A Multi-Scale Model of Hepcidin Promoter Regulation Reveals Factors Controlling Systemic Iron Homeostasis
[['Hepcidin is a humoral polypeptide that plays a central role in systemic iron homeostasis ( reviewed in [1] ) .', 'One main function of hepcidin is to maintain constant levels of iron circulating in the blood despite imbalances in external iron availability: Iron overload in the blood stimulates hepcidin transcription in hepatocytes .', 'Hepcidin in turn blocks intestinal iron uptake and macrophage iron release into the blood by binding to the iron exporter ferroportin and triggering its degradation .', 'Thus , hepcidin is part of a negative feedback circuit that stabilizes the iron blood concentration .', 'Negative feedback is known to play a key role for the robustness and homeostasis of biochemical networks ( reviewed in [2] ) .', 'Biochemical negative feedbacks have been shown to compensate for various perturbations including stochasticity in gene expression [3] , [4] , mutations [5] and pharmacological inhibition [6] , [7] .', 'In systemic iron homeostasis , iron diet content affects the iron concentration in the blood [8] , [9] , suggesting that the hepcidin feedback loop only partially compensates for perturbations .', 'Nevertheless , genetic perturbations of iron-dependent hepcidin regulation result in hemochromatosis , a common hereditary disease [10] .', 'At the molecular level , hereditary hemochromatosis ( HH ) is caused by inappropriately low hepcidin expression and an inability to compensate changes in iron blood levels .', 'HH patients are characterized by chronic iron overload that causes organ damage such as liver fibrosis .', 'Hepcidin expression is primarily controlled at the transcriptional level .', 'Information about iron blood levels is transduced from the hepatocyte cell membrane to the nucleus by the bone morphogenetic protein ( BMP ) signalling pathway [11] , [12]: Increasing iron blood concentrations are sensed by Hfe and Tfr2 , two transmembrane proteins mutated in HH [13] , [14] .', 'The signal is transmitted by the BMP co-receptor HJV and BMP receptor 1 to trigger the phosphorylation and nuclear translocation of SMAD1/5/8 transcription factors ( referred to as SMADs hereafter ) .', 'BMP6 is regulated by hepatic iron levels and plays a critical role in this process [15] , [16] .', 'The hepcidin promoter contains two BMP-responsive elements ( BRE1 and BRE2 ) that are recognized by the SMADs ( sometimes also abbreviated as BMP-RE1 and BMP-RE2 ) [17]–[19] .', 'Mutations in the BRE1 promoter element and in the BMP signalling pathway are associated with HH [20] .', 'Hepcidin expression is also regulated by inflammatory cytokines and hypoxia [21] , [22] .', 'Inflammatory cytokines such as IL6 activate the STAT3 signaling pathway in hepatocytes .', 'Phosphorylated STAT3 transcription factors ( TFs ) are directly recruited to a STAT binding site ( STATBS ) in the hepcidin promoter , thereby enhancing hepcidin expression and reducing iron blood levels [23] , [24] .', 'Chronic inflammation causes an iron-related disorder , known as anemia of inflammation ( AI ) , because the persistent lack of iron availability blocks erythropoiesis [10] .', 'This indicates that the integration of BMP and IL6 signals at the level of hepcidin expression plays a key role in systemic iron homeostasis .', 'Combinatorial gene regulation by binding of multiple different transcription factors to the same promoter is a recurrent aspect of transcription networks .', 'Thermodynamic modeling employs methods from statistical thermodynamics to describe combinatorial binding of transcription factors and RNA polymerase ( RNAP ) to the promoter , depending on the protein concentrations and binding energies .', '[25]–[32] .', 'The modeling framework focusses on transcription initiation and is based on the assumption that gene activity is determined by RNAP recruitment to the transcription start site ( TSS ) .', 'Thermodynamic modeling has been shown to accommodate various modes of signal integration on a promoter [25] , [26] , [29] , some of which have been confirmed experimentally for bacterial and yeast promoters [33]–[36] .', 'More recently , the framework was extended to aspects of eukaryotic gene regulation , including nucleosome positioning effects [37] , [38] .', 'In this work , we combined experimental measurements and thermodynamic modeling to quantitatively analyze how the iron-sensing BMP and inflammatory IL6 pathways coordinately