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. 2015 Jun 23;43(11):5318-30.
doi: 10.1093/nar/gkv423. Epub 2015 May 1.

Signal Integration by the CYP1A1 Promoter--A Quantitative Study

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Free PMC article

Signal Integration by the CYP1A1 Promoter--A Quantitative Study

Pascal Schulthess et al. Nucleic Acids Res. .
Free PMC article

Abstract

Genes involved in detoxification of foreign compounds exhibit complex spatiotemporal expression patterns in liver. Cytochrome P450 1A1 (CYP1A1), for example, is restricted to the pericentral region of liver lobules in response to the interplay between aryl hydrocarbon receptor (AhR) and Wnt/β-catenin signaling pathways. However, the mechanisms by which the two pathways orchestrate gene expression are still poorly understood. With the help of 29 mutant constructs of the human CYP1A1 promoter and a mathematical model that combines Wnt/β-catenin and AhR signaling with the statistical mechanics of the promoter, we systematically quantified the regulatory influence of different transcription factor binding sites on gene induction within the promoter. The model unveils how different binding sites cooperate and how they establish the promoter logic; it quantitatively predicts two-dimensional stimulus-response curves. Furthermore, it shows that crosstalk between Wnt/β-catenin and AhR signaling is crucial to understand the complex zonated expression patterns found in liver lobules. This study exemplifies how statistical mechanical modeling together with combinatorial reporter assays has the capacity to disentangle the promoter logic that establishes physiological gene expression patterns.

