Fig. 6: NFATc2 and JunB regulate RORγt expression in ILC3s. | Nature Communications

Fig. 6: NFATc2 and JunB regulate RORγt expression in ILC3s.

From: Dysregulation of Wnt/β-catenin signaling contributes to intestinal inflammation through regulation of group 3 innate lymphoid cells

Fig. 6

a–c ILC3s from Ctnnb1ex3fl/wt (Ctrl) and CreERT2Ctnnb1ex3fl/wt (KO) mice were treated with 4-hydroxytamoxifen. The chromatin accessible regions, RNA polymerase II (pol II) binding regions, and histone modification marks (H3K4me3 and H3K27ac) were detected by CUT&Tag assay and sequencing. a Intergrative genomics viewer (IGV) browser view around Rorc gene locus is shown. P1–P6 indicate chromatin-accessible regions surrounding the Rorc gene locus. b Sequences of P1–P4 were selected and the potential binding transcription factors (TFs) were predicted by MotifFinder. Venn diagram comparing the predicted TFs which have the potential to bind to P1–P4 (gray), transcriptionally upregulated (red) and downregulated (blue) genes by RNA-seq in CHIR-treated ILC3s in Fig. 4. c The upregulated transcription factors (red) and downregulated transcription factors (blue) which have the potential to bind to P1–P4 are shown. d Luciferase activity in 293T cells transfected with vector alone (pGL3-vec) or vector containing P3 (pGL3-P3), together with empty vector (pcDNA-vec) or vector expressing KLF7 (pcDNA-KLF7), NFATc2 (pcDNA-NFATc2) and JunB (pcDNA-JunB), respectively. The results represent the relative firefly luciferase activities normalized to the corresponding Renilla luciferase activities. e ILC3s from Ctnnb1ex3fl/wt (Ctrl) and CreERT2Ctnnb1ex3fl/wt (KO) mice were treated with 4-hydroxytamoxifen. The NFATc2 and JunB binding peaks at Rorc gene locus were detected by CUT&Tag assay and sequencing, as shown in the IGV browser view. f ILC3s isolated from Rag1−/− mice were treated with DMSO and CHIR-99021 (CHIR) for 24 hours in vitro. Immunoblotting (IB) of NFATc2, β-catenin, JunB, and GAPDH are shown. g, h Flow cytometry analysis of RORγt expression and relative mRNA expression of Rorc in ILC3s transfected with Ctrl siRNA and Nfatc2 siRNA (g) or Junb siRNA (h). Each dot represents one individual replicate (n = 4 in d, n = 3 in g, h). Error bars represent the SEM. Statistical significance was tested by two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons (d) and unpaired two-sided Student’s t-test (g, h). Data are representative of three independent experiments (d, f, g, h).

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