control hepcidin expression .'], ['Systemic iron homeostasis is maintained by an auto-regulatory negative feedback loop that involves the transcriptional induction of hepcidin in the liver: elevated iron levels in the blood induce hepcidin expression by activating the BMP signaling pathway in the liver .', 'Released hepcidin in turn induces the degradation of intestinal iron transporters , thereby lowering the blood iron level .', 'We analyzed a conceptual mathematical model of this circuitry to understand how systemic iron homeostasis can be maintained despite imbalances in iron availability and consumption ( Supplemental Text S1 ) .', 'The model suggests that the regulatory loop most potently balances iron blood levels if hepcidin expression responds in a steep , nonlinear manner to alterations in iron blood levels .', 'Robust homeostasis further requires that the hepcidin promoter is able to sense and to respond to a broad range of iron blood concentrations .', 'We therefore reasoned that data of dose-dependent hepcidin promoter regulation , and its modulation by inflammatory IL6 stimulation , could provide valuable insights into the regulation of iron homeostasis .', 'Hepatic cell culture systems do not directly respond to stimulation with extracellular iron [39] .', 'BMP6 is involved in hepatic iron-sensing in vivo , and is commonly used as an external stimulus to characterize how hepcidin expression responds to changes in iron blood levels in vitro [15] , [16] .', 'We performed co-stimulation experiments with BMP6 and IL6 in human hepatoma ( HuH7 ) cells , and measured the activity of a luciferase reporter gene driven by the hepcidin promoter 24 h after stimulation .', 'The hepcidin expression response of HuH7 cells was characterized in previous studies , and it was concluded that HuH7 cells reflect known features of hepcidin expression in vivo ( see Discussion ) .', 'Reporter gene assays were performed in HuH7 cells transiently expressing luciferase constructs under control of wildtype ( WT ) or mutant hepcidin promoters ( Fig . 1B and C ) .', 'The WT promoter spans 3 kb upstream of the transcription start site , and contains a proximal STAT-binding site ( STATBS ) , a nearby BMP-responsive element ( BRE1 ) , and a distal BMP-responsive element ( BRE2 ) .', 'We and others previously showed that these sequence motifs are necessary and sufficient for responsiveness towards BMP and IL6 stimulation ( see Discussion ) .', 'In each of the three promoter mutants , one of these transcription factor binding sites is non-functional: The BRE1m and BRE2m constructs exhibit point mutations in the corresponding BMP-responsive elements , while STATdel is characterized by a complete deletion of the STAT-binding site .', 'For simplicity , we will generally refer to transcription binding site mutations even when discussing the deletion of the STATBS .', 'The raw luciferase activity reads of at least four biological replicates were processed ( see Methods ) and the merged data is shown in Fig . 1C ( using the same arbitrary concentration units for all heatmaps ) .', 'BMP6 and IL6 mono-stimulation both increased the luciferase activities of the WT promoter construct .', 'Maximal IL6 stimulation enhanced luciferase activities by 20-fold , while a 450-fold increase was observed upon maximal BMP stimulation .', 'The IL6 response saturated within the concentration range used , because IL6 concentrations beyond 6 ng/ml hardly increased expression any further .', 'Thus , BMP6 increased expression much more efficiently than IL6 in terms of maximal possible inducibility .', 'Co-stimulation with IL6 and BMP further enhanced the luciferase activity of the WT promoter compared to mono-stimulation .', 'We confirmed by qPCR measurements that this co-stimulation response quantitatively reflects the expression of endogenous hepcidin mRNA ( Supplemental Protocol S1 , Supplemental Fig . S1 ) .', 'As expected , the STATdel promoter fails to respond to IL6 stimulation , but is still sensitive to BMP treatment .', 'The luciferase activities of the BRE1m construct are much lower than WT for both basal and induced conditions ( Fig . 1C ) .', 'However , the BRE1m promoter is qualitatively similar to the WT promoter in terms of stimulus inducibility , because maximal BMP stimulation induces a large increase in luciferase activity , while the maximal IL6 dose has a much lesser impact .', 'The co-stimulation response of the BRE2m construct is qualitatively different from that of the WT and BRE1m promoters .', 'The BRE2m promoter resembles a coincidence detector ( ‘logical AND gate’ ) : Mono-stimulation with either BMP or IL6 raises luciferase activity to intermediate levels only , and co-stimulation with both ligands is required for maximal expression ( Fig . 