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rsif.royalsocietypublishing.org
ReportCite this article: Fagerlund R, Behar M,Fortmann KT, Lin
YE, Vargas JD, Hoffmann A.
2015 Anatomy of a negative feedback loop:
the case of IkBa. J. R. Soc. Interface 12:20150262.
http://dx.doi.org/10.1098/rsif.2015.0262
Received: 25 March 2015
Accepted: 3 August 2015
Subject Areas:systems biology
Keywords:negative feedback, gene regulation,
NFkB, IkBa, oscillation
Author for correspondence:Alexander Hoffmann
e-mail: [email protected]
†Present address: Department of Biomedical
Engineering, The University of Texas at Austin,
Austin, TX 78712, USA.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsif.2015.0262 or
via http://rsif.royalsocietypublishing.org.
& 2015 The Author(s) Published by the Royal Society. All
rights reserved.
Anatomy of a negative feedback loop:the case of IkBa
Riku Fagerlund1, Marcelo Behar1,†, Karen T. Fortmann1, Y. Eason
Lin1,2,3,Jesse D. Vargas1,2,3 and Alexander Hoffmann1,2,3
1Department of Chemistry and Biochemistry, University of
California, San Diego, La Jolla, CA 92093, USA2Department of
Microbiology, Immunology, and Molecular Genetics, and 3Institute
for Quantitative andComputational Biosciences, University of
California, Los Angeles, CA 90095, USA
The magnitude, duration and oscillation of cellular signalling
pathwayresponses are often limited by negative feedback loops,
defined as an‘activator-induced inhibitor’ regulatory motif. Within
the NFkB signallingpathway, a key negative feedback regulator is
IkBa. We show here that,contrary to current understanding,
NFkB-inducible expression is not sufficientfor providing effective
negative feedback. We then employ computationalsimulations of NFkB
signalling to identify IkBa molecular properties thatare critical
for proper negative feedback control and test the resulting
predic-tions in biochemical and single-cell live-imaging studies.
We identifiednuclear import and nuclear export of IkBa and the
IkBa–NFkB complex, aswell as the free IkBa half-life, as key
determinants of post-induction repressionof NFkB and the potential
for subsequent reactivation. Our work emphasizesthat negative
feedback is an emergent systems property determined bymultiple
molecular and biophysical properties in addition to the
required‘activator-induced inhibitor’ relationship.
1. IntroductionNegative feedback control is a ubiquitous
regulatory motif in many biologicalsystems, critical to the
maintenance of proper homeostasis, dynamic control inresponse to
perturbations, or oscillatory patterns [1]. The defining feature of
a nega-tive feedback motif is an activator–inhibitor pair in which
the activator inducesexpression or activity of the inhibitor.
Indeed, many studies focus on the molecularmechanism(s) that
provide(s) inducibility, often characterized by the fold changeand
any intrinsic delay. However, actual molecular circuits within
cells are incom-pletely described by the activator-inducible
inhibitor paradigm, as they may needto contend with physical
realities within the cell such as the biochemistry of mol-ecular
interactions, sub-cellular compartmentalization or protein
half-life. Thus,proper functioning of a negative feedback circuit
may depend on biochemicalproperties other than the
activator-responsive control of the inhibitor.
IkBa is a prominent negative feedback regulator in the NFkB
signalling system[2,3]. IkBa directly controls the dynamics of the
transcription factor NFkB, a centralregulator of inflammatory and
immune response gene expression [4,5]. Through itsreversible
sequestration of NFkB in the cytoplasm, IkBa not only controls the
dur-ation of NFkB activity [4,6] but also enables reactivation that
can result in oscillatorydynamics observed both in population
studies [4] and in single cells [7].Mathematical models were shown
to recapitulate these dynamic features [8,9],and reduced models
have identified NFkB-responsive expression of IkBa as a
keydeterminant of oscillatory dynamics [10–12]. The dynamics of
NFkB signallingare stimulus-specific, and a critical determinant of
inflammatory and immunegene expression programmes [13,14],
prompting pioneering work to focusdrug-targeting strategies on
dynamical features to achieve superior specificity [15].
Here, we examine the molecular properties that confer IkBa’s
ability to con-trol NFkB dynamics. We find that while inducible
expression of IkBa isrequired for proper NFkB dynamics [16],
inducible expression is not sufficientas inducible expression of
another IkB family member, IkBb, is unable to
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Figure 1. (Caption overleaf.)
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support normal dynamical control of NFkB. This findingprompts us
to characterize other IkBa properties that arerequired for proper
negative feedback control of NFkB. Our
study delineates how several molecular properties combineto
produce the emergent systems property of dynamicnegative feedback
control of NFkB.