Figures

Figure 1.
Figure 1.
Cross-talk between AhR and Wnt/β-catenin signaling pathways in zonal expression of CYP1A. (A) Hepatic zonation schematic of β-catenin activity and CYP1A expression in response to TCDD treatment. Area of cells displaying high β-catenin activity is thought to remain invariant to presence of TCDD while a larger amount of cells express CYP1A after TCDD exposure. (B) Schematic representation of the two signaling pathways that converge on the CYP1A1 promoter. TCDD can bind to the AhR and Arnt that is then assumed to recruit free β-catenin. β-catenin is activated (dashed arrow) through the binding of Wnt to its surface receptors and forms a TF complex with TCF. The CYP1A1 promoter possesses five functional TFBS within 1.2 kbp, four for TCDD/AhR/Arnt (rectangular binding sites C, D, E, F) and one for β-catenin/TCF (elliptical binding site T). The double-headed and numbered arrows depict the two reactions used in the signaling model. The C- and D-DRE are color coded as in subsequent figures. (C) β-Catenin modulates CYP1A1 expression independently of the β-catenin/TCF binding site. Relative luciferase activity of a promoter construct in which the β-catenin/TCF binding site was inactivated by point-mutation over a series of TCDD concentrations treated with DMSO (gray circles) or 10 μM of the β-catenin inhibitor iCRT3 (blue triangles). (D) The interaction between AhR and β-catenin is increased upon TCDD treatment. Top: Western analysis of AhR expression in lysates from 55.1c cells. Co: solvent control; T: 250 nM TCDD for 1 h; T+i: 250 nM TCDD + 10 μM iCRT3. Bottom: western analysis of β-catenin after immunoprecipitation with an anti-AhR antibody (AhR AB ‘+’). The two bands show the wild-type (wt) and exon 3-deleted (mut) versions of β-catenin, i.e. the constitutively active β-catenin present in the cells.
Figure 2.
Figure 2.
A mathematical model recapitulates cross-talk and cooperativity between binding sites in the control of a synthetic promoter constructed from elements of the CYP1A1 promoter. (A) Cooperativity between TFs binding to DREs on the natural promoter. Relative luciferase activity of three natural promoter constructs over a series of TCDD concentrations was measured. On the three promoter constructs only the following TFBS combinations are present as WT sequence while the others are inactivated through point-mutation: square: β-catenin/TCF and D-DRE; circle: β-catenin/TCF and C-DRE; triangle: β-catenin/TCF, C- and D-DRE. (B) Schematic depiction of the synthetic promoter construct library. Six constructs hold one to six copies of the sequence of the C-DRE (turquois), three constructs hold one to three copies of the sequence of the D-DRE (red), and differently long non-TF responsive sequences (19, 49, 156, 292 bp) were inserted between two C-DREs for four constructs. (C) Scheme explaining the parameters used in the thermodynamic model. Ki represent the association constants of the TFs to the C and T TFBS as well as between the RNAP and the DNA. εC and εT are the binding energies between the TFs and the RNAP while εTC is the interaction term between the TFs. (D) TCDD concentration series of the synthetic promoter constructs can be explained with a thermodynamic model. TCDD concentration series of nine synthetic constructs as well as one β-catenin titration series where the relative luciferase activity was measured (gray dots and blue triangles). The upper seven graphs represent the C-DRE constructs while the lower three graphs represent D-DRE constructs. The graph in the top right corner shows a β-catenin titration series measured at 250 nM TCDD concentration. Error bars represent one standard deviation of 6–10 biological replicates. The black curves show the fits of the thermodynamic model. (E) Cooperative binding dominates the synthetic promoter constructs. Binding energies resulting from fits are displayed on the promoter construct holding six copies of the C-DRE. Arrows depict significant binding events. Their colors represent binding strength where lower values represent stronger association. All binding events were present in the model. Those equal or close to 0 are not depicted as arrows. (F) Stimulus-response curves for constructs with different distances between DREs confirm reduced cooperativity for longer distances. Relative luciferase activity was measured over increasing TCDD concentrations for four promoter constructs with different distances (19, 49, 156, 292 bp) between their C-DREs. (G) Model simulations showing why three C-DRE binding sites show maximal induction. Top: The average number of occupied binding sites is plotted over the number of present binding sites in the synthetic constructs. Bottom: The probability that the first DRE, i.e. the one closest to the RNAP is occupied by TFs is shown. Note that these values were calculated for 250 nM of TCDD. (H) Thermodynamic model correctly predicts and experimental data and confirms an AND gate relationship between the Wnt/β-catenin and the AhR signaling pathway on the 3× C-DRE construct. Left: prediction of the promoter activity of the synthetic construct holding three C-DREs by the thermodynamic model through variation of the β-catenin activity parameter. Right: corresponding measurements of the relative luciferase activity of the 3× C-DRE construct where the inhibitor iCRT3 modulated the β-catenin activity. The cells were stimulated with increasing TCDD concentrations.
Figure 3.
Figure 3.
Promoter logic of the human CYP1A1 promoter dissected by a mathematical model. (A) Schematic representation of the reporter library for the natural CYP1A1 reporter. Rectangular binding sites are DREs while elliptical binding sites are targets of β-catenin/TCF. Gray symbols depict TFBS that were inactivated by point-mutation while the WT binding sequence is represented by black symbols. (B) The response of the natural promoter constructs to TCDD concentration series can be explained with a thermodynamic model. Relative luciferase activity was measured over increasing TCDD concentrations and an additional β-catenin titration for 18 natural promoter constructs (gray dots and blue triangles). The β-catenin titration was measured at 250 nM TCDD. Error bars represent one standard deviation of 4–10 biological replicates. The black solid curves show the fits of the mathematical model. (C) Binding between the RNAP and the TFs dominates the natural promoter constructs. Binding energies resulting from fits displayed on the natural promoter. Arrows depict existing binding events. Their colors represent binding strength where lower values represent stronger association. Only short range binding events were considered in the model, those equal or close to zero are not depicted as arrows. (D) Predicted response to Wnt/β-catenin and AhR signaling pathway for three different natural promoter constructs. Left: prediction of the promoter activity of three natural constructs (C-DRE and β-catenin/TCF TFBS present; β-catenin/TCF TFBS inactivated through point-mutation; WT construct) by the mathematical model through variation of the β-catenin activity parameter. Right: Corresponding measurements of the relative luciferase activity of three constructs where the β-catenin activity was modulated by the iCRT3 inhibitor. The cells were simultaneously stimulated with increasing TCDD concentrations. (E) Hill Coefficients confirm more switch-like behavior of synthetic promoter constructs. From the double-stimulated datasets of the synthetic 3× C DRE and the natural WT promoter construct the subsets for 100 % β-catenin activity was fitted to Hill functions. Relative luciferase activity for both constructs is represented by turquoise (synthetic 3× C DRE construct) and black (natural WT construct) dots. The dashed lines depict the corresponding Hill functions. The resulting Hill coefficients for the two constructs are shown.
Figure 4.
Figure 4.
Model predicts spatial expression of CYP1A1 as a result of its promoter logic. Top: Predictions of the thermodynamic model for the WT construct for selected TCDD concentrations. Each hexagon represents an idealized hepatic lobule that is exposed to a β-catenin activity gradient from central vein (high) to portal vein (low). Bottom: Representative immunostainings of mouse liver for CYP1A for different concentrations of 3-MC.

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