Repression of the IFNB1 promoter by NFAT5 is mediated by an overlapping NFAT-IRF site. (A) Activity of a luciferase reporter driven by the human IFNB1 promoter (IFNB1-Luc) in unstimulated or poly(I:C)-stimulated (6 h) RAW 264.7 macrophage cells transfected with shNFAT5 or shGFP (left), or with plasmids encoding the DBD of NFAT5 fused to GFP (NFAT5-DBD) or GFP alone (right). (B) Activity of the 3× PRDIII-I Luc (left) and 2× PRDII-Luc (right) reporters in unstimulated or poly(I:C)-stimulated (6 h) RAW 264.7 cells transfected with shNFAT5 or shGFP. (C) Western blot of NFAT5 in poly(I:C)-treated RAW 264.7 cells transfected with shGFP or shNFAT5. (D) Mutagenesis strategy to target the consensus binding sites for NFAT5, IRF3 (PRDIII and PRDI), and NF-κB (PRDII) in the IFNB1 enhanceosome. (E–G) Activity of the corresponding IFNB1-Luc reporter mutants. In all experiments, luciferase activity was normalized for the efficiency of electroporation using a Renilla-expressing plasmid cotransfected in the same cells. Normalized luciferase units in each experiment are shown relative to a reference sample (with value 100) of cells transfected with control shGFP plasmid or GFP-encoding plasmid and stimulated with 10 µg/ml poly(I:C). Error bars in A, B, and E–G show the mean ± SEM. Results in A are from 10 independent transfection experiments in the left panel and 3 in the right panel. Results in B are from six independent transfection experiments in the left panel and four in the right panel. Western blots in C are representative of two independent experiments. Results in E and G are from six independent transfection experiments and from three independent experiments in F. Statistical significance was determined with a one-sample t test for comparisons with the reference sample or with an unpaired t test for comparisons between the other samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Flow cytometry assay to assess the competition between the DBDs of NFAT5 and IRF proteins at the PRDIII element of the IFNB1 enhanceosome. The schematic diagram depicts a GST-tagged DBD of NFAT5 (GST–NFAT5-DBD) bound to a biotin-tagged, double-strand PRDIII oligonucleotide (IFNB1 PRDIII oligo) and captured on glutathione-Sepharose beads. The IFNB1 PRDIII oligo is detected by flow cytometry with eFluor 450–labeled streptavidin. Competition between IRF and NFAT5 was assessed by preincubating the IFNB1 PRDIII oligo with recombinant IRF-DBD. The flow cytometry dot plot shows the forward scatter (FSC) and side scatter (SSC) profiles in logarithmic scale (log10) of the glutathione-Sepharose beads, and the histograms on the right show the eFluor 450 signal on NFAT5-coated beads for a mutant IFNB1 PRDIII oligo with the NFAT5 site disrupted (NFAT5mut oligo, used to determine the nonspecific binding background), and for the wild-type IFNB1 PRDIII oligo in the absence or presence of competitor IRF3 (wild-type and DNA binding mutant) and IRF7. The bars show the quantification of the flow cytometry assays. Results are from two independent experiments, each one with two technical replicates for each binding reaction. Statistical significance was determined with an unpaired t test. **, P < 0.01.
Figure 7.

Repression of the IFNB1 promoter by NFAT5 is mediated by an overlapping NFAT-IRF site. (A) Activity of a luciferase reporter driven by the human IFNB1 promoter (IFNB1-Luc) in unstimulated or poly(I:C)-stimulated (6 h) RAW 264.7 macrophage cells transfected with shNFAT5 or shGFP (left), or with plasmids encoding the DBD of NFAT5 fused to GFP (NFAT5-DBD) or GFP alone (right). (B) Activity of the 3× PRDIII-I Luc (left) and 2× PRDII-Luc (right) reporters in unstimulated or poly(I:C)-stimulated (6 h) RAW 264.7 cells transfected with shNFAT5 or shGFP. (C) Western blot of NFAT5 in poly(I:C)-treated RAW 264.7 cells transfected with shGFP or shNFAT5. (D) Mutagenesis strategy to target the consensus binding sites for NFAT5, IRF3 (PRDIII and PRDI), and NF-κB (PRDII) in the IFNB1 enhanceosome. (E–G) Activity of the corresponding IFNB1-Luc reporter mutants. In all experiments, luciferase activity was normalized for the efficiency of electroporation using a Renilla-expressing plasmid cotransfected in the same cells. Normalized luciferase units in each experiment are shown relative to a reference sample (with value 100) of cells transfected with control shGFP plasmid or GFP-encoding plasmid and stimulated with 10 µg/ml poly(I:C). Error bars in A, B, and E–G show the mean ± SEM. Results in A are from 10 independent transfection experiments in the left panel and 3 in the right panel. Results in B are from six independent transfection experiments in the left panel and four in the right panel. Western blots in C are representative of two independent experiments. Results in E and G are from six independent transfection experiments and from three independent experiments in F. Statistical significance was determined with a one-sample t test for comparisons with the reference sample or with an unpaired t test for comparisons between the other samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (H) Flow cytometry assay to assess the competition between the DBDs of NFAT5 and IRF proteins at the PRDIII element of the IFNB1 enhanceosome. The schematic diagram depicts a GST-tagged DBD of NFAT5 (GST–NFAT5-DBD) bound to a biotin-tagged, double-strand PRDIII oligonucleotide (IFNB1 PRDIII oligo) and captured on glutathione-Sepharose beads. The IFNB1 PRDIII oligo is detected by flow cytometry with eFluor 450–labeled streptavidin. Competition between IRF and NFAT5 was assessed by preincubating the IFNB1 PRDIII oligo with recombinant IRF-DBD. The flow cytometry dot plot shows the forward scatter (FSC) and side scatter (SSC) profiles in logarithmic scale (log10) of the glutathione-Sepharose beads, and the histograms on the right show the eFluor 450 signal on NFAT5-coated beads for a mutant IFNB1 PRDIII oligo with the NFAT5 site disrupted (NFAT5mut oligo, used to determine the nonspecific binding background), and for the wild-type IFNB1 PRDIII oligo in the absence or presence of competitor IRF3 (wild-type and DNA binding mutant) and IRF7. The bars show the quantification of the flow cytometry assays. Results are from two independent experiments, each one with two technical replicates for each binding reaction. Statistical significance was determined with an unpaired t test. **, P < 0.01.

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