Fig 2: Intersection of immune disease risk loci, open chromatin regions, and TF-binding interactions with the genome. (A–C) Heatmaps for inflammatory bowel disease (A), multiple sclerosis (B), and allergic disease, asthma, hay fever, or eczema (C) risk loci are shown. The x axis displays disease-associated loci. An asterisk means that the genomic coordinates of the ChIP-seq peaks measuring open chromatin regions or TF binding (at 5 h) significantly intersect with the disease risk loci at a RELI-corrected P value threshold of 10-6. A colored box indicates that the given locus contains at least one disease-associated variant located within an open chromatin region or a ChIP-seq peak for the given TF. (D) The genome browser screenshots shows the ATAC-seq in naive and activated T cells, ChIP-seq (cFOS, JUNB, and NFAT1 at 5 h activation), and heatmap of the H3K4me2 level in Th2 cells of patients with asthma and healthy controls in the IL2RA and PHF19 loci. Red bars indicate differentially enriched loci (FDR is shown under the bars), and a pink background indicates EORs. Representative data were pooled for A–C from one to three independent experiments using a total of three donors.

Fig 3: SRC2 recruited by NFAT1 binds to the promoter and active gene expression of Nr4a2.(A) Schematic representation of the six regions on Nr4a2 promoter covered by P0 to P5 primers, the locations of identified NFAT1-binding sites, and the region deleted using CRISPR-Cas9. (B and C) ChIP-qPCR analysis of SRC2 binding (B) or NFAT1 binding (C) to the Nr4a2 promoter (P0 to P5 regions) in CD4+ cells from indicated genotypes under Treg polarization at the indicated time points (n = 3 per genotype per group). (D) Immunoblot analysis of NFAT1 among anti-SRC2 antibody immunoprecipitated (IP) SRC2 complexes from SRC2fl/fl CD4+ cells 24 hours after Treg polarization. Bottom blots, whole-cell lysate input control. (E) PCR analysis of the abundance of NFAT1/SRC2 binding region on Nr4a2 promoter in CD4+ cells transduced with virus expressing nontarget control (NTC) or the region containing NFAT1/SRC2-binding guiding RNAs shown in (A) (crNr4a2) and polarized under Treg condition for 40 hours. (F) Immunoblot analysis of Nr4a2 in naive CD4+ cells or CD4+ T cells transduced with virus expressing NTC and crNr4a2 shown in (E) and polarized under Treg conditions for 40 hours. The number in the bottom of the blots is the relative mean intensity of each band, and the right panel is the summary of the relative mean intensity. (G) Representative flow cytometric analysis of Foxp3 (left panels), percentages of Foxp3+ cells (middle panels), and MFI for Foxp3 (right panels) among GFPhiCD4+ cells transduced with virus expressing NTC, crFoxp3, and crNr4a2 guiding RNAs and polarized under Treg condition at the indicated time points (n = 3 per treatment per group). Data are from three experiments (B, C, and F, right panels; G, middle and right panels; presented as means ± SD) or are from one representative of three independent experiments (D to G, left panels). *P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t test). MW, molecular weight; bp, base pairs.

