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From noise to synthetic nucleoli: can synthetic


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Volume 8 Number 4 April 2016 Pages 371578

Integrative
Biology
Interdisciplinary approaches for molecular and cellular life sciences
www.rsc.org/ibiology

Themed issue: Biological Insights from Synthetic Biology


ISSN 1757-9694
lin d in

REVIEW ARTICLE
e!
ed xe

Mark Isalan et al.


M nde

From noise to synthetic nucleoli: can synthetic biology achieve new


insights?
I
Integrative Biology
REVIEW ARTICLE

From noise to synthetic nucleoli: can synthetic


biology achieve new insights?
Cite this: Integr. Biol., 2016,
8, 383
Marta Ciechonska, Alice Grob and Mark Isalan*

Synthetic biology aims to re-organise and control biological components to make functional devices.
Along the way, the iterative process of designing and testing gene circuits has the potential to yield
many insights into the functioning of the underlying chassis of cells. Thus, synthetic biology is
converging with disciplines such as systems biology and even classical cell biology, to give a new level
of predictability to gene expression, cell metabolism and cellular signalling networks. This review gives
Received 28th October 2015, an overview of the contributions that synthetic biology has made in understanding gene expression, in
Accepted 27th December 2015 terms of cell heterogeneity (noise), the coupling of growth and energy usage to expression, and
DOI: 10.1039/c5ib00271k spatiotemporal considerations. We mainly compare progress in bacterial and mammalian systems, which
have some of the most-developed engineering frameworks. Overall, one view of synthetic biology can
www.rsc.org/ibiology be neatly summarised as creating in order to understand.

Insight, innovation, integration


Synthetic biology involves the engineering of life either to construct functional biological devices or to test the functioning of biological systems, thus achieving
countless insights into the inner workings of the cells. Here, we provide an overview of the impact that the field of synthetic biology has had in the areas of gene
expression, cell heterogeneity (noise), coupling of growth and energy usage to expression, and spatiotemporal dynamics of cellular machinery. We compare
bacterial and mammalian systems, which currently provide some of the most-developed engineering frameworks. Over the last decade, the many insights that
have arisen from synthetic biology have triggered an exciting transition from creating in order to understand towards creating in order to cure.

1. Introduction insights into inter- and intra-cellular behaviour, from accurate


temporal quantification of transcription and translation, controlled
The application of synthetic biology, via the study of engineered by defined stimuli, to rewiring or de novo reconstruction of
genetic circuits and networks, has led to many important insights native circuits, to directed evolution of synthetic networks and
into native gene network functions, gene expression (including the manipulation of multicellular interaction, via modulation
transcriptional and translational noise), cell physiology (including of multi-circuit dynamics.
growth and metabolism), and the elucidation of basic biological Many synthetic biologists have been inspired by a statement
design principles that are analogous to fundamental laws in from Richard Feynman, found after his death and written in
physics. The influence of engineering principles in biology, and chalk on his blackboard: What I cannot create, I do not understand.
recent advances in DNA synthesis and sequencing technologies, This has served both as a rallying call for synthetic biology and
has additionally led to the construction of predictive mathe- as a central unifying feature for a field that encompasses diverse
matical models based on experimental data, which have, in disciplines. Here, we will explore bacterial and mammalian
turn, guided the design of more robust, predictable, and even systems in particular, with a view to seeing the limits of what
entirely novel networks. we can engineer and understand, particularly for synthesising
In this review, rather than focusing on the technological and predictable spatiotemporal gene expression.
engineering advances in synthetic biology, we will concentrate
on seeing how re-engineering biology has given us new under-
standing. Synthetic biology has been instrumental for gaining 2. Bacterial gene circuits: from
synthesis to insight
Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.
E-mail: m.isalan@imperial.ac.uk Some of the first developments in synthetic biology included
Authors contributed equally. the generation of simple synthetic gene expression circuits,

This journal is The Royal Society of Chemistry 2016 Integr. Biol., 2016, 8, 383--393 | 383
Review Article Integrative Biology

