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. 2018 Nov 5;215(11):2901–2918. doi: 10.1084/jem.20180314

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© 2018 Buxadé et al.

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Figure 4. — Binding of NFAT5 to regulatory regions of MHCII-related genes and identification of a remote NFAT5-bound region upstream Ciita. (A) qChIP analysis of NFAT5 binding to promoters of MHCII genes and promoter I of Ciita in untreated or IFNγ-stimulated (400 U/ml, 4 h) wild-type (W) and NFAT5-deficient (KO) BMDMs (n = 3). Results shown are the mean ± SEM of three independent experiments with BMDMs from wild-type and NFAT5-deficient littermate mice. (B) ChIP-seq analysis of wild-type and NFAT5-deficient BMDMs showing the position of NFAT5-binding site peak A 47 kb upstream of the Ciita locus. Two independent experiments are shown. For experiment 1 (accession no. GSE107948), sequences were aligned to mouse chromosome 16 sequence in UCSC Genome Browser NCBIm37/mm9, and for experiment 2 (accession no. GSE107950), the sequence used was mouse chromosome 16 in UCSC Genome Browser GRCm38/mm10. Positions of genes Nubp1 and Tvp23a (Fam18a; the latter overlapping with Ciita peak A) are shown. (C) qChIP analysis of NFAT5 binding to peak A upstream of Ciita in macrophages left untreated (–) or stimulated with IFNγ (100 or 400 U/ml, 4 h; n = 5, except n = 3 for IFNγ 100 U/ml). Results show the mean ± SEM of four independent experiments comparing five pairs of BMDM cultures from wild-type and NFAT5-deficient littermate mice, all including unstimulated cells and treatment with 400 U/ml IFNγ, and three of them also including IFNγ 100 U/ml. (D) qChIP analysis of NFAT5 binding to peak A in conventional myeloid DCs (cDCs) in comparison with BMDMs and peritoneal macrophages (pMs). Binding of NFAT5 to peak A region in cDCs and pMs is represented as relative to its binding in wild-type BMDMs. qChIP with anti-NFAT5 antibodies in NFAT5-deficient BMDMs and with a preimmune serum are shown as negative controls. Results show the mean ± SEM of three independent experiments. (E) mRNA expression of Ciita, Tvp23a, and Nubp1 in wild-type and NFAT5-deficient BMDMs. RNA samples were extracted from three independent pairs of BMDM cultures from wild-type and NFAT5-deficient littermates. The left panels show the comparison between Ciita and Tvp23a or Nubp1 mRNA expression levels, and the right panels show the lack of responsiveness of Tvp23a and Nubp1 to IFNγ stimulation (400 U/ml, 24 h). Statistical significance in A and C–E was determined by an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., not significant.