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Figure 1. (Overleaf.) NFkB-dependent transcriptional control is
not sufficient for IkB negative functions. (a) Schematic diagram of
pBabe and 5xkB retroviralexpression constructs consisting of a
tandem repeat of 5xkB sites driving the expression of IkBa. (b)
Electrophoretic mobility shift assay (EMSA) and immunoblotanalysis
of wt, IkBa2/2, IkBa2/2þ pBabe_IkBa, and IkBa2/2þ 5xkB_ IkBa murine
embryo fibroblast (MEF) cell lines. EMSA indicates NFkB
activityover a 120 min time course after stimulation with 1 ng ml21
of TNF; NFY binding was used as an EMSA control. Western blot shows
protein abundances for IkBa,IkBb and IkBe with a-tubulin as a
loading control. (c) IkBa2/2 MEFs reconstituted with NFkB-inducible
IkBa or IkBb were treated with 1 ng ml21 of TNFand nuclear extracts
analysed by EMSA for NFkB binding activity; NFY binding was used as
an EMSA control. Immunoblots of corresponding cytoplasmic
fractionswere probed with antibodies specific for IkBa, IkBb and
IkBe ; a-tubulin was used as a loading control. Densitometric
quantification of NFkB is presented as abar graph below. (d ) EMSA
and immunoblot analysis of wild-type MEFs and MEFs that have the
endogenous coding region for IkBa replaced by IkBb knock-in(AKBI).
Cells were treated with 1 ng ml21 of TNF and nuclear extracts
analysed by EMSA for NFkB-binding activity; NFY binding was used as
an EMSA control.Immunoblots of corresponding cytoplasmic fractions
were probed with antibodies specific for IkBa, IkBb and IkBe ;
a-tubulin was used as a loading control.Densitometric
quantification of NFkB is presented as a bar graph below. (e,f )
NFkB nuclear localization at the single-cell level.
IkBa2/2b2/2e2/2RelA2/2
MEFs reconstituted with AcGFP1-RelA and NFkB-inducible IkBs. (e)
IkBa cells were treated with 10 ng of TNF and fluorescent images
were captured every 5 minand cellular localization of AcGFP1-RelA
was measured and plotted as a normalized nuclear to cytoplasmic
ratio individually (bottom left colour traces) and as acombined
average (bottom right black trace). ( f ) IkBb cells were treated
with 10 ng of TNF and fluorescent images were captured every 5 min
and cellularlocalization of AcGFP1-RelA was measured and plotted as
a normalized nuclear to cytoplasmic ratio individually (bottom left
colour traces) and as a combinedaverage (bottom right black
trace).
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2. Results2.1. NFkB-responsive transcriptional control
is necessary but not sufficient for IkBanegative feedback
Studies of NFkB dynamic control by IkB family membershave
identified IkBa as the key negative feedback regulatordue to its
highly inducible NFkB-responsive promoter [2–4].To characterize the
role of NFkB-inducible expression, we com-plemented IkBa-deficient
murine embryo fibroblasts (MEFs)with retroviral plasmids that
express IkBa from either a constitu-tive (pBabe) or an
NFkB-inducible (5xkB) promoter (figure 1a).Unlike
pBabe-reconstituted cells, 5xkB_IkBa reconstituted cellsshowed
dynamic resynthesis profiles similar to endogenousIkBa in wild-type
cells following stimulation with tumour necro-sis factor (TNF)
(figure 1b). Importantly, when we examined thecontrol of NFkB
activity by electrophoretic mobility shift assay(EMSA), we found
that 5xkB cells showed post-induction repres-sion and the transient
trough of NFkB activity characteristic ofwild-type cells,
correcting the misregulation in IkBa-deficientcells, whereas cells
constitutively expressing IkBa were unableto capture this response
(figure 1b).
To test whether NFkB-inducible control was not onlyrequired but
also sufficient for NFkB dynamic control, wecomplemented
IkBa-deficient cells with a 5xkB retrovirusexpressing IkBb, a
highly homologous IkB family membercapable of inhibiting NFkB but
not normally providing nega-tive feedback. Interestingly, these
cells did not show properdynamic control of NFkB even though IkBb
expression wasunder NFkB control similar to IkBa (figure 1c;
electronic sup-plementary material, figure S1A). However, when
anotherknown IkB negative feedback regulator, IkBe [17], was
linkedto this promoter, NFkB activity did show
post-inductionrepression (electronic supplementary material, figure
S1B).These results indicate that, despite the high degree of
sequencehomology, IkBa and IkBb have distinct molecular
propertiesthat, along with differential gene expression control,
renderIkBa an effective negative feedback regulator but not IkBb.In
order to confirm the validity of this conclusion, we
obtainedfibroblasts from a genetic knock-in mouse in which the
IkBbcoding region was engineered to replace the IkBa open
readingframe such that IkBb expression was under the control of
theendogenous IkBa promoter [18]. Remarkably, these so-calledAKBI
cells also failed to show proper NFkB post-induction
attenuation despite highly inducible IkBb expression(figure 1d;
electronic supplementary material, figure S1c).
In order to examine translocation dynamics in singlecells, and
without the confounding contributions of other IkBfamily members,
we generated IkBa2/2b2/2e2/2RelA2/23T3 cells that lack all three
classical NFkB inhibitors and RelA,and reconstituted them with a
constitutively expressed fluor-escent GFP-RelA and
NFkB-responsively expressed IkBa orIkBb. Whereas reconstitution
with IkBa resulted in transientNFkB activation in response to TNF
treatment, defined by atrough at about 60 min, followed by a second
phase insome cells (figure 1e), reconstitution with
NFkB-inducibleIkBb resulted in sustained NFkB activation showing
only slowand incomplete post-induction repression (figure 1f ). Of
note,in both conditions, the mean RelA nuclear localization
profileof the collection of individual cells (figure 1e,f, black
trace) clo-sely resembled the population level in biochemical
studies(figure 1c,d). These data clearly indicate that when
expressionof IkBa or IkBb is driven by the same NFkB-responsive
promo-ter, resulting in ostensibly similar expression profiles,
only IkBacan provide effective dynamic negative feedback control
onNFkB. Thus, inducible inhibitor expression in and of itself isnot
sufficient for proper negative feedback control of NFkB.
