sleep–wake cycle.
Thus, reconstruction of the dynamic properties of the
SCN will be among the next applications of synthetic
approaches for the mammalian circadian clock.
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560 Systems biology

Figure 3

A synthetic approach toward creating the gating property of the SCN. (a) A phase-dependent sensitivity to external stimuli such as light is one of thefundamental properties of the circadian clock. Photic information from the retina is delivered through glutamate and PACAP of the RHT. VIP is also
proposed as an intrinsic SCN factor mediating the photic signaling. These neurotransmitters bind to the receptors expressed at the surface of SCN
cells and activate intracellular signaling pathways. The signaling pathways may be regulated by the circadian rhythm to display phase-dependent
sensitivity to the extracellular signals. In fact, the photic signal transduction system selectively delivers the information of light only at night and it
entrains the circadian clock. (b) In a synthetic approach, researchers may be able to implement a designed transcriptional circuit composed of thesignaling molecules regulated by CCEs into a non-SCN cell type (e.g., fibroblast) to execute the property of the phase-dependent resetting of circadianclock by external stimuli.

One of the fundamental dynamic properties of the central
circadian clock is gating, a phase-dependent response toexternal stimuli. For example, exposure to light during
subjective night-time, but not during subjective dayCurrent Opinion in Biotechnology 2010, 21:556–565

time, can effectively entrain the central circadian clock[3] (Figure 3a). In the photic response program, glutamate and pituitary adenylate cyclase activating peptide(PACAP) in the retinohypothalamic tract (RHT) arewww.sciencedirect.com

Challenges in designing mammalian circadian clocks Susaki, Stelling and Ueda 561

thought to be the main neurotransmitters that deliver
photic information to the SCN; they convey criticalinformation for photic entrainment [4,5,6,23]. Morespecifically, glutamate binds and activates the NMDA
receptors of SCN neurons and it induces intracellular
calcium-dependent signaling pathways, which in turnupregulate Per1 and Per2 in the SCN cells[3,4,6,23] (Figure 3a). PACAP binds to G-protein
coupled receptors (PAC1-receptor and VPAC2) of SCN
cells [23] (Figure 3a). Intrinsic neurochemical signaling
among the SCN neurons, mediated by vasoactive intestinal polypeptide (VIP) and its receptor VPAC2, has also
been proposed as an important factor in the photic
entrainment of the mammalian clock [23,24](Figure 3a). Mathematical models of photic entrainment
can help predict the efficiency of multiple control targets
and their combinations for circadian phase resetting, as
studied by Bagheri and colleagues [25].
These extracellular stimuli activate several intracellularsignaling molecules. It is not clear how the gating process
is implemented in the signal transduction pathways
associated with these components; however, several ofthese proposed factors, including Ryr2 mRNA, phosphorylated MAPK, cGMP levels, and PGK activity, havebeen reported to exhibit oscillatory behaviors[3,4,23,26–28], and the oscillation of these molecules along with the circadian rhythm can prompt cells
to exhibit gating of the photic response. Again, synthetic
cyclic transcriptional regulation of these signal transduction pathways may provide insight into the underlying
molecular mechanisms for gating in the SCN neurons.
Electrophysiological oscillations are another fundamental
dynamic property of the SCN. A single SCN neuron can
express circadian rhythmicity in its electrophysiologicalactivity [4,6,7,23,29]. Recent studies provide evidence that electrophysiological oscillation in harmony
with the circadian rhythm does indeed regulate changes
in such physiological functions as the osmosensory reaction during late sleep [30]. Both experimental analyses
and mathematical modeling suggest that this rhythmicity
is partly mediated by several kinds of Ca+ or K+ channels,
including L-type voltage-dependent Ca2+ channels, fastdelayed rectifier K+ channels (Kv3.1b and Kv3.2), or aCa2+-activated Bk channel (Kcnma1) [23,29,31,32].
Intriguingly, the electrophysiological oscillations areassociated with daily expression changes of these channels, such as the fast-delayed rectifier K+ channels (highin day-time) and the Bk channel (high in night-time)[23,31,32]. Mutations in circadian clock genes —including a Tau mutation in casein kinase I epsilon ora dominant negative mutation of the Clock gene — change
the SCN’s electrophysiological rhythms in mice [29]. A
recent report suggested that the Per1 gene is involved insome electrophysiological features within SCN cells
www.sciencedirect.com[33], further supporting the direct interactions between
the clock gene network and electrophysiological components. However, the exact nature of the interactions
remains elusive. Synthetic design and implementation of
cyclic electrophysiological activity in non-SCN cells mayhelp reveal the sufficient minimal components requiredfor cyclic electrical outputs from the SCN neurons.
A third fundamental property of SCN neurons is theirability of synchronized oscillations—they form a coupledoscillator at the tissue level against inevitable noise. Morespecifically, the SCN consists of 20,000 neurons andtheir circadian activity is unequivocally synchronized[2,6]. Synchronization is indispensable to the SCN
function as a central pacemaker as was revealed throughthe study of the singularity phenomenon [34]. Furthermore, the SCN derived from several clock gene knockout mice showed sustained circadian oscillation althoughthese clock genes were required for the circadian oscillation at a single-cell levels, suggesting compensation of
the circadian behavior by the coupling [35]. An impairment in the intercellular communication among the SCN
cells results in desynchronization and arrhythmicity[36,37].
What are the underlying mechanism(s) for synchronization? Our understanding of collective synchronization incoupled non-linear oscillators has been derived mainly bystudying simplified phase models such as the Kuramotomodel [38]. More realistic subsequent theoretical studies
have refined the picture [39–44,45]. For example, mathematical modeling of the intercellular coupling of noiseresistant circadian oscillators highlighted the importanceof oscillatory factor(s) that are secreted at a specificcircadian time and can induce light- or dark-pulse-typephase shifts in neighboring cells [43]. More recently,
Bernard and colleagues developed a realistic model thatcomprises a heterogenous set of damped cellular oscillators and a coupling agent. Connectivity was simulated in
the three-dimensional in vivo SCN or two-dimensionalsliced SCN with separate core and shell compartment of
the tissue [45]. This model emphasized the importanceof population size, number of oscillators, and connectivity
for the synchronization. Besides the SCN, to understandthe basic principle of synchronized oscillatory behavior at
the population level, Danino and colleagues recently
developed an engineered gene network that enable synchronized oscillations in a growing population of E. coli
[14], and highlighted the importance of a small secretory
molecule and an appropriate cell density as well. Similar
principles may also establish synchronization in the SCN,
given that SCN cells are packed into a small region of the
hypothalamus with intercellular connectivity [23].
Regarding specific biological mechanisms, for instance,
the VIP-VPAC2 signaling pathway is thought to mediate
intercellular communication among the SCN neurons[6,37,46]. These signaling pathways thus may provide
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562 Systems biology

