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. 2021 Jan 20;12:481. doi: 10.1038/s41467-020-20786-w

Fig. 1. Identification and characterization of a novel active enhancer of the RORγt gene.

Fig. 1

a IGV browser views of H3K4me2 profiles of RORCE (chr3: 94,169,746-94,173,149), which is indicated by red box, in Th17 cells, Th1 cells, and ILCs. RORγt and its isoform RORγ are encoded by Rorc locus through the activation of alternative promoters RORγt promoter (RORγt p) and RORγ promoter (RORγ p), respectively. b RORCE1 (chr3: 94,169,746-94,170,945), RORCE2 (chr3: 94,170,946-94,172,113), RORCE3 (chr3: 94,172,215-94,173,149), RORCE1/2 (chr3: 94,169,746-94,172,113), RORCE2/3 (chr3: 94,170,946-94,173,149), and full-length RORCE (RORCEf, chr3: 94,169,746-94,173,149) were cloned upstream of RORγt promoter (RORγt p) in pGL3 vectors. c–e Dual-luciferase assays of the above six reporter constructs (b) in EL4 (c), 293T cells (d), or Th17-polarized cells (e). 293T, EL4, and Th17-polarized cells were transfected with indicated plasmids and cultured at 37 °C. After 24 h of transfection, cells were stimulated with PI for 4–5 h. The cells were harvested, lysed, and assessed with dual-luciferase reporter system. Renilla luciferase activity of pRL-TK was used for normalization. f RORCE1, RORCE2, RORCE3, RORCE1/2, RORCE2/3, and RORCEf were cloned downstream of the luciferase gene in pGL3-RORγt p vectors, and then Dual-luciferase assays of various reporter constructs in Th17-polarized cells were performed. Mean ± SEM are shown, n = 5 independent experiments, unpaired two-tailed Student’s t-test (c–f). Experimental mice were between 8 and 12 weeks of age, with no preference to gender and were maintained on a C57BL/6 background. Source data are provided as a Source Data file.