2.2. Mathematical modelling identifies multiplemolecular
properties of IkBa contributingto the negative feedback control of
NFkB
IkBa has several characteristics—other than
NFkB-dependentsynthesis—that in principle may contribute to its
negative feed-back function, e.g. its nuclear import and export
properties, aswell as constitutive and signal-induced degradation
of free andNFkB-bound IkBa (figure 2a). Here, we use a previously
estab-lished in silico model of NFkB regulation to investigate
thecontributions of each of these processes to the control
ofdynamic NFkB signals. When normalized for maximumactivity, we
confirmed that partial inhibition of the NFkB-dependent synthesis
of IkBa potently impaired post-inductionattenuation, with 10%
inhibition resulting in a 30% increase inthe signalling level at 70
min (figure 2b, row 1, and 2c). How-ever, we also found that
partial inhibition of IkBa nuclearimport had a similar effect with
a 10% inhibition causing an11% increase in signalling at 70 min
(figure 2b, row 3). Weakinhibition of the degradation of free IkBa
had little effect onpost-attenuation induction, whereas stronger
inhibition shifted
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Figure 2. Modelling IkBa properties contributing to negative
feedback control of NFkB. (a) Schematic illustrates negative
feedback control of NFkB by IkBs.Numbers indicate potential
reactions that may contribute to dynamic regulation of NFkB. (b)
Normalized nuclear NFkB concentration is shown for
unperturbedmodels (black) and models in which the indicated
reactions are partially inhibited (blue-red lines reflect various
degrees of inhibition). Reaction numbers as in (a).(c) Sensitivity
analysis of three temporal phases of the NFkB with respect to
changes in IkBa regulation. ((i) – (iii)) Sensitivity ratio
(nucNFkBperturbed 2 nucNFkBunperturbed)/nucNFkBunperturbed in per
cent units for each reaction (numbers as in (a)). (iv) Global
sensitivity (RMSD) integrated over 120 min. Results are shown
forthree levels of inhibition (10%, twofold and 10-fold).
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the peak of NFkB to later times resulting in a modest increase
inlate activity (90% inhibition caused 14% increase in signallingat
120 min, figure 2b, row 2). Partial inhibition of nuclearexport or
of signal-induced degradation of NFkB-bound IkBaalso reduced the
post-attenuation reactivation, with a 10% inhi-bition causing 1%
and 9.5% decreased signalling at 120 min,respectively (figure 2b,
rows 4 and 5).
To compare the contribution of these processes, we deter-mined
the sensitivity of NFkB activity at three specific
timesrepresenting early, post-induction attenuation and late
partsof the signal to various perturbations (figure 2c). We also
quan-tified the global sensitivity to each perturbation as the
rootmean square deviation (RMSD) of the perturbed and unper-turbed
signals over 120 min, sampled at 1 min intervals
(figure 2c(iv)). This analysis posits that
IkBa-mediatedpost-induction attenuation of NFkB activity (figure
2c(ii)) is afunction not only of the NFkB-dependent synthesis
ratebut also of IkBa’s nuclear import, as well as its
constitutivedegradation. It also predicts that nuclear export and
IKK-dependent degradation of NFkB-bound IkBa are importantfor late
post-attenuation signalling (figure 2c(iii)).
2.3. Experimental testing of model predictions: multipleIkBa
properties contribute distinct characteristicsto NFkB dynamic
control
To test the computational predictions, we pursued a
geneticperturbation approach. Previous work showed nuclear
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import of IkBa to be mediated by an unconventional NLSsequence
[19,20]. Using this information, we reconstitutedIkBa-deficient
cells with an IkBa NLS mutant (IkBaNLSm:L110A,L115A,L117A,L120A).
In DNA binding studies ofIkBaNLSm cells, TNF induced NFkB
activation comparableto that of wild-type IkBa cells (figure 3a),
and although theresynthesis of IkBaNLSm protein was effectively
inducedby NFkB, the IkBaNLSm cells were defective for the
rapidpost-induction repression of NFkB. At 70 min, the signalin
IkBaNLSm cells was 2.9-fold higher than in wild-typeIkBa cells
(considering the different basal levels). This is con-sistent with
the threefold increase predicted by the modelwhen the corresponding
parameter is reduced to 35% of itswild-type value (figure 3a;
electronic supplementary material,figure S2a). Consistent with
population-level biochemicalstudies, IkBa2/2b2/2e2/2RelA2/2 cells
expressing GFP-RelAshowed that IkBaNLSm was defective in
post-inductionrepression and cytoplasmic relocalization of NFkB in
singlecells (figure 3b). Despite the substantial heterogeneity
inRelA cytoplasmic re-localization, the population mean
closelyresembles the population-level results obtained by
EMSA.These data clearly show that the nuclear localization of
IkBais indispensable for proper termination of NFkB activity.