1C ) .', 'This suggests that BRE1 and BRE2 fulfill very distinct functions in hepcidin regulation: The BRE2m promoter shows only a slight reduction in basal luciferase activity compared to the WT promoter .', 'This reduction in basal activity did not reach statistical significance ( paired t-test ) , and was much less pronounced than the effect of a BRE1 mutation .', 'The promoter responsiveness to IL6 mono-stimulation was significantly reduced by the BRE1 mutation but not by the BRE2 mutation ( paired t-test ) .', 'Thus , BRE2 has lesser impact on the IL6-induced fold-change in hepcidin expression than BRE1 .', 'The most prominent feature of the BRE2m promoter is the reduced ability to respond to BMP stimuli compared to the WT promoter ( 0 . 001<p<0 . 0025 , paired t-test ) : maximal BMP stimulation enhances luciferase activity by only 80-fold in BRE2m , when compared to 400-fold in WT and BRE1m .', 'The loss of BRE1 hardly affects the promoter responsiveness to maximal BMP doses , although it has some impact at intermediate BMP doses .', 'Taken together , these data raise the interesting question of how two transcription factor binding sites with very similar sequence can show qualitatively distinct behavior in hepcidin expression regulation .', 'The coordinated regulation of hepcidin expression by BMP and IL6 may involve crosstalk at the level of signal transduction .', 'We analyzed signaling pathway interactions by measuring the phosphorylation of STAT3 and SMAD1/5/8 after stimulation with BMP and/or IL6 using quantitative immunoblotting .', 'Cells were stimulated for 12 h after starvation , and phosphorylated SMAD1/5/8 and STAT3 were detected in the cell lysate using phospho-specific antibodies ( Supplemental Fig . S2 ) .', 'The signals were quantified by densitometry and the results of biological duplicates were merged ( see Methods ) .', 'SMAD1/5/8 but not STAT3 showed basal phosphorylation in unstimulated cells which is consistent the role of BRE1 in controlling basal luciferase activities ( Fig . 1C ) .', 'As expected , stimulation with BMP or IL6 alone resulted in dose-dependent increases of SMAD1/5/8 and STAT3 phosphorylation , respectively ( Fig . 1D ) .', 'Co-stimulation with a saturating dose of IL6 appeared to slightly reduce BMP-induced SMAD1/5/8 phosphorylation ( Fig . 1D , bottom ) , but the effect is not statistically significant ( paired t-test ) .', 'High doses of BMP had significant inhibitory effects on IL6-mediated STAT3 phosphorylation ( p<0 . 001 , paired t-test ) .', 'However , the effect was moderate and typically resulted in a less-than two-fold reduction in phospho-STAT3 levels ( Fig . 1D , top ) .', 'Thus , while co-stimulation with BMP and IL6 enhances hepcidin expression relative to mono-stimulation , a slight cross-inhibition is observed at the level of transcription factor phosphorylation .', 'We integrated these measurements at different levels into a mathematical model to quantitatively understand the determinants of hepcidin expression regulation .', 'Our model describes luciferase activity as a function of the extracellular IL6 and BMP concentrations , and consists of two modules: The signaling module describes SMAD and STAT TF phosphorylation in response to BMP and IL6 stimulation , while the promoter module characterizes combinatorial TF binding to the promoter and gene expression .', 'The kinetic parameters of the model as well as the regulatory details at the promoter level were unknown .', 'We therefore estimated the parameters by model fitting to TF phosphorylation and luciferase activity data ( Figs . 1C and D ) , and systematically compared the ability of different promoter variants to fit the data in an unbiased modeling approach .', 'The signaling module assumes that the dose-response curves of IL6-induced STAT phosphorylation and BMP-induced SMAD phosphorylation are of sigmoidal shape , as suggested by previous studies [4] , [40] .', 'Dose-response curves of TF phosphorylation are therefore represented using sigmoidal Hill equations ( Supplemental Text S3 ) .', 'Inhibitory crosstalk between SMAD and STAT proteins was modeled by assuming that the phosphorylation degree of one TF affects the Hill equation parameters describing the other pathway .', 'The thermodynamic promoter model describes luciferase activity as a function of the phosphorylated TF concentrations , and assumes that TFs affect the occupancy of the promoter: If the pSMAD and pSTAT concentrations are zero , the promoter will either be completely empty or RNA Polymerase II ( RNAP ) may bind to the transcription start site ( TSS ) at a basal level ( Fig . 