NFAT5 binds to the Ifnb1 promoter and limits recruitment of IRF3. (A) ChIP and quantitative PCR (qChIP) analysis of NFAT5 binding to the Ifnb1 promoter in NFAT5-deficient (Nfat5−/−) and wild-type (Nfat5+/+) BMDMs left untreated (−) or stimulated with poly(I:C) (+; 1 µg/ml, 2.5 h). (B) qChIP analysis of H4ac and total H4 in the Ifnb1 promoter in untreated or poly(I:C)-treated Nfat5−/− and Nfat5+/+ BMDMs. (C) qChIP analysis of p65 and IRF3 recruitment to the Ifnb1 promoter in untreated or poly(I:C)-treated Nfat5−/− and Nfat5+/+ BMDMs. Results show the mean ± SEM of six BMDM cultures of each genotype analyzed in four independent experiments in A; three independent experiments in B, each with cells from one NFAT5-deficient mouse and one wild-type littermate; and five to six BMDM cultures of each genotype analyzed in C from three independent (p65) and four independent (IRF3) experiments. Statistical significance was determined with an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Alignment of the IFNB1 gene promoter regions in different mammalian species. Alignment of the indicated genomic regions was done with Jalview. The positions of IRF-binding sites PRDIII and PRDI and the NF-κB–binding site PRDII are marked with colored boxes. The NFAT5 consensus binding motif is shown in red letters. The position of a conserved NFAT5 consensus binding site that partially overlaps with PRDIII is indicated with red boxes. The NFAT5-binding site in PRDIII is lost in opossum and sheep but is found further downstream in PRDI (5′-TGGAAA-3′; marked with small red boxes). The transcription start sites (TSS) in human and mouse genomes are indicated. The position of the TSS in human IFNB1 in current databases has been updated with respect to the position originally used in the IFNB1 promoter reporter in Fig. 7.
Figure 6.

NFAT5 binds to the Ifnb1 promoter and limits recruitment of IRF3. (A) ChIP and quantitative PCR (qChIP) analysis of NFAT5 binding to the Ifnb1 promoter in NFAT5-deficient (Nfat5−/−) and wild-type (Nfat5+/+) BMDMs left untreated (−) or stimulated with poly(I:C) (+; 1 µg/ml, 2.5 h). (B) qChIP analysis of H4ac and total H4 in the Ifnb1 promoter in untreated or poly(I:C)-treated Nfat5−/− and Nfat5+/+ BMDMs. (C) qChIP analysis of p65 and IRF3 recruitment to the Ifnb1 promoter in untreated or poly(I:C)-treated Nfat5−/− and Nfat5+/+ BMDMs. Results show the mean ± SEM of six BMDM cultures of each genotype analyzed in four independent experiments in A; three independent experiments in B, each with cells from one NFAT5-deficient mouse and one wild-type littermate; and five to six BMDM cultures of each genotype analyzed in C from three independent (p65) and four independent (IRF3) experiments. Statistical significance was determined with an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Alignment of the IFNB1 gene promoter regions in different mammalian species. Alignment of the indicated genomic regions was done with Jalview. The positions of IRF-binding sites PRDIII and PRDI and the NF-κB–binding site PRDII are marked with colored boxes. The NFAT5 consensus binding motif is shown in red letters. The position of a conserved NFAT5 consensus binding site that partially overlaps with PRDIII is indicated with red boxes. The NFAT5-binding site in PRDIII is lost in opossum and sheep but is found further downstream in PRDI (5′-TGGAAA-3′; marked with small red boxes). The transcription start sites (TSS) in human and mouse genomes are indicated. The position of the TSS in human IFNB1 in current databases has been updated with respect to the position originally used in the IFNB1 promoter reporter in Fig. 7.

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