IkBa also relies on a nuclear export sequence (NES) forthe
efficient nuclear export of NFkB [21,22]. Mathematicalmodelling
suggested strong inhibition of nuclear exportwould result in
reduced late NFkB activity. To assess therole of IkBa nuclear
export on the sub-cellular localizationcontrol of NFkB, we
generated an NES mutant (IkBaNESm;L45A,L49A,I52A). Reconstituted
cells expressing IkBaNESmwere unable to produce the post-repression
reactivation ofNFkB characteristic of wild-type protein-controlled
NFkB sig-nalling (figure 3c; electronic supplementary material,
figureS2b). The activity is qualitatively similar to the model
predic-tion for a fivefold attenuation in the
correspondingparameter. These results indicate that the nuclear
export func-tion of IkBa is crucial for the post-repression
activation ofNFkB signalling. Interestingly, the single-cell
studies withthe NES mutant revealed seemingly contradictory
results(figure 3d ), as most cells displayed sustained RelA
nuclearlocalization. This apparent discrepancy is resolved by
recog-nizing that: (1) the mutant localizes to the nucleus
causinginhibition of NFkB activity but does not allow for
NFkBexport and reactivation, and (2) the biochemical assay
detectsDNA binding activity of free NFkB, whereas the
single-cellimaging is a readout of NFkB localization only.
IkBa is known to have a very short half-life that is exten-ded
approximately twofold by an IkBa5M mutant
(S283A,S288,T291A,S293A,T296A) [23,24]. When expressed inIkBa2/2
cells from an NFkB-responsive promoter, IkBa5Machieved effective
post-induction repression of NFkB activity(figure 3e; electronic
supplementary material, figure S2c). How-ever, the re-activation of
NFkB was undetectable, consistent withcomputational predictions
that indicated a signal close to basallevel at 120 min when the
corresponding parameter was reducedto 35% of its wild-type value.
Similarly, in single live-cell studies,IkBa5M mediated efficient
relocalization of RelA to the cyto-plasm with a complete absence of
late-phase activity (figure 3f ).
3. DiscussionGiven the well-documented role of NFkB activity in
vitalcellular processes, a number of mechanisms have evolved
to ensure precise regulation of its activity. The IkBa nega-tive
feedback loop is a prominent NFkB regulatorymechanism, allowing for
both post-induction repressionand repeated or oscillatory bursts of
activity, and is criticalfor providing complex dynamic control
which is thoughtto mediate specificity in NFkB’s pleiotropic
physiologicalfunctions. Prior studies have established that the
NFkB-responsive IkBa promoter is critical for this negative
feedbackcontrol [16], but it has remained unclear whether specific
charac-teristics of the IkBa protein may be important as well.
Indeed,biochemical studies presented here using MEFs derived
frommice in which IkBb was engineered into the IkBa locus to(AKBI
MEFs) clearly demonstrate that, even when IkBb isunder
NFkB-transcriptional induction, it is unable to provideproper
negative feedback. These data motivated our characteriz-ation of
IkBa protein properties that contribute to propernegative feedback
function. Our strategy was to complementIkBa2/2 cells with
retroviral transgenes providing for kB-responsive expression of
engineered IkBa variants defective inspecific molecular
characteristics.
In addition to traditional biochemical approaches to studyNFkB
response and regulation, we examined NFkB responsedynamics in
single cells. Recent studies have characterizedNFkB dynamics in
single cells, but, to date, no studies haveemployed gene knock-out
cells to probe underlying molecu-lar mechanisms. Thus, regulatory
control mechanismsidentified at the biochemical/population level
have yet tobe reconciled with single-cell microscopy tracking
studiesthat boast high temporal resolution and individual
cellularhistories. In this work, we employed a cell line
lackingRelA and all canonical IkB proteins (IkBa2/2IkBb2/2
IkBe2/2RelA2/2 cells), which we then reconstituted
withNFkB-inducible IkB variants and fluorescent RelA repor-ter in
order to examine the contributions of specific IkBprotein
characteristics.
Although the IkB proteins were first identified as cyto-plasmic
inhibitors, it has become clear that they play amajor role in
regulating nuclear NFkB. IkBa has beenshown to be efficiently
transported into the nucleus whereit binds active NFkB dimers on
the promoters of NFkB-activated genes and facilitates the
dissociation of thetranscription factor from DNA. Comparing mutants
withwild-type IkB proteins, we were able to show the contri-butions
of IkB inducible synthesis, nucleo-cytoplasmictransport and
degradation control to the various aspectsof the prototypical NFkB
response; namely, duration andamplitude of initial NFkB activation,
post-activation repres-sion and post-repression re-activation of
NFkB signalling.Our biochemical assays, together with single-cell
studies,demonstrated that IkBa nuclear localization is
indispensablefor the rapid termination of NFkB activity.
