Journal: Nature Communications

Article Title: NFAT primes the human RORC locus for RORγt expression in CD4 + T cells

doi: 10.1038/s41467-019-12680-x

Figure Lengend Snippet: NF-kB cooperates with NFAT in activating RORC 2 transcription. a NF-kB proteins bind to the RORC 2 promoter in vitro. DNA affinity capture assay was performed in Jurkat E6.1 cells with biotinylated oligos encompassing the NFAT binding sites (see Fig. ). Western blot of the DNA-bound proteins was probed with anti-p65 and anti-p50 antibodies. IL - 2 prom : NFAT binding site from the IL - 2 promoter, neg : sequence in the RORC locus with no NFAT binding site. b NF-kB binds to the human RORC locus in stimulated CD4 + T cells in vivo. CD4 + T cells from cord blood were stimulated for 20 h in the indicated conditions. ChIP was performed with an anti-p65 antibody, followed by RT-qPCR of the RORC promoters and CNS-1.8 kb enhancer region. Data are shown as percent of input, after subtraction of the irrelevant IgG background and represent average and SD of 3 experiments. n.d.: not detected. c NF-kB cooperates with NFAT to activate transcription from the human RORC 2 promoter. HEK293T cells were transfected with expression vectors for p65 and p50 (NF-kB) or the indicated NFAT transcription factors, together with a RORC2 promoter construct. Normalized transcriptional activity is represented as fold changes over the activity of the RORC 2 promoter alone (first bars). Data shown represent average and SD of 11, 6 and 10 independent experiments (for left, middle and right graphs respectively). d NFATc3 activates transcription from the RORC 2 promoter. HEK293T cells were transfected with the RORC 2 promoter in the presence or absence of NFATc3. Luciferase activity was measured after 20 h and represented as fold change relative to activity of the RORC 2 promoter alone (first bar). Plotted are average and SD from 14 transfections. e NFATc3 binds to the RORC locus. Extracts from sorted DPint and SP CD4 + thymocytes were subjected to chromatin immunoprecipitation with anti-NFATc3 antibodies (left panel, average and SD of 2–4 replicates for the different regions) and anti-NFATc1 antibodies (right panel, one representative experiment). NFAT enrichment is represented as percentage of input after background subtraction. Shown are the p-values from two-tailed t -tests performed between the indicated conditions. Source data are provided as a

Article Snippet: Cells were transfected in a 48 well plate with 100 ng of pGL3 reporter vectors, 5 ng of pRL-TK vector (Renilla luciferase, to normalize for transfection efficiency), and 180 ng of the following expression vectors: pEF6-NFATc2 expression vector (a kind gift from O. Kaminuma), pEGZ-NFATc1A and pEGZ-NFAT1C (a kind gift from F. Berberich-Siebelt), pCMV4-p50 (purchased from Addgene) and pCDNA-p65 (a kind gift from G. Natoli).

Techniques: In Vitro, Binding Assay, Western Blot, Sequencing, In Vivo, Quantitative RT-PCR, Transfection, Expressing, Construct, Activity Assay, Luciferase, Chromatin Immunoprecipitation, Two Tailed Test