2A , bottom row ) .', 'For increasing TF concentrations the promoter will be occupied by pSMAD , pSTAT or RNAP , or a combination of these , giving rise to multiple promoter states .', 'The presence of three specific binding TF sites and RNAP binding to the TSS yields to 24\u200a=\u200a16 promoter states ( Fig . 2A ) .', 'In which of these states the promoter exists depends in a complex manner on the concentration of phosphorylated TFs , their binding affinity for DNA and may also be altered by TF/TF or TF/RNAP interactions on the promoter .', 'Equations that describe the probability of each promoter state as a function of protein concentrations and binding affinities can be derived based on principles of statistical thermodynamics ( Supplemental Text S2 , Supplemental Protocol S2 ) .', 'The promoter states in the model are linked to gene expression by assuming that RNAP-bound promoters are transcriptionally active , while promoters devoid of RNAP binding are silent ( Fig . 2A ) .', 'A transcription initiation rate can thus be calculated from the probabilities of the promoter states ( Supplemental Text S2 , Supplemental Protocol S2 ) .', 'We neglected gene regulation at the levels of transcription elongation and post-transcriptional processing in our model .', 'The experimentally measured luciferase activity is therefore assumed to be proportional to the simulated initiation rate .', 'All variants of the promoter module comprised well-known aspects of promoter regulation such as pSTAT/pSMAD binding to the promoter and RNAP recruitment by TFs ( grey arrows in Fig . 2B ) .', 'We additionally allowed cooperative TF binding , implying that TFs may mutually enhance their recruitment to the promoter ( red arrows in Fig . 2B ) .', 'Given three possible cooperative TF interactions alone or in combination , we considered 8 promoter module variants in total ( Fig . 2B ) .', 'Model fitting was done by minimizing the χ2 metric which allows for larger difference between model and experiment if the experimental error is large ( see Methods ) .', 'Model variants with different numbers of parameters were compared with respect to goodness-of-fit to the training data using the Akaike information criterion ( see Methods ) .', 'Based on these measures , the data were best explained by a promoter module containing a single cooperative interaction among SMAD and STAT TFs bound to BRE1 and STATBS , respectively ( model 4 in Fig . 2B ) .', 'More complex models also containing the cooperative interaction between BRE1 and STATBS ( models 6—8 in Fig . 2B ) did not fit the data better than the selected model .', 'Model variants lacking the BRE1-STATBS cooperativity ( models 1—3 and 5 in Fig . 2B ) fitted the experimental data less well than the selected model from a quantitative point of view .', 'Moreover , they qualitatively failed to explain why IL6 mono-stimulation induces a lesser fold-change in the BRE1m construct when compared to WT ( Supplemental Fig . S3 , Fig . 1C , [19] ) .', 'The best-fit model ( Figs . 2C and D ) comprises 19 kinetic parameters and describes the data with an accuracy close to experimental measurement noise ( χ2\u200a=\u200a124 , N\u200a=\u200a80 ) .', 'Nine model parameters enter the sigmoidal Hill functions which describe the stimulus-induced TF phosphorylation , and signaling crosstalk between transcription factors .', 'The remaining 10 parameters enter the promoter module , and describe the TF affinity for DNA binding sites , the TF interaction strength with RNAP , and the cooperativity of TF binding to DNA .', 'The nature of the model parameters is described in detail in Supplemental Protocol S2 , and a list of best-fit parameter values can be found in Supplemental Table S1 .', 'Not all model parameters could be unequivocally identified based on the experimental data , implying that multiple parameter sets yield a comparable fit to the training data ( Supplemental Fig . S4 ) .', 'This non-identifiability of parameters gives rise to uncertainties in the model predictions .', 'We therefore performed all model analyses for many measurement-compliant parameter sets ( with χ2<135 ) , not only for the best-fit solution ( see Methods ) .', 'The model predictions in Fig . 