Specifically, weshowed that an NES-deficient form of IkBa supported
effi-cient induction and post-induction repression of NFkBDNA
binding activity, but not the characteristic re-activationand
second phase NFkB activity. The deficiency in NES func-tion
prevents the protein from efficiently returning NFkB tothe
cytoplasm for the next round of activation, maintainingan inactive
IkBaNESm-bound pool of NFkB in the nucleus.Finally, by employing an
IkBa harbouring five mutationsthat confer stability, increasing the
half-life of the normallyrapidly turned over uncomplexed/free
protein, we wereable to show the importance of such rapid turnover
ingenerating characteristic NFkB temporal profiles. In cells
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3t3 abe–/–RelA–/– + 5xkB_IkBa NESm + GFP-RelA
3t3 abe–/–RelA–/– + 5xkB_IkBa5M + GFP-RelA
Figure 3. Multiple IkBa properties contribute distinct
characteristics to NFkB control. Nuclear localization of IkBa is
required for the termination of NFkB activity (a,b).Nuclear export
function of IkBa is required for post-repression activation of NFkB
activity (c,d ). IkBa protein half-life control is critical for
sustained NFkB dynamics (e,f ).IkBa2/2 MEFs were reconstituted with
NFkB-inducible wild-type or NLS mutant (a,b), NES mutant (c,d ) or
the 5M mutant (e,f ) form of IkBa. The cells were treatedwith 1 ng
ml21 of TNF and nuclear extracts were analysed by EMSA for NFkB
activity and corresponding cytoplasmic extracts subjected to
western blotting with indicatedantibodies (a,c,e). Bar graphs show
quantification of EMSA; curves are modelling the result of NFkB
activity in single cells upon stimulation with 10 ng ml21 of
TNF.Real-time fluorescent images of IkBa2/2b2/2e2/2RelA2/2 MEFs
reconstituted with AcGFP1-RelA and NFkB-inducible IkBa NLS mutant
(b), NES mutant (d ) orthe 5M mutant ( f ) IkBa (showing cellular
localization of RelA at indicated time points). Below the
fluorescent images, single-cell traces show the ratio of nuclear
tocytoplasmic localization of AcGFP1-RelA in fluorescent images
(left) as well as the average curve and standard deviation of the
single-cell traces (right).
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expressing this IkBa5M form, we found a complete absenceof
second-phase activation, indicating that a low level offree IkBa
protein (ensured by a short half-life) is requiredfor this aspect
of the response.
Our results demonstrate not only that IkBa feedbackis dependent
on NFkB-inducible synthesis but also thatseveral other processes
dependent on the molecular charac-teristics of the protein itself,
for example import, exportand half-life control, must be tuned in a
coordinatedmanner to generate the hallmark features of NFkB
signal-ling, namely post-induction repression and reactivation.By
contrast, the IkBb protein does not support proper nega-tive
feedback control even when expressed from IkBa’spromoter; we
speculate that substantially reduced nucleo-cytoplasmic transport
[25,26] may be a key underlyingreason; a second characteristic that
may play a role isIkBa’s but not IkBb’s ability to strip NFkB off
the DNA[27]. Indeed, these properties are not required for theNFkB
dimer stabilization/chaperone function recentlyascribed to IkBb
[28]. Our results illustrate the more generalpoint: that negative
feedback regulation in cells is a complexprocess that depends on
multiple molecular propertiesbeyond activator-induced expression
[29,30]. These findingsmay well extend to other transcriptional
networks asnuclear transport is a defining feature of many gene
regulat-ory networks. Understanding the specific contributions
ofeach process as well as their characteristic time scales isan
important step for identifying effective druggable targetsthat may
allow for correction of dynamic misregulation incells associated
with pathology [15].
4. Material and methods4.1. Computational modellingThe response
of the NFkB regulatory module was simulated usingthe computational
ODE-based model described in [31]. In order tofocus on regulatory
mechanisms involving IkBa, the other IkBfamily members were
removed. Following equilibration, TNFresponses were simulated as in
[31]. Time-course curves infigure 2 were generated by applying
multipliers to the kinetic par-ameters corresponding to the
reactions in figure 2a. The multipliervalues were:
223,22.5,22,21.5,21,20.5 (reactions 1, 2, 3 and 5)
and226,25,24,23,22,21 (reaction 4), reflecting different
sensitivities forreaction 4. NFkB time courses are normalized to
their peakvalue. Sensitivity ratios sr(t) at a particular time ti
are definedas: (nucNFkBperturbed 2
nucNFkBunperturbed)/nucNFkBunperturbed,where
nucNFkBperturbed/unperturbed are the normalized
nuclearconcentrations of NFkB at time ti obtained with a model
with/without a multiplicative factor (0.9, 0.5, 0.1) for the
indicatedkinetic rate parameter (values shown in per cent units).
Theglobal average sensitivity in figure 2c was calculated as the
RMSof the sr(t) sampled at 1 min intervals between 1 and 120
minpost-stimulation.
4.2. DNA constructsNFkB-inducible IkBa and IkBb constructs were
generated in theself-inactivating (SIN) retrovirus backbone
(HRSpuro) modifiedto express the IkBa or IkBb transgene under the
control of fivetandem kB sites upstream of a minimal promoter. IkBa
mutantforms were produced using site-directed mutagenesis. For
live-cell studies, AcGFP1 was fused to the N-terminus of RelA
andthe resulting construct was sub-cloned into the
constitutivelyexpressing retroviral plasmid pBabe-Hygro.