Figure 4

Synthetic approach toward designing electrophysiological oscillation and coupling in the SCN. SCN cells exhibit characteristics such as oscillatory
electrophysiological activity and synchronized circadian oscillations (coupling), which are critical for SCN function as a central pacemaker.
Researchers may incorporate a designed transcriptional circuit regulated by CCEs, and composed of (a) ion channels or their modifiers or (b) secretory
molecules and their receptors, into a non-SCN cell type (e.g., fibroblast) to create the desired properties of an oscillated electrical activity orintercellular coupling.

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Challenges in designing mammalian circadian clocks Susaki, Stelling and Ueda 563

first targets for future synthetic approaches to reveal theunderlying molecular mechanisms of synchronization
among SCN neurons.
Taken together, we propose that a key to designing SCN
function is to use and modify the mechanisms describedabove to generate designed outputs from the SCN(Figures 3 and 4). A possible synthetic approach is toimplement these oscillatory networks in cells and reproduce the desired property. This is analogous to thestudy of proof-by synthesis of circadian phase where adesigned transcriptional network in which the gene
expression is regulated by CCEs, and thus regulated
along with the circadian rhythm, is incorporated into cells[17] (Figures 3 and 4). Observing whether the desired
property is executed properly will help indicate thesufficiency of the system. These synthetic approaches
at the individual network level will help to elucidate therelationship between the circadian clock and other networks in the brain that function to regulate various
behavioral outputs such as the sleep–wake cycle.

Conclusions
The integration of systems and synthetic approaches willplay a critical role in improving our understanding ofdynamic physiological functions and help in the designof systems that show desired properties. We discussed thisapproach in the specific context of the circadian rhythms.
Application of the synthetic approach to studies of the
mammalian circadian rhythm will enhance not only thischallenging research field, but for clinical medicine as well.
In particular, people nowadays usually have to live in theartificial environment without the natural day–night cycleon the earth. Furthermore, recent social and medical
problems such as aging of the human population, a partof which are accompanied by circadian rhythm disorders, isa key concern worldwide. Thus, the synthetic approach
may become one of the best model cases that can informbiological engineering, with a potential for the analysis andtreatment of many diseases.

Acknowledgements
The authors are supported by the Research Fellowships of the Japan
Society for the Promotion of Science for Young Scientists, the Mitsubishi
Foundation, Research Program of Innovative Cell Biology by Innovative
Technology, intramural Grant-in-Aid from the RIKEN Center for
Developmental Biology (CDB), and Director’s Fund from CDB.

References and recommended reading
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564 Systems biology

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Current Opinion in Biotechnology 2010, 21:556–565