3 were therefore formulated as a range corresponding to the simulation runs with the highest and lowest predicted luciferase activity .', 'In most cases , reliable model predictions were possible despite non-identifiability of individual parameter values .', 'In our model , hepcidin expression was mostly determined by the dynamics of TF binding to the promoter , while inhibitory signaling crosstalk played only a minor role: Elimination of signaling crosstalk did not significantly change the simulated luciferase activities , and this conclusion held true for all measurement-compliant parameter sets ( Supplemental Fig . S5 ) .', 'Moreover , we compared the ability of model variants with and without signaling crosstalk to fit the luciferase activity data ( Fig . 1C ) .', 'The fit of the crosstalk-less model to the training data was significantly better as judged by the Akaike information criterion ( Supplemental Fig . S5 ) .', 'We therefore focused our model validation on regulation events at the promoter level , and neglected the relatively weak signaling crosstalk effects .', 'We sought to verify our model by an independent set of experiments not used for model calibration .', 'The formulation of model predictions was focused on double mutant promoters which simultaneously lack two TF binding sites ( Fig . 3A ) .', 'One central promoter mechanism predicted by the model is the cooperative interaction between pSMAD and pSTAT TFs , bound to BRE1 and STATBS , respectively .', 'The double mutant promoter lacking functional BRE1 and STAT elements ( BRE1mSTATdel promoter , Fig . 3A ) was employed to independently confirm the cooperativity effect .', 'The BRE1STATdel promoter shows a ∼30-fold reduced expression relative to WT upon stimulation with 2 ng/ml IL6 ( Fig . 3B , red bar ) .', 'The corresponding STATBS and BRE1 single deletions reduce expression by ∼3 and ∼30-fold , respectively ( Fig . 3B , blue and green bars ) .', 'Thus , the BRE1m and BRE1mSTATdel promoters exhibit similar expression levels .', 'This is consistent with BRE1-STATBS cooperativity , because the single BRE1m single deletion already eliminates the cooperativity effect , and thereby a large part of the STATBS contribution to expression .', 'In contrast , a transcription model lacking the cooperativity would predict an independent contribution of both sites , implying that the expression reduction in the BRE1mSTATdel promoter equals the product of the single deletion fold-changes ( Fig . 3B , top ) .', 'Thus , the double mutant data qualitatively supports the model with cooperativity between BRE1 and STATBS , also for higher doses of IL6 ( Fig . 3B ) .', 'BRE1 and BRE2 play different roles in hepcidin expression with respect to basal expression , BMP inducibility and co-stimulation response .', 'One difference between the two sites is the above-mentioned cooperative interaction with the STATBS that is specific to BRE1 .', 'How do the BMP-responsive elements differ beyond this interaction ?', 'The model predicted that BRE1 has higher affinity for phosphorylated SMAD than BRE2 , explaining why BRE1 plays a predominant role under basal conditions .', 'Upon sufficiently strong BMP stimulation both sites are predicted to activate RNAP with comparable efficiency .', 'In conclusion , the model suggested that BRE1 and BRE2 should behave similarly in the absence of cooperative promoter interactions .', 'This prediction can be tested by co-stimulation of BRE2mSTATdel and BRE1mSTATdel promoters which solely contain BRE1 and BRE2 , respectively ( Fig . 3A ) .', 'The experimental data was in good qualitative agreement with model predictions: Both mutants showed very similar co-stimulation heatmaps and primarily responded to BMP stimulation ( Fig . 3C , bottom row ) .', 'Maximal luciferase activity at high BMP levels was comparable for both constructs , indicating that BRE1 and BRE2 indeed drive RNAP activation with similar efficiency ( Figs . 3C and D ) .', 'Basal activity was approximately 10-fold higher in the BRE2STATdel promoter , suggesting that the isolated BRE1 has indeed a higher pSMAD affinity than BRE2 ( Fig . 3D ) .', 'Quantitative model predictions for the BRE2mSTATdel and BRE1mSTATdel heatmaps were only possible up to a certain range of absolute luciferase activities owing to non-identifiability of model parameters ( Fig . 