4.3. Cells and cell cultureImmortalized IkBa2/2 MEFs were
previously described [4] andIkBa2/2b2/2e2/2RelA2/2 MEFs were
produced by interbreed-ing of the four individual mouse knock-out
strains andharvesting E13.5 embryos, subjecting primary MEFs to the
3T3protocol of repeated passage until a stably proliferating cell
cul-ture emerged. AKBI MEFs were a generous gift from BingBingJiang
(Boston University). MEFs were cultured in Dulbecco’smodified
Eagle’s medium supplemented with 100 U penicillin/streptomycin
(10378016; Life Technologies), 0.3 mg ml21 gluta-mine and 10% fetal
calf serum (complete medium). Plat-E cells[32] were maintained in
complete medium containing blasticidin(10 mg ml21) and puromycin (1
mg ml21).
4.4. Retrovirus-mediated gene transductionNFkB-inducible IkB and
AcGFP1-RelA constructs were trans-fected into Plat-E packaging
cells pre-conditioned in antibiotic-free complete medium using
poly(ethylenimine). Supernatantwas collected 48 h
post-transfection, filtered and used to infecttarget cells with 4
mg ml21 polybrene to enhance infection effi-ciency (Sigma).
Infected cells were selected with puromycinhydrochloride (Sigma)
for IkBs and/or with hygromycin B (Invi-voGen) for the AcGFP1-RelA.
Murine TNF (Roche) was used at 1or 10 ng ml21.
4.5. Biochemical analysesWhole-cell extracts were prepared in
radioimmunoprecipitationassay buffer with protease inhibitors and
normalized for totalprotein before immunoblot analyses. Cytoplasmic
and nuclearextracts for immunoblot analyses and EMSA, respectively,
wereprepared as previously described [4,24]. IkBa was probed
withsc-371, IkBb with sc-945 and a-tubulin with sc-5286.
Allantibodies were from Santa Cruz Biotechnology.
4.6. MicroscopyCells were plated onto 35 mm glass bottom dishes
(MatTek) oriBidi eight-well chambers (iBidi) 24 h prior to
stimulation andimmediate imaging. Images were acquired on an Axio
ObserverZ1 inverted microscope (Carl Zeiss Microscopy GmbH,Germany)
with a 40�, 1.3 NA oil-immersion, or 20�, 0.8 NAair-immersion
objective to a Coolsnap HQ2 CCD camera (Photo-metrics, Canada)
using ZEN imaging software (Carl ZeissMicroscopy GmbH, Germany).
Environmental conditions weremaintained in a humidified chamber at
378C, 5% CO2 (Pecon,Germany). Quantitative image processing was
performed usingthe FIJI distribution of IMAGE J (NIH). All cells of
each frame inthe microscope imaging experiments were measured for
totalfluorescence intensity. Time-course data were normalized bythe
minimum and maximum values to account for the varyingoverall
intensities of different cells. The single-cell traces wereaveraged
and error bars in the mean curves are the standarddeviation from
the mean.
Authors’ contributions. R.F. performed the experimental work,
assisted byK.T.F., Y.E.L. and J.D.V. M.B. performed the
computational model-ling. R.F., J.D.V. and A.H. wrote the
manuscript.Competing interests. We have no competing
interests.Funding. The work was supported by grants to A.H. from
the NIH: R01GM071573 and P01 GM071862. R.F. was a Sigrid Juselius
Foundationand Saatioiden postdoctoral fellow and M.B. was a Cancer
ResearchInstitute postdoctoral fellow.Acknowledgements. We thank
Bingbing Jiang (Boston University) for thegenerous gift of AKBI
MEFs and acknowledge Santa Cruz Biotech-nology for their
support.
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8
References
rsif.royalsocietypublishing.orgJ.R.Soc.Interface
12:20150262
1. Alon U. 2007 An introduction to systems biology:design
principles of biological circuits. Boca Raton,FL: Chapman and
Hall/CRC.
2. Scott ML, Fujita T, Liou HC, Nolan GP, Baltimore D.1993 The
p65 subunit of NF-kappa B regulates Ikappa B by two distinct
mechanisms. Genes Dev. 7,1266 – 1276.
(doi:10.1101/gad.7.7a.1266)
3. Chiao PJ, Miyamoto S, Verma IM. 1994 Autoregulationof I kappa
B alpha activity. Proc. Natl Acad. Sci. USA 91,28 – 32.
(doi:10.1073/pnas.91.1.28)
4. Hoffmann A, Levchenko A, Scott ML, Baltimore D.2002 The
IkappaB-NF-kappaB signaling module:temporal control and selective
gene activation.Science 298, 1241 – 1245.
(doi:10.1126/science.1071914)
5. Hoffmann A, Baltimore D. 2006 Circuitry of nuclearfactor
kappaB signaling. Immunol. Rev. 210,171 – 186.
(doi:10.1111/j.0105-2896.2006.00375.x)
6. Shih VF, Kearns JD, Basak S, Savinova OV, Ghosh G,Hoffmann A.
2009 Kinetic control of negativefeedback regulators of
NF-kappaB/RelA determinestheir pathogen- and cytokine-receptor
signalingspecificity. Proc. Natl Acad. Sci. USA 106, 9619 –
9624.(doi:10.1073/pnas.0812367106)
7. Nelson DE et al. 2004 Oscillations in NF-kappaBsignaling
control the dynamics of gene expression.Science 306, 704 – 708.