Fig. 1 Synthetic and natural bacterial gene circuits. (A) One of the first synthetic circuits, the repressilator, is composed of three repressors that are
arranged in a feedback loop, and results in oscillatory behaviour. (B) An improved synthetic oscillator, the relaxation oscillator, including both positive and
negative feedback loops, results in stable and robust oscillations. (C) Bacillus subtilis energy stress response frequency modulation (FM) pulse control. A
noise-activated rise in the fluctuation of the phosphatase RsbQP levels results in de-phosphorylation of RsbV, activating the alternative sigma factor sB,
leading to upregulation of expression of the operon genes and resulting in operon feedback. When RsbW kinase activity overcomes phosphatase activity,
the pulse is terminated. Increased stress results in higher phosphatase levels and results in more frequent pulsing. (D) Transient dierentiation to
competence is controlled by noise-related fluctuation of the master regulator ComK in Bacillus subtilis. Expression noise controls competence initiation,
with a rise in the fluctuating levels of ComK resulting in positive autoregulation of transcription and negative feedback through control of the ComK
inhibitor ComS. (E) Multicellular patterning: a bacterial senderreceiver system generating two-dimensional pattern formation. The circuit contains
sender (yellow) and receiver (green and red) cells. Adapted from the Vibrio fischieri motility network, sender cells secrete the enzyme LuxI, that generates
the messenger acyl homoserine lactone (AHL). AHL diffuses through the medium away from the sender population (yellow triangle), is internalized, and
binds to LuxR in receiver cells, thus activating the expression of cI and LacIMI in a circuit engineered to act as a band detection filter (band detection high
and low thresholds are indicated by blue stars). Two populations of receiver cells are plated together, expressing green or red fluorescent protein
expression (GFP or RFP). Repression of GFP or RFP results from differences in LacI stability, which controls the distance of the ring from the sender
population. (F) Multicellular patterning: a dual-module density-sensing and motility-directing circuit generates radiating ring patterns away from the
centrally placed AHL signal. AHL is generated via expression of LuxI. LuxR, activated by AHL, turns on the expression of cI, which represses the motility
regulator cheZ.

such as the repressilator and the toggle switch.1,2 These networks biology, transforming our understanding of how, when, and
contained sets of inducible promoters and transcription repressors, why specific genes are expressed.
with fluorescent markers serving as quantitative outputs, and Gene expression in isogenic bacterial populations varies
were developed based on electric circuit design rules. Primarily, from cell-to-cell, even when the population reaches a stable
they served as a proof of principle that non-native genetic average expression level or steady state. This is due to variations
circuits can be expressed and stably maintained in bacteria. in the number of hardware units, such as transcriptional
In a sense, the field has expanded from simple models to more translational machinery and regulatory molecules (resulting in
complex ones (Fig. 1). Nonetheless, the concepts of using extrinsic noise), as well as the inherent stochasticity attributed
controllable transcriptional and translational regulation, as to the random nature of single-molecule kinetics (resulting in
well as employing quantifiable readouts for gene expression, intrinsic noise). These two measures of stochasticity contribute
still form the building blocks of many complex synthetic networks greatly to the phenotypic heterogeneity documented in genetically
today.3 The development of such circuits has been reviewed identical populations.6,7 Moreover, fluctuations resulting from noise
extensively elsewhere.4,5 Here, we will rather focus on exploring seem to be an intrinsic property of gene expression, as seen in
two questions: artificial cells composed of cell membrane-mimetic vesicles,
! What are the biological insights that synthetic approaches containing only transcription and translation machinery.8
have achieved? Noise-dependence also plays a prominent role during dynamic
! How do they help us to understand underlying cellular cell responses to changing environmental conditions; this involves
processes? pulses in activation and deactivation of regulatory factors, in a
stochastic manner within a population.9 Occurrences of such
pulse regulation include the control of the energy stress
2.1 Lessons from bacteria I: the role of noise response, entry into competence, and the sporulation of Bacillus
Initially considered a major roadblock to overcome, stochastic subtilis.1012 For example, activation of the energy stress response
gene expression, or noise, has arguably become one of the involves frequency modulation (FM) pulsing of the alternative
main biological insights contributed by the field of synthetic sigma factor dB, with increasing stress positively correlating