3C , top and middle row ) .', 'The experimentally observed luciferase activities were within the predicted range ( Fig . 3C ) .', 'The model suggested that saturating RNAP binding to the TSS limits hepcidin expression upon strong stimulation , whereas signaling pathway saturation plays only a minor role .', 'Saturation implies that single deletions promoters should exhibit expression levels relatively similar to WT , because the remaining TF binding sites maintain near-complete occupancy of the TSS with RNAP .', 'This buffering effect should be abrogated upon a combined TF binding site deletion , leading to the prediction that hepcidin double mutant promoters exhibit very low expression compared to the corresponding single mutants .', 'We confirmed this prediction by measuring the activity of the BRE1mBRE2m double mutant promoter ( Fig . 3E ) : The double deletion of BRE1 and BRE2 reduces the luciferase activity by up to ∼1000-fold compared to WT ( red bars ) .', 'The corresponding single deletions typically reduce expression by ∼10-fold or less , and are thus much more similar to the WT promoter ( blue and green bars ) .', 'This behavior indicates TSS saturation , because the fold-change by a double deletion equals the product of the corresponding single deletion fold-changes in thermodynamic transcription models without saturation .', "The validity of our model is further supported by the quantitative agreement of the models' predictions with the data: the buffering of single deletions is more pronounced for strong ( co- ) stimulation ( Fig . 3E ) , where promoter saturation is particularly prominent .", 'We find similar , albeit less pronounced , buffering effects between STATBS and BRE2 ( Supplemental Fig . S6 ) and conclude that saturating RNAP binding is an important aspect of promoter behavior .', 'Promoter saturation also explains why the BRE1 single mutation strongly reduces expression at intermediate BMP concentrations , while having lesser impact at basal and strong stimulation conditions ( Fig . 3E , blue bars , grey corridor ) .', 'The BRE1 deletion has a strong effect on expression at intermediate BMP doses , because the high-affinity BRE1 is already fully occupied by pSMAD .', 'Higher BMP concentrations alleviate the impact of BRE1 deletions , because low-affinity pSMAD binding to the BRE2 saturates the promoter , thereby buffering the loss of BRE1 .', 'We investigated in silico how the simultaneous presence of two BREs affects the behavior of the hepcidin promoter , and found that this promoter design enhances the steepness of the BMP dose-response curve: A certain increase in phospho-SMAD levels induces a larger expression fold-change in the WT promoter when compared to BRE1m and BRE2m promoters ( Fig . 4A ) .', 'The hepcidin promoter only contains a single STAT binding site as opposed to two BREs .', 'Accordingly , the model predicts that the hepcidin promoter specifically responds with high sensitivity towards BMP stimulation , and is much less sensitive towards IL6 stimulation ( Fig . 4B ) .', 'The initial stimulation experiments used for model training ( Fig . 1C ) were based on three BMP and IL6 concentrations , and therefore did not allow for conclusions concerning the steepness of the BMP and IL6 response .', 'To verify the model prediction by an independent set of experiments , we performed detailed dose-response measurements with multiple doses of IL6 and BMP , respectively ( Fig . 4C ) .', 'These data confirm that the BMP response is much steeper than the IL6 response , and thereby validate the model .', 'Systemic iron homeostasis is maintained by an auto-regulatory negative feedback loop in which hepcidin expression levels depend on and control the circulating iron levels in the blood ( Fig . 4D ) .', 'A high BMP sensitivity of the promoter may allow the iron-BMP signaling axis to sense minor changes in iron blood levels , and to maintain systemic iron homeostasis .', 'We simulated iron homeostasis in the living animal using an extended model with feedback ( Fig . 4D ) .', 'Iron blood levels were described by the species Feb , whose levels are controlled by influx and efflux reactions .', 'The iron influx rate is proportional to the intestinal iron concentration ( species Fei ) which reflects the dietary iron content .', 'Iron blood levels control the activity of the BMP signaling pathway , and thus hepcidin expression .', 'Negative feedback regulation was considered in the model by assuming that the iron influx is negatively influenced by hepcidin ( Supplemental Text S4 ) .', 'Hepcidin expression regulation by BMP and IL6 in the model was described using the previously derived best-fit model ( Figs . 