(doi:10.1126/science.1099962)
8. O’Dea E, Hoffmann A. 2010 The regulatory logic ofthe
NF-kappaB signaling system. Cold Spring Harb.Perspect. Biol. 2,
a000216. (doi:10.1101/cshperspect.a000216)
9. Basak S, Behar M, Hoffmann A. 2012 Lessons frommathematically
modeling the NF-kappaB pathway.Immunol. Rev. 246, 221 – 238.
(doi:10.1111/j.1600-065X.2011.01092.x)
10. Krishna S, Jensen MH, Sneppen K. 2006 Minimalmodel of spiky
oscillations in NF-kappaB signaling.Proc. Natl Acad. Sci. USA 103,
10 840 – 10 845.
11. Hayot F, Jayaprakash C. 2006 NF-kappaB oscillationsand
cell-to-cell variability. J. Theor. Biol. 240,583 – 591.
(doi:10.1016/j.jtbi.2005.10.018)
12. Mothes J, Busse D, Kofahl B, Wolf J. 2015 Sourcesof dynamic
variability in NF-kappaB signaltransduction: a mechanistic model.
BioEssays 37,452 – 462. (doi:10.1002/bies.201400113)
13. Werner SL, Barken D, Hoffmann A. 2005 Stimulusspecificity of
gene expression programs determined
by temporal control of IKK activity. Science 309,1857 – 1861.
(doi:10.1126/science.1113319)
14. Behar M, Hoffmann A. 2010 Understanding thetemporal codes of
intra-cellular signals. Curr. Opin.Genet. Dev. 20, 684 – 693.
(doi:10.1016/j.gde.2010.09.007)
15. Behar M, Barken D, Werner SL, Hoffmann A. 2013The dynamics
of signaling as a pharmacologicaltarget. Cell 155, 448 – 461.
(doi:10.1016/j.cell.2013.09.018)
16. Werner SL, Kearns JD, Zadorozhnaya V, Lynch C, O’DeaE,
Boldin MP, Ma A, Baltimore D, Hoffmann A. 2008Encoding NF-kappaB
temporal control in responseto TNF: distinct roles for the negative
regulatorsIkappaBalpha and A20. Genes Dev. 22, 2093 –
2101.(doi:10.1101/gad.1680708)
17. Kearns JD, Basak S, Werner SL, Huang CS, HoffmannA. 2006
IkappaBepsilon provides negative feedbackto control NF-kappaB
oscillations, signalingdynamics, inflammatory gene expression. J.
CellBiol. 173, 659 – 664. (doi:10.1083/jcb.200510155)
18. Cheng JD, Ryseck RP, Attar RM, Dambach D, BravoR. 1998
Functional redundancy of the nuclear factorkappa B inhibitors I
kappa B alpha and I kappa Bbeta. J. Exp. Med. 188, 1055 – 1062.
(doi:10.1084/jem.188.6.1055)
19. Sachdev S, Bagchi S, Zhang DD, Mings AC, HanninkM. 2000
Nuclear import of IkappaBalpha isaccomplished by a ran-independent
transportpathway. Mol. Cell Biol. 20, 1571 – 1582.
(doi:10.1128/MCB.20.5.1571-1582.2000)
20. Sachdev S, Hoffmann A, Hannink M. 1998 Nuclearlocalization
of IkappaB alpha is mediated by thesecond ankyrin repeat: the
IkappaB alpha ankyrinrepeats define a novel class of cis-acting
nuclearimport sequences. Mol. Cell Biol. 18, 2524 – 2534.
21. Huang TT, Kudo N, Yoshida M, Miyamoto S. 2000 Anuclear
export signal in the N-terminal regulatorydomain of IkappaBalpha
controls cytoplasmiclocalization of inactive
NF-kappaB/IkappaBalphacomplexes. Proc. Natl Acad. Sci. USA 97,1014
– 1019. (doi:10.1073/pnas.97.3.1014)
22. Huang TT, Miyamoto S. 2001 Postrepressionactivation of
NF-kappaB requires the amino-terminal nuclear export signal
specific toIkappaBalpha. Mol. Cell Biol. 21, 4737 –
4747.(doi:10.1128/MCB.21.14.4737-4747.2001)
23. Mathes E, O’Dea EL, Hoffmann A, Ghosh G. 2008NF-kappaB
dictates the degradation pathway ofIkappaBalpha. EMBO J. 27, 1357 –
1367. (doi:10.1038/emboj.2008.73)
24. O’Dea EL, Kearns JD, Hoffmann A. 2008 UV as anamplifier
rather than inducer of NF-kappaB activity.Mol. Cell 30, 632 – 641.
(doi:10.1016/j.molcel.2008.03.017)
25. Chen Y, Wu J, Ghosh G. 2003 KappaB-Ras binds tothe unique
insert within the ankyrin repeat domainof IkappaBbeta and regulates
cytoplasmic retentionof IkappaBbeta�NF-kappaB complexes. J.
Biol.Chem. 278, 23 101 – 23 106. (doi:10.1074/jbc.M301021200)
26. Malek S, Chen Y, Huxford T, Ghosh G. 2001IkappaBbeta, but
not IkappaBalpha, functionsas a classical cytoplasmic inhibitor of
NF-kappaBdimers by masking both NF-kappaB nuclearlocalization
sequences in resting cells. J. Biol.Chem. 276, 45 225 – 45 235.