384 | Integr. Biol., 2016, 8, 383--393 This journal is The Royal Society of Chemistry 2016
Integrative Biology Review Article

with increased pulse frequency.10 Synthetic rewiring of the native core and two adjustable components that contribute to cell
circuit involved in this response revealed that noise activates a growth: ribosomal proteins and proteins responsible for the
phosphorylation pulse switch that can be propagated, or turned influx of nutrients. These components are in constant flux with
off, by a combination of a mixed positive and delayed negative respect to each other. The insertion of synthetic circuitry, and
transcriptional feedback loop, thus allowing for amplification and the addition of extraneous protein expression, is thought to
subsequent pulse termination.10 appropriate ribosomal resources from the adjustable proteome
In another example of pulse regulation, rewiring the ComS-ComK fraction and thus exert a negative eect on the growth rate.20,21
competence-control circuit of B. subtilis demonstrated that stochastic The cellular contribution to gene expression is therefore not
gene expression plays multiple roles leading up to, and during, limited to regulatory mechanisms and noise, but also to a global
dierentiation to this transient state. With fluctuation in ComK eect on expression based on the physiological constraints of
expression controlling the initiation of competence, noise plays the system.22,23
a central role in driving its expression and accumulation. The quantitative nature of this kind of work has contributed
Additionally, noise provides a wider range of response duration, to the concept of introducing bacterial growth laws, by applying
which is controlled by the degradation of ComS; as ultra low analogies from Kirchos and Ohms laws to predict the relation-
levels of ComS are necessary for exit from competence, the system ship of gene expression and growth rate.24 However, while
becomes more sensitive to stochastic fluctuations of this regulator.11 dierences in growth rate and ribosome availability of E. coli
A dierent type of noise-related pulsing is a phenomenon strains are certainly factors of gene expression, strain-specific
encountered during transcriptional bursting, which describes nitrogen and carbon metabolism capacity add another layer of
stochasticity involved in global gene expression, particularly complexity in predicting circuit output.25 Additionally, the
in highly-expressed bacterial genes. This bursting pattern of function of a given protein product can also exert an eect on
transcription was identified in various synthetic circuits, however, the growth rate and on other cellular factors. For example, not
its molecular mechanism has only recently been characterized: it only can stochastic expression of catabolically active enzymes be
results from the reversible binding and dissociation of gyrase to propagated and cause growth fluctuations, noise can also be
resolve the buildup of highly supercoiled DNA.6,13,14 The latter transmitted to other genes via cell growth.26 Heterogeneity in protein
system serves as an excellent example of how cellular behaviours, expression, resulting in metabolic stochasticity, can be propagated
such as stochastic gene expression, are brought into focus via and can ultimately influence phenotypic heterogeneity.26,27
synthetic biology approaches and can subsequently lead to the The cellular growth rate can itself aect the dynamics of a
identification of novel mechanistic insight. gene circuit, and may be responsible for selecting favourable
While noise appears to be a global variable in gene expression, phenotypes, such as the development of bistability for persistence
it does not aect all genes equally. In E. coli, highly-expressed under antibiotic challenge.28,29 Conversely, synthetic circuits can
genes have been shown to contain sequences associated with also aect the growth rate, as they place an energetic burden onto
higher extrinsic noise levels,15 while promoters associated with the chassis, or host organism, with greater output generally
functionally important genes were characterized by decreased associated with decreased expression capacity and growth rate.30
noise.16 Additionally, a recent analysis of a synthetic promoter However, additional protein cost may be normalized after several
library evolved de novo revealed that, while initial promoter generations, as the cell adjusts its ribosomal content to new
noise levels are generally low, selective pressure placed on these steady-state levels.31 Interestingly, construct output is not simply
regulatory sequences routinely results in increased expression inversely correlated with host capacity, and circuit design plays
noise. Promoter-associated noise is further propagated by noise an important role in maximizing eciency, with strong RBS-
associated with regulators, and may act as the primary step to containing circuits being least ecient.32 This is perhaps not
the evolution of more finely tuned regulation.17 surprising as the availability of cellular translation machinery
Overall, noise contributes to many levels of gene and network plays a decisive role in cell growth rate.33
regulation, and has been studied through elegant synthetic circuit The application of synthetic biology to develop robust
design. Thus, we are finally beginning to unravel and control the growth and expression models, via the generation of quantitative
molecular mechanisms responsible for this phenomenon. datasets in multiple bacterial species and conditions, promises to
transform our predictive understanding of gene expression,
2.2 Lessons from bacteria II: growth laws and metabolic cost with global cell properties being equally important to signalling
An equally important insight from the field of synthetic biology is via transcription factors.
the concept of cellular energy allocation. While it may now seem
intuitive, the quantitative co-dependent relationships of bacterial 2.3 Lessons from bacteria III: multicellular synthetic gene
gene expression, metabolism and growth have only recently begun circuits for complex environments
to emerge. The introduction of synthetic tools, such as inducible Synthetic biology approaches have been applied to understanding
promoters and tunable circuits, has allowed for the quantification the principles of not only intra-, but also inter-bacterial signalling
of the cellular cost of producing various component parts, thus networks, involving spatial as well as temporal dimensions.
defining a relationship between expression and growth.18,19 Understanding how networks have gained their physiological
One phenomenological theory of bacterial growth suggests function, discovering multiple solutions to the generation of
that the cellular proteome may be subdivided into one unchangeable identical phenotypes, and analysing the evolutionary decisions