2C and D ) .', 'The remaining parameters of the model describe the iron influx and efflux , hepcidin degradation , and the strength of hepcidin-mediated feedback on the iron influx .', 'Homeostasis was analyzed by assessing how the iron blood level in the model ( Feb ) responds to a change dietary iron content ( Fei ) , efficient homeostasis implying that a given fold-change in Fei elicits a much lesser fold-change in Feb .', 'The key assumption we made was that hepcidin-mediated feedback regulation has a very strong impact on the iron influx .', 'For the limit of strong feedback , it can be shown analytically that the degree of iron homeostasis is solely determined by the steepness of the hepcidin promoter response , and independent of the remaining model parameters ( Supplemental Text S1 ) .', 'The range of intestinal iron concentrations for which homeostasis is observed is determined by the range of BMP concentrations that can be sensed by the hepcidin promoter ( Supplemental Text S1 ) .', 'The model thus allowed us to quantitatively analyze how the hepcidin promoter architecture affects systemic iron homeostasis , although the remaining model parameters were unknown .', 'The simulations in Fig . 4E show that a model with the WT hepcidin promoter efficiently maintains systemic iron homeostasis , as the iron blood levels remain essentially constant over a broad range of intestinal iron concentrations .', 'Models with BRE1m and BRE2m promoters perform less well , as the perturbation-response curves are steeper and homeostasis is restricted to a narrower range of influx rates ( Fig . 4E , green and red curves ) .', 'This suggests that the simultaneous presence of two BMP-responsive elements in the promoter indeed optimizes the performance of the systemic iron homeostasis loop .', 'One important question is why IL6 stimulation reduces iron blood levels and induces anemia of inflammation even though the homeostasis loop should effectively buffer IL6-induced perturbations in hepcidin expression .', 'Simulations of the extended feedback model show strongly diminished iron levels and a loss of homeostasis if high IL6 levels are assumed ( Fig . 4E , blue dashed line ) .', 'This effect can be understood by considering the BMP dose-response curve of the best-fit promoter model for varying IL6 concentrations ( Fig . 4F ) : increasing IL6 levels reduce the sensitivity of the BMP dose-response curve due to ( partial ) saturation of the TSS with RNAP .', 'Moreover , significant changes in hepcidin expression are restricted to a narrower range of phospho-SMAD levels .', 'The hepcidin promoter therefore responds less efficiently to changes in the iron/BMP signaling in the presence of IL6 .', 'This impairs the iron sensing capability of the hepcidin promoter in vivo , and leads to a breakdown of feedback homeostasis .', 'Iron blood levels are chronically elevated in HH , in most cases due to inactivating mutations in the iron-sensing BMP signaling axis .', 'One unexplored question is why HH is commonly associated with inactivating mutations in the SMAD signaling pathway , while mutations in the BRE1 promoter element are rare and BRE2 mutations have not been identified yet [20] .', 'The iron homeostasis model predicts that a BRE1 deletion affects the iron blood levels more strongly than the BRE2 deletion in the WT homeostasis range ( compare green and red lines in Fig . 4E , respectively ) .', 'The more critical role of BRE1 may explain why only BRE1 mutations have been associated with HH , and can be explained by its higher phospho-SMAD affinity when compared to BRE2 .', 'The model further predicts strong buffering of BRE1 and BRE2 single deletions: Single mutations in either site have much weaker effects than a complete feedback ablation by a BRE1mBRE2m double mutation ( light blue line in Fig . 4E ) .', 'The very strong effect of a BRE double deletion in cultured cells ( Fig . 