(doi:10.1074/jbc.M105865200)
27. Bergqvist S, Alverdi V, Mengel B, Hoffmann A,Ghosh G,
Komives EA. 2009 Kinetic enhancement ofNF-kappaBxDNA dissociation
by IkappaBalpha. Proc.Natl Acad. Sci. USA 106, 19 328 – 19 333.
28. Tsui R, Kearns JD, Lynch C, Vu D, Ngo KA, Basak S,Ghosh G,
Hoffmann A. 2015 IkappaBbeta enhancesthe generation of the
low-affinity NFkappaB/RelAhomodimer. Nat. Commun. 6, 7068.
(doi:10.1038/ncomms8068)
29. Nguyen LK, Kulasiri D. 2009 On the functionaldiversity of
dynamical behaviour in genetic andmetabolic feedback systems. BMC
Syst. Biol. 3, 51.(doi:10.1186/1752-0509-3-51)
30. Nguyen LK. 2012 Regulation of oscillation dynamicsin
biochemical systems with dual negative feedbackloops. J. R. Soc.
Interface 9, 1998 – 2010. (doi:10.1098/rsif.2012.0028)
31. Mukherjee SP, Behar M, Birnbaum HA, Hoffmann A,Wright PE,
Ghosh G. 2013 Analysis of the RelA:CBP/p300 interaction reveals its
involvement inNF-kappaB-driven transcription. PLoS Biol.
11,e1001647. (doi:10.1371/journal.pbio.1001647)
32. Morita S, Kojima T, Kitamura T. 2000 Plat-E: anefficient and
stable system for transient packagingof retroviruses. Gene Ther. 7,
1063 – 1066. (doi:10.1038/sj.gt.3301206)
http://dx.doi.org/10.1101/gad.7.7a.1266http://dx.doi.org/10.1073/pnas.91.1.28http://dx.doi.org/10.1126/science.1071914http://dx.doi.org/10.1126/science.1071914http://dx.doi.org/10.1111/j.0105-2896.2006.00375.xhttp://dx.doi.org/10.1073/pnas.0812367106http://dx.doi.org/10.1126/science.1099962http://dx.doi.org/10.1101/cshperspect.a000216http://dx.doi.org/10.1101/cshperspect.a000216http://dx.doi.org/10.1111/j.1600-065X.2011.01092.xhttp://dx.doi.org/10.1111/j.1600-065X.2011.01092.xhttp://dx.doi.org/10.1016/j.jtbi.2005.10.018http://dx.doi.org/10.1002/bies.201400113http://dx.doi.org/10.1126/science.1113319http://dx.doi.org/10.1016/j.gde.2010.09.007http://dx.doi.org/10.1016/j.gde.2010.09.007http://dx.doi.org/10.1016/j.cell.2013.09.018http://dx.doi.org/10.1016/j.cell.2013.09.018http://dx.doi.org/10.1101/gad.1680708http://dx.doi.org/10.1083/jcb.200510155http://dx.doi.org/10.1084/jem.188.6.1055http://dx.doi.org/10.1084/jem.188.6.1055http://dx.doi.org/10.1128/MCB.20.5.1571-1582.2000http://dx.doi.org/10.1128/MCB.20.5.1571-1582.2000http://dx.doi.org/10.1073/pnas.97.3.1014http://dx.doi.org/10.1128/MCB.21.14.4737-4747.2001http://dx.doi.org/10.1038/emboj.2008.73http://dx.doi.org/10.1038/emboj.2008.73http://dx.doi.org/10.1016/j.molcel.2008.03.017http://dx.doi.org/10.1016/j.molcel.2008.03.017http://dx.doi.org/10.1074/jbc.M301021200http://dx.doi.org/10.1074/jbc.M301021200http://dx.doi.org/10.1074/jbc.M105865200http://dx.doi.org/10.1074/jbc.M105865200http://dx.doi.org/10.1038/ncomms8068http://dx.doi.org/10.1038/ncomms8068http://dx.doi.org/10.1186/1752-0509-3-51http://dx.doi.org/10.1098/rsif.2012.0028http://dx.doi.org/10.1098/rsif.2012.0028http://dx.doi.org/10.1371/journal.pbio.1001647http://dx.doi.org/10.1038/sj.gt.3301206http://dx.doi.org/10.1038/sj.gt.3301206
Anatomy of a negative feedback loop: the case of
I[kappa]B[alpha]IntroductionResultsNF[kappa]B-responsive
transcriptional control is necessary but not sufficient for
I[kappa]B[alpha] negative feedbackMathematical modelling identifies
multiple molecular properties of I[kappa]B[alpha] contributing to
the negative feedback control of NF[kappa]BExperimental testing of
model predictions: multiple I[kappa]B[alpha] properties contribute
distinct characteristics to NF[kappa]B dynamic control
DiscussionMaterial and methodsComputational modellingDNA
constructsCells and cell cultureRetrovirus-mediated gene
transductionBiochemical analysesMicroscopyAuthors’
contributionsCompeting interestsFunding
AcknowledgementsReferences