This journal is The Royal Society of Chemistry 2016 Integr. Biol., 2016, 8, 383--393 | 385
Review Article Integrative Biology

taken to arrive at particular natural networks are necessary for probed the entire design space of 3-node gene networks that
the elucidation of common design principles, if they exist, and can form a central stripe in response to a chemical signalling
for the design of novel networks and behaviours. gradient.30 Synthetic biology therefore allows the systematic
Multicellular spatiotemporal patterns have been observed analysis of a whole network design space and can lead to the
during bacterial swarming and chemotaxis, and have been identification of common design principles of circuit and net-
recognised as self-organising behaviours.34,35 The autonomous work function.
nature of the formation of these spatiotemporal patterns was Reconstruction of network dynamics through circuit engineering
recapitulated using synthetic circuitry that coupled motility must also consider the context of the native mechanism. While an
with cell density. Stripes with variable periodicity were obtained alternative synthetic network may simulate the behaviour of a
by changing expression levels of the motility-associated signal naturally evolved one, it may not be as stable or robust. For example,
within the circuit.36 Such proof-of-concept reconstruction provides an engineered circuit with an alternative architecture was compared
an excellent tool for the analysis of mechanistically complex and to the dierentiation to competence circuit in B. subtilis.39 While the
uncharacterised natural stripe-forming phenotypes, and may be desired phenotype was reconstituted, the circuits diered in noise
extrapolated to other periodic population phenomena, such as profiles, which, in turn, resulted in distinct competence dynamics.39
reactiondiffusion, where spatiotemporal dynamics are controlled Dierences in circuit response may point to selection between high
by a diffusible signal.36 versus low variability in environmental cues, and circuit noise must
Another excellent model for the study of spatial and temporal be considered during network design and phenotype analysis.
signalling is the generation of stripe patterns in bacterial The competence circuit has also been used to demonstrate
populations.37 A recent and powerful approach is to consider that dynamical behaviour results from the integration of multiple
all potential network designs that a given circuit may adopt in inputs aecting distinct circuit locations, as opposed to two inputs
order to form a stable phenotype. This carries the benefit of acting on the same circuit element.40 This brings to light the idea
discovering multiple, equally attractive solutions, including that quantities of environmental cues may also play a role in
ones that are not found in native biological systems, giving bacterial circuit dynamics. Exploring transcription network
new mechanistic insight (Fig. 2).30,38 Thus, by adopting a inputoutput rewiring on a large scale, via the construction of
computational approach to generate a genotypephenotype net- a combinatorial library of promoters and transcription or s-factor
work map, and by experimentally evaluating prediction results genes, has shown that E. coli can tolerate, and indeed take
of theoretically-derived stripe-forming networks, Schaerli et al. advantage of, extensive rewiring regimes.41 Additionally, the
surprising finding that expression of most newly constructed
promotergene combinations has little effect on growth, is
indicative of an underlying mechanism responsible for the
buffering of changes in host genetics.41
Combining systems and synthetic biology approaches can
lead to the determination of specific rules for network stability
within a complex cellular environment. The randomisation of
entire circuits followed by evolutionary selection has shown
that fitness decreases with the increasing number of added
genes, high expression levels, and repeated sequences.42 This
randomisation and selection approach was superior to rational
circuit design by yielding a robust circuit with low gene expression
and no repeated sequences. Moreover, an expression threshold
maximum was established for ensuring increased evolutionary
stability of the circuit.42 Overall, it is clear that synthetic biology is
changing our view of the control of gene expression in bacteria, by
showing that transcription factor network topologies alone are
insucient to give us a predictive understanding of cell pheno-
types. As we will see in the next section, similar conclusions are
likely to hold for eukaryotic cells.