3E ) is thus predicted to be preserved in vivo .', 'These simulations may explain why BMP signaling pathway mutations that simultaneously inactivate expression regulation via BRE1 and BRE2 are by far the most common cause of HH .', 'Taken together , the steepness of the hepcidin promoter response appears to be a key parameter controlling how well the systemic iron homeostasis loop compensates for fluctuations in iron diet content .'], ['The fine-tuned expression of hepcidin plays a central role in systemic iron homeostasis , and is deregulated in two major clinical settings , HH and anemia of inflammation .', 'Here , we comprehensively characterized the gene regulatory function of the hepcidin promoter using systematic promoter mutagenesis and co-stimulation experiments .', 'We employed a multi-scale modeling approach capturing signaling and gene expression , and discriminated various promoter regulation scenarios .', 'This approach complements existing strategies linking signaling and gene expression events [41] , [42] , and may be extended in future studies to model global transcription patterns based on mRNA expression profiles , TF binding information and mRNA half-life data .', 'Gene expression may be a gradual or a binary event at the single cell level [43] .', 'Our mathematical model assumes that TF phosphorylation and reporter gene expression measurements in a cellular ensemble reflect the behavior of single cells , and thus presumes a gradual mode of hepcidin expression .', 'Experimental studies indicate that BMP-induced target gene expression is indeed a gradual event at the single-cel
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Systemic iron homeostasis involves a negative feedback circuit in which the expression level of the peptide hormone hepcidin depends on and controls the iron blood levels .', 'Hepcidin expression is regulated by the BMP6/SMAD and IL6/STAT signaling cascades .', 'Deregulation of either pathway causes iron-related diseases such as hemochromatosis or anemia of inflammation .', 'We quantitatively analyzed how BMP6 and IL6 control hepcidin expression .', 'Transcription factor ( TF ) phosphorylation and reporter gene expression were measured under co-stimulation conditions , and the promoter was perturbed by mutagenesis .', 'Using mathematical modeling , we systematically analyzed potential mechanisms of cooperative and competitive promoter regulation by the transcription factors , and experimentally validated the model predictions .', 'Our results reveal that hepcidin cross-regulation primarily occurs by combinatorial transcription factor binding to the promoter , whereas signaling crosstalk is insignificant .', 'We find that the presence of two BMP-responsive elements enhances the steepness of the promoter response towards the iron-sensing BMP signaling axis , which promotes iron homeostasis in vivo .', 'IL6 co-stimulation reduces the promoter sensitivity towards the BMP signal , because the SMAD and STAT transcription factors compete for recruiting RNA polymerase to the transcription start site .', 'This may explain why inflammatory signals disturb iron homeostasis in anemia of inflammation .', 'Taken together , our results reveal why the iron homeostasis circuit is sensitive to perturbations implicated in disease .']
['The nutritional iron uptake is tightly regulated because the body has limited capacity of iron excretion .', 'Mammals maintain iron homeostasis by a negative feedback loop , in which the peptide hepcidin senses the iron blood level and controls iron resorption .', 'Molecular perturbations in the homeostasis loop lead to iron-related diseases such as hemochromatosis or anemia of inflammation .', 'Quantitative studies are required to understand the dynamics of the iron homeostasis circuitry in health and disease .', 'We investigated how the biological activity of hepcidin is regulated by combining experiments with mathematical modeling .', 'We present a multi-scale model that describes the signaling network and the gene promoter controlling hepcidin expression .', 'Possible scenarios of hepcidin regulation were systematically tested against experimental data , and interpreted using a network model of iron metabolism in vivo .', 'The analysis showed that the presence of multiple redundant regulatory elements in the hepcidin gene promoter facilitates homeostasis , because changes in iron blood levels are sensed with high sensitivity .', 'We further suggest that inflammatory signals establish molecular competition at the hepcidin promoter , thereby reducing its iron sensitivity and leading to a loss of homeostasis in anemia of inflammation .', 'We conclude that quantitative insights into hepcidin expression regulation explain features of systemic iron homeostasis .']
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