Fig 4: NFATs are relevant in human oligodendrocytes. a NFAT expression in human iOLs (grey bars) and NPCs (open bars) by qrtPCR after normalization to GAPDH. Average NFAT levels in NPCs were set to 1 (NFATc1: 1.4 ± 0.3; NFATc2: 14.6 ± 1.9; NFATc3: 1.6 ± 0.3 and NFATc4: 0.9 ± 0.2). b Immunocytochemistry of MBP-positive (red) iOL with antibodies directed against NFATs (white in upper row, green in lower row); counterstaining with Hoechst dye (blue). c–g Analysis of differentiating iOL cultured 21 (c, e, h) or 35 days (d, f, g, i, j) in absence (Ctr, open bars) or presence of 0.1 (light grey bars) to 0.5 µM (dark grey bars) VIVIT (c–g, n = 4) or 1 (light grey bars) to 10 µM (dark grey bars) FK506 (h–j, n = 3–4). VIVIT/FK506 incubation was continuous (c–f, h, i) or from days 21–35 (g, j). Cultures were stained with anti-O4 (green, c) and anti-MBP antibodies (green, d). From staining and FACS analysis (see Suppl. Fig. 7c, d) the fraction of O4-positive cells after 21 days (e, h) and MBP-positive cells after 35 days (f, i) was determined. For treatment during days 21–35, the fraction of FACS-sorted O4-positive cells was determined that had reached a MBP-positive stage (g, j). The relative number of marker-positive cells under control conditions was set to 1 and used to normalize in pairwise fashion (values: 1 for control, 0.97 ± 0.01 for 21 days 0.1 µM VIVIT, 0.68 ± 0.06 for 21 days 0.5 µM VIVIT, 0.86 ± 0.10 for 35 days 0.1 µM VIVIT, 0.60 ± 0.10 for 35 days 0.5 µM VIVIT, 0.82 ± 0.05 for 0.1 µM VIVIT from days 21–35, 0.86 ± 0.04 for 0.5 µM VIVIT from days 21–35, 0.99 ± 0.21 for 21 days 1 µM FK506, 0.39 ± 0.17 for 21 days 10 µM FK506, 1.12 ± 0.36 for 35 days 1 µM FK506, 0.55 ± 0.09 for 35 days 10 µM FK506, 0.83 ± 0.17 for 1 µM FK506 from days 21–35 and 0.86 ± 0.10 for 10 µM FK506 from days 21–35). k–n NFAT immunohistochemistry in human brain tissue. Antibodies directed against NFATc3 and NFATc4 (red in k and l, black in m) were combined with anti-NOGOA antibodies (green in k and l, red in m). Arrows mark double positive cells, arrowheads NOGOA-positive oligodendrocytes without NFATc4. n Percentage of NOGOA-positive oligodendrocytes with nuclear NFATc4 in periplaque white matter (86 ± 4%), active and demyelinating lesions (41 ± 8%), active and post-demyelinating lesions (41 ± 8%), active and remyelinating plaques (59 ± 19%), and control CNS tissue (99 ± 1%). o Model of oligodendroglial Nfat action. Statistical significance was determined by two-tailed Student’s t-test (a) or Bonferroni-corrected one-way ANOVA (e–j, n) (*P = 0.05; **P = 0.01; ***P = 0.001). Scale bars, 50 µm

Fig 5: AS stimulates NFATc2 expression by activating p53.(A) HCT116 cells left untreated (Untreated), treated with AS 10 µM (AS), or infected with lentivirus carrying control shRNA for 96 hours (Control shRNA), then left untreated or treated with AS for 24 hours (Control shRNA + AS), or infected with lentivirus carrying p53 shRNA for 96 hours, then left untreated (p53 shRNA) or treated with AS for 24 hours (p53 shRNA + AS), and blotted with the antibodies as indicated. (B) RT-PCR of NFATc2 of HCT116 cells with the identical treatment. Data represents the result of one of the three similar experiments. *p < 0.05 indicates statistically significant difference between (Control shRNA + AS) and (p53 shRNA + AS). (A). (C) Western blot of NFATc2 and p53 in HCT116 cells left untreated or treated with AS, or PFTa 60 µM, or AS plus PFTa for 24 hours. PFTa was added one hour before AS. (D) NFATc2 promoter was separated into ten different segments by PCR as indicated and subjected to ChIP with anti-p53 antibody. In untreated HCT116 cells (-AS), p53 bond to the DNA sites on -2481 to -1564 (segments 2, 3, and 4) and -360 to +220 (segments 9 and 10). Upon AS stimulation (+AS), p53 bond to the sites on -1882 to -1564 (segment 4), and -669 to +220 (segments 8, 9 and 10). TSS, transcription start site. Numbers indicate relative position from the TSS. L = ladder. (E) Luciferase assay to determine p53 responsive sites on the NFATc2 promoter. HCT116 cells were transfected in triplicate with pCDNA3.1-p53 plasmid and an empty pGL3B vector as control (C), or pGL3B containing promoter sequence from -2481 to +220, from -2481 to -66, from -1882 to +220, from -1563 to +220, from -2481 to -670, and from -2481 to +220 with segment 4 deleted. Data represented the results without (-AS) or with (+AS) the treatment of AS for 12 hours. kDa, molecular weight. Error bar represents the confidence interval of the triplicate results. Nearly identical results were obtained with a repeated experiment.