Fig. 2 Exploring the design space of possible dynamical mechanisms of


stripe-forming 3-node gene regulatory networks (GRNs). GRN complexity 3. Rise of the mammals
decreases vertically, with all combinations derived computationally and thus
converged onto stalactites representing the simplest minimal networks 3.1 Sub-cellular organisation and stability of mammalian
that can execute a function. The design space of functional stripe-forming synthetic circuits
networks divides into four minimal networks of incoherent feed-forward
(IFF) loops (third tier, I-1-4), which converge on a two-node archetype
There is great potential in mammalian systems not only to engineer
I-zero. Nodes I3 and I4 are novel arrangements previously unreported both useful functions or devices, but also to gain insights into the
in nature and in synthetic biology studies.30 mechanisms behind native cell systems. Whereas bacterial synthetic

386 | Integr. Biol., 2016, 8, 383--393 This journal is The Royal Society of Chemistry 2016
Integrative Biology Review Article

biologists have worked hard to engineer and standardize


modularity43 as well as component orthogonality4446 in gene
circuits, so far there have not been similar concerted attempts
to standardize parts for mammalian systems on a large scale.
Nonetheless, mammalian synthetic biology is progressing
rapidly (reviewed in ref. 5 and 47), benefiting directly from
the large amount of pioneering gene circuit engineering in
bacteria (reviewed in ref. 4). For example, Francois Jacob and
Jacques Monods study of the lactose operon in E. coli48 enabled
the first synthetic inducible promoters in mammalian cells.4951
Since then, toggle switches,52 Boolean logic gates,53 hysteretic
switches,54 oscillators,5557 and even electrically-induced tran-
scriptional switches58 or light-induced opto-genetic switches5962
have been successfully developed in mammalian cells (reviewed
in ref. 47, 63 and 64). Despite this progress, mammalian
synthetic circuits can be less reliable, more context sensitive,
and necessitate longer design-testing iteration cycles than their
bacterial counterparts. At the most extreme, this implies making
stable cell lines to test each new gene circuit reliably in a fixed
genomic context (e.g. in ref. 65).
A key consideration is the subcellular compartmentalisation
of eukaryotic cells, which creates its own engineering demands.
Fig. 3 Functional compartmentalisation of human nucleoli. Functional
For example, eukaryotic cells have highly compartmentalised nucleoli are tripartite. Their inner FCs (green box) contain rDNA and RNA
nuclei, which tightly pack their genomes into chromatin and polymerase I (pol I) machinery unengaged in transcription. Pol I machinery
chromosome territories. Thus, nuclei are functionally organised (UBF, SL1, Rrn3 and pol I) is dedicated to rDNA transcription and is active at
into domains such as the nuclear lamina, nucleoli, speckles, the interface between FCs and DFCs (white box). Primary transcripts, 47S
histone locus bodies (HLBs), PML bodies and Cajal bodies pre-rRNAs, are produced into the DFCs. Processing of these transcripts
into mature 18S, 58S and 28S rRNAs requires a succession of coordinated
(CBs).6668 Unpredictable long-range genome interactions (revealed cleavages (green arrows) and modifications. While early steps of this
by HiC studies), occurring at the border of chromosome territories, process occur within the DFCs (yellow box), the later steps and assembly
are responsible for spatial effect variegation and stochasticity of the ribosomal subunits 40S and 60S occur within the GC (red box).
observed in the mammalian transcriptome.69 The resulting FCs are at the core of nucleoli, surrounded by DFCs and further embedded
expression modulation provided by the nuclear locus makes into the GC.

