XB-ART-51689Nucleic Acids Res. April 20, 2016; 44 (7): 3045-58.
Quantification of transcription factor-DNA binding affinity in a living cell.
The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element.
PubMed ID: 26657626
PMC ID: PMC4838337
Article link: Nucleic Acids Res.
Genes referenced: ar foxa1 nr3c1
Article Images: [+] show captions
|Figure 1. Apparent dissociation constant (Kd) analysis in vivo using three different concentrations of specific DNA sites in Xenopus oocytes. (A) Experimental design. (B) The pBS(HRE)x7 harbors seven repeats of the DNA segment, the partially palindromic HRE with its two binding elements (BE) highlighted in gray and a FoxA1 binding site is indicated by a rectangle. The protected guanines within the HRE are highlighted by empty circles and the direction of primer extension is shown by a black arrow. (C) Upper diagram, analysis of amount of [3H]-Dex per oocyte nucleus in double samples after oocyte injection with indicated amount of GR mRNA (ng). Four samples were incubated with [3H]-Dex supplemented with excess of unlabeled Dex. Stippled line indicates background that was subtracted. Lower diagram: nuclear GR concentration as a function of GR mRNA injection. (D) Autoradiogram showing the pattern of dimethylsulphate (DMS) methylation at increasing GR concentrations using 0.05 μM of HRE as shown in 1E, double samples for each GR concentration. Gray arrows show borders of the seven repeats, The HRE is indicated by two gray boxes with the protected bands as empty circles for the first repeat (to the right). Scans and columns illustrate quantification of methylation of corresponding guanines with error bars showing the average deviation of double samples. (E) GR-HRE binding analyzed by DMS methylation protection for three different concentrations of HRE as determined by primer extension (See also Supplementary Figure S1A). The concentration of nuclear GR required for 50% GR-HRE binding or DMS methylation protection, is highlighted by arrows pointing at the x-axis. The 50% binding of the 0.5 μM GRE curve was extrapolated from 47.2% binding at the highest GR concentration (dotted line). (F) Graphic estimation of Kd: when total nuclear GR concentration at 50% HRE binding is plotted as a function of the concentration of GR:HRE complex at 50% saturation then the free GR concentration present in absence of any GR:HRE complex is equal to the apparent Kd.|
|Figure 2. GR binding affinity to a complete HRE or a half HRE. (A) The DNA constructs used, the binding elements for GR and AR (BE1 and BE2) are highlighted in gray and the FoxA1 site indicated by a rectangle. These constructs are referred to in the text as HRE1/1 + Fox and HRE1/2 + Fox. (B) Nuclear GR concentration analyzed by [3H]-Dex quantification in nuclear extracts. (C) Autoradiogram showing the pattern of DMS methylation, empty circles signify partially protected bands, unprotected bands used as reference are marked with black dots. Columns in the diagrams to the left and right illustrate quantification of DMS methylation with error bars showing the average deviation of double samples. (D) GR-HRE binding based on DMS methylation protection plotted as a function of nuclear GR concentration. (E) Binding activity at the FoxA1 site based on DMS methylation protection, presumably by a DNA binding protein of endogenous origin.|
|Figure 3. GR binding affinity to a complete HRE (HRE1/1 + Fox) and a half HRE (HRE1/2 + Fox) in presence of FoxA1 protein. Oocytes were injected with increasing amounts of GR mRNA, 3.5–8 ng, mixed with a constant amount of FoxA1 mRNA, 0.46 ng, resulting in 0.4 μM nuclear FoxA1 quantified as described (Supplementary Figure S3C). (A) Pattern of DMS methylation, as in Figure 2C. (B) GR-HRE binding based on DMS methylation protection of BE1 plotted as a function of nuclear GR concentration. (C) FoxA1-DNA binding based on DMS methylation protection plotted as a function of nuclear GR concentration.|
|Figure 4. Comparison of GR-HRE binding without or with FoxA1. (A) The DNA construct HRE1/1ΔFox with a triple-point mutated FoxA1 site. (B) Oocytes injected with GR mRNA, 1.4–7 ng mRNA, followed by injection of ssHRE1/1ΔFox, 4 ng, were analyzed as in Figure 1A, the diagram shows the GR-DNA binding monitored by DMS methylation protection of corresponding guanines in BE1 and BE2 at lower strand as a function of nuclear GR concentration. A Hill plot is for the same data is shown to the right. (C) Oocytes injected with increasing GR mRNA, 1.5–9.2 ng and constant FoxA1 mRNA, 0.35 ng, followed by 4 ng ssDNA HRE1/1 + Fox injection and analyzed as in 4B. The oocytes contained 0.12 μM nuclear FoxA1 based on [14C]-lysine labeled oocytes analyzed as described in Supplementary Figure S3C. (D) A Hill plot based on data in Figure 4C after introducing Facc = 0.59 in order to determine the Kd for the accessible HRE sites.|
|Figure 5. Comparison of AR- and GR-HRE binding to the HRE1/1 + Fox and in presence of FoxA1. (A) Xenopus oocytes were injected with 0.46 ng FoxA1 mRNA mixed with increasing amount of mRNA coding for either AR, 0.3–1.2 ng, or GR, 2.3–7.8 ng, followed by 4 ng of HRE1/1 + Fox ssDNA injection (Figure 2A). The intranuclear AR and GR concentration plotted as a function of injected mRNA, the inset diagram shows an expanded version of the AR diagram. Error bars signify average deviation of double samples. (B) HR-HRE binding based on DMS methylation protection at the HRE1/1 + Fox and plotted as a function of nuclear concentration of indicated HRs. The arrows show the graphically determined apparent Kd, i.e. the receptor concentration at 50% saturation of the HRE site. The nuclear HRE concentration was 0.12 μM (analyzed as in Supplementary Figure S1A). (C) FoxA1-DNA binding from the same experiment as shown in Figure 5A and B. (D) AR-DNA binding as a function of total nuclear AR concentration is shown, 0.2–0.69 μM of nuclear AR was expressed together with ∼0.6 μM FoxA1 and then 10 ng of ssDNA containing HRE1/1 + Fox was injected resulting in 0.24 μM of nuclear HRE. (E) Experiment described in Figure 5D. AR-HRE1/1 + Fox binding depicted as Fb/Facc plotted as function of Cf (i.e. nuclear AR not specifically bound to DNA). The stippled arrow indicates the graphically determined Cf at 50% GR-HRE binding, i.e the apparent Kd.|
|Figure 6. FoxA1 drastically enhances AR binding to HRE1/1 + Fox and HRE1/2 + Fox constructs. Groups of oocytes were mRNA injected to express AR or AR together with FoxA1 followed by nuclear injection of HRE1/1 + Fox or HRE1/2 + Fox constructs. (A) AR-HRE binding based on DMS methylation protection plotted as a function of nuclear AR concentration. (B) FoxA1-DNA binding based on DMS methylation protection plotted as a function of nuclear AR concentration.|