it difficult to plug-and-play gene expression functions, such as


inducible promoters for synthetic circuits.
Moreover, mammalian nuclei are highly dynamic with nuclear de novo formation of nucleoli inner centers, i.e., synthetic fibrillar
components constantly diusing passively through the nucleo- centers (FCs) called pseudo-NORs (Fig. 3 and 4).77,78 XEn arrays
plasm70 and architectural disruption upon every cellular division.71,72 constitute strong binding sites for nucleolar HMG box transcription
In the past, our understanding of the tight spatiotemporal regulation factor UBF (Upstream binding factors). Interestingly, during mitosis,
of gene expression has relied largely on the lac operator (LacO)/ the resulting UBF-loaded chromatin forms a prominent chromo-
lac repressor (LacI) system, which allowed the tethering of somal feature, i.e. a secondary constriction of under-condensed
genes to specific sub-nuclear domains and/or the visualisation chromatin, constituting the first step in nucleolar formation.77,78
of gene movement using GFP-tagged LacI.73 These studies have As sites of ribosome biogenesis, nucleoli are functionally
highlighted that the location of genes and their chromatin structured around arrays of ribosomal genes (rDNA) into three
states are strongly connected,74 and that the mammalian genome distinct compartments: the fibrillar centers (FCs), the dense
is regulated in 3D.75 Generally, silenced heterochromatin is found fibrillar components (DFCs) and the granular component (GC)
at the nuclear and nucleolar periphery, while active euchromatin is (Fig. 3).79,80 While the role of FCs in ribosome biogenesis
more central. remained uncertain for organisms having DFC/GC bipartite
Synthetic biology is now allowing us to investigate comprehen- nucleoli,81 synthetic pseudo-NORs now suggest that FCs are
sively how the critical organisation of mammalian nuclei is essential for the cell cycle propagation of mammalian nucleoli
established and propagated through open mitosis. Indeed, through open mitosis (Fig. 4).66,78 They contain unengaged
de novo biogenesis of synthetic nuclear domains demonstrates our transcription factors and are devoid of processing factors.
knowledge of the basic mechanisms involved: tethering of a CB Transcription of rDNA only occurs at the interface between
constituent, fused with LacI, to an ectopic array of LacO appears FCs and DFCs, producing pre-rRNA into the DFCs where early
to be sucient to nucleate ectopic CBs via proteinprotein processing takes place. Subsequent late processing of pre-rRNA
interactions.76 Similarly, using ectopic arrays of Xenopus enhancer and assembly of ribosome subunits occurs within the GC. In
(XEn) from ribosomal genes (rDNA) in human cells resulted in the humans, rDNA arrays are found within the short arms of the

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Review Article Integrative Biology

Fig. 4 Lessons from synthetic neonucleoli. (A) Nucleoli are formed around nucleolar organizer regions or NORs, located, in humans, on the short arms
of acrocentric chromosomes. NORs contain rDNA arrays in a specific conserved chromosomal context formed of the telomeric Distal Junction (DJ) and
centromeric Proximal Junction (PJ). rDNA are constituted of an intergenic spacer (IGS) and a transcription unit. (B) Synthetic neo-NORs revealed the
existence of NOR territories within nucleoli, reminiscent of the chromosome territories (white dashed outline). These NOR territories are overlay with
functional compartmentalization into FC, DFC and GC; in a similar way that chromosome territories are overlay with nuclear bodies, such as nucleoli or
HLBs. In humans, nucleoli are typically surrounded by perinucleolar heterochromatin (dark blue) derived from the DJs (white spots). While nucleoli
disassemble during mitosis, NORs competent in nucleolar formation remain undercondensed and retain components of the pol I machinery. This results
in a secondary (21) constriction, a prominent feature of metaphase acrocentric chromosomes. Synthetic pseudo-NORs, arrays of XEn UBF binding sites
(green boxes), resulted in the formation of novel mitotic 21 constrictions and interphase bodies reminiscent of nucleolar FCs, arguing that 21 constrictions
are the mitotic counterpart of interphase FCs. Synthetic neo-NORs, formed of XEn arrays interspersed with rDNA transcription units, drove the formation
of mitotic 21 constrictions and fully functional compartmentalized neonucleoli. Upon UBF depletion, synthetic pseudo-NORs and neonucleoli are lost.
(C) During open mitosis, even though pol I transcription is repressed, nucleolar FC components (green spots on the blue metaphase plate) remain
associated with competent NORs, while DFC and GC components (red) relocate at the chromosomes periphery. At the exit from mitosis, rDNA
transcription resumes and DFC/GC processing factors are recruited to the newly synthesized pre-rRNAs, thus reforming functional nucleoli.
Construction of pseudo-NORs, neo-NORs and neonucleoli revealed that UBF induces the formation of a specific chromatin state ensuring a mitotic
bookmarking that is essential but not sucient for nucleolar formation. Thus, cell cycle inheritance and biogenesis of nucleoli is a staged process, where
UBF-dependent bookmarking of competent NORs ensure an early resumption of rDNA transcription and pre-rRNA-dependent reassembly of nucleoli.

five acrocentric chromosomes and nucleoli can derive from eciency of their transcription.78 Moreover, pseudo-NORs,
more than one acrocentric chromosome (Fig. 4). Thus, nucleoli neo-NORs and neonucleoli provide compelling evidence that
are both dynamically and functionally compartmentalised and nucleolar biogenesis and propagation through open mitosis is
at the convergence of multiple chromosomes. As such, they are a staged process, where UBF-dependent mitotic bookmarking
the ultimate paradigm of nuclear functional organisation.80 precedes transcript-dependent nucleolar assembly (Fig. 4C).
Therefore, it was reasonable to ask the question: do we under- Similarly, the generation of ectopic HLBs, speckles, paraspeckles
stand nucleoli well enough to engineer them? and nuclear stress bodies absolutely requires the synthesis or
Pseudo-NORs reproduced the first step in the formation of tethering of an RNA constituent.8385 Thus, de novo construction
artificial nucleoli and their mitotic memory.66,78 However, the of nuclear bodies has highlighted the key role of architectural RNAs
de novo construction of fully functional synthetic nucleoli, in nuclear self-organisation. The memory of such organisation
termed neonucleoli, required not only UBF binding sites but also and its rapid re-establishment after cell division could be driven by
rDNA transcription units assembled in arrays called neo-NORs78 mitotic bookmarking.66 Such epigenetic marks retain the memory
(Fig. 4). Their construction revealed that a chromosomal context, of active genes through mitosis to ensure an early G1 reactivation
ensuring perinucleolar heterochromatin,82 is implicated in of essential genes and/or the maintenance of cell lineage
maintaining the genomic stability of rDNA arrays and the transcriptomes and phenotypes.86

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Integrative Biology Review Article

The development of synthetic biology has also enabled a more engineering synthetic signalling pathways, thus developing
systematic study of epigenetic memory and spatial regulation of mammalian receiver cells.65,9496 Such populations of cells,
gene expression by nurturing the development of designed DNA capable of sensing environmental metabolites, constitute powerful
binding factors such as zinc fingers, TALEs and CRISPR/Cas9- tools to better understand the molecular spatiotemporal dynamics
derived systems, and their application in editing the epigenome of essential biological processes, including embryonic pattern for-
(Fig. 5).8790 mation, paracrine or autocrine signalling and cellular migration.
The rise of mammalian synthetic biology has highlighted Inspired by the extensive synthetic biology work on spatio-
the crucial role of spatial and epigenetic regulation of gene temporal patterning in bacteria (e.g. in ref. 30, 37 and 97)
circuits for their stability. One strategy toward more network mammalian analogues have appeared. For instance, the classical
reliability is to target the genomic integration of synthetic French flag model of stripe formation98 has been a common
circuits to genome safe harbours to minimise undesired inter- engineering goal in both bacterial37 and mammalian synthetic
ference.91 For ultimate reliability and predictability, synthetic biology. In such systems, cells in a field receive high, middle or
circuits may even require the construction of well-characterised low concentrations of a morphogen diusion gradient, and
mammalian artificial chromosomes (MACs). The first MACs express flag-like stripe genes accordingly. Thus, a mammalian
were formed in cells transfected with a DNA cocktail including concentration detection network has successfully been engineered
a centromeric a-satellite array, genomic DNA, and telomeric in response to a tetracycline gradient.99 Furthermore, a toolkit
sequences.92 However, MACs were only observed after uncontrolled to establish morphogen diusion gradient in cysts has been
rearrangement events, resulting in MACs larger than the input developed to extend the study of pattern formation to 3D
DNA.93 Next-generation MACs could benefit from the inclusion cultures in collagen.65 Building artificial senderreceiver systems
of rDNA chromosomal context, providing a protective hetero- in cells also allows the use of information theory to study
chromatin shell, since isolation from the surrounding nucleo- communication between cells quantitatively (reviewed in ref. 100).
plasm and interfering activities would improve the eciency Next-generation networks will likely provide sophisticated
and reliability of mammalian synthetic circuits. Moreover, progress synthetic therapeutics, with high precision control devices
in the development of MACs will certainly continue improving our coupling sensing and delivery mechanisms. Already, mammalian
understanding of kinetochores and centromeres. Indeed, while synthetic biology has provided circuits oriented toward thera-
mammalian systems have been challenging for synthetic biology, peutics. RNA-based multi-input Boolean logic cell classifiers can
its principles and methodology have provided the most powerful specifically recognize and kill cancer-derived HeLa cells, rather
means of understanding the basics of mammalian spatiotemporal than HEK293 and MCF7 cells, according to their miRNA expression
genomic organisation. patterns.101,102 Refinement of the cell classifiers using RNA
With the means of understanding and overcoming challenges replicons and RNA-binding proteins provides an even more
like spatiotemporal stability, mammalian synthetic biology is a robust and finely tuneable system.102
rapidly expanding field now turning to synthetic signalling Another complex device with therapeutic potential consists
pathways and exciting multicellular networks. of sender and receiver cells, contained in alginate-poly-L-lysine-
alginate capsules, circulating as prosthetic delivery units in the
mouse bloodstream, and responding to an acetaldehyde signal.103
3.2 Multicellular mammalian synthetic networks and
These units form multicellular networks, which behave as
synthetic therapies
hormone-like information-processing devices: exogenous signals
In the process of reducing noise and coordinating gene expression trigger quorum-sensing cross-talk to finely tune the blood level
at a cell population level, synthetic biologists have turned to of a critical protein, the human placental secreted alkaline
phosphatase.103
Mammalian synthetic therapies can also take advantage of
lymphocyte T cells, which can be collected and genetically
engineered before being injected back into patients. Thus,
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rerouted to specifically recognize and kill cancer cells.104,105
Finally, perhaps the most exciting development in the last
decade has been that of CRISPR/Cas systems for genome
editing and a wide range of other applications.106 Already,
Fig. 5 Epigenome editing. Introduction of ectopic arrays of UBF binding
CRISPR systems have been successfully incorporated in the
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marking. Ectopic binding sites of other natural DNA-binding factors (light term, these systems are likely to revolutionise ex vivo therapies,
blue), like LacI or TetR, can also be used for epigenome editing following the where cells are removed, modified and reimplanted, such as in
fusion of these factors with a chromatin-modifying domain (grey). Fusion with
pioneering HIV treatment.109 In the longer term, in vivo therapies
a zinc finger protein (yellow), a TALE protein (dark blue) or a nuclease-deficient
dCas9 protein (red) can also be used to target chromatin-modifying factors to
should become well established, as long as specificity and host
a specific locus. These synthetic DNA-targeted factors can either activate immunity complexities are overcome.106,110 Widespread genome
gene expression (e.g. VP16) or repress it (e.g. KRAB). editing will transform therapeutics and genetics, likely leading

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