Fig. 5: Activated β-catenin signaling influences ILC3s via epigenetic regulation mechanisms. | Nature Communications

Fig. 5: Activated β-catenin signaling influences ILC3s via epigenetic regulation mechanisms.

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

Fig. 5

a Living ILC3s isolated from Rag1–/– mice were treated with DMSO and CHIR-99021 (CHIR) for 24 hours in vitro. The immunoprecipitation (IP) was performed with IgG or anti-β-catenin antibody. Immunoblotting (IB) of β-catenin, TCF-1, and GAPDH was shown. b ILC3s from Ctnnb1ex3fl/wt (Ctrl) mice and CreERT2Ctnnb1ex3fl/wt (KO) mice were isolated and treated with 4-hydroxytamoxifen. The TCF-1 binding regions were detected by CUT&Tag assay and sequencing at day 9. Intergrative genomics viewer (IGV) browser view of control and TCF-1 binding peaks at Rorc gene locus is shown. c Luciferase activity in 293T cells transfected with vector alone (pGL3-vec) or vector containing Rorc promoter (pGL3-R1), together with empty vector (pcDNA-vec) or vector expressing TCF-1 (pcDNA-TCF-1). The results represent the relative firefly luciferase activities normalized to the corresponding Renilla luciferase activities. d–f ILC3s from Ctnnb1ex3fl/wt (Ctrl) and CreERT2Ctnnb1ex3fl/wt (KO) mice were treated with 4-hydroxytamoxifen. The histone modification marks and chromatin accessible regions were detected by CUT&Tag assay and sequencing on day 9. d Venn diagram comparing genes that gained less H3K4me3 modification in CreERT2Ctnnb1ex3fl/wt ILC3s (green), genes that gained less H3K27ac modification in CreERT2Ctnnb1ex3fl/wt ILC3s (red) and transcriptionally down regulated genes in CHIR-treated ILC3s (blue). GO enrichment analysis of Venn diagram overlapping genes is also shown. e RNA expression heatmap of indicated Venn diagram overlapping genes (n =  3 biological replicates for each group) are shown. f De novo transcription factor–binding motif analysis (HOMER) of more accessible regions in Ctrl and KO ILC3s. The most highly significantly enriched motifs and corresponding P values are listed. Each dot represents one individual replicate (n = 4 in c). Error bars represent the SEM. Statistical significance was tested by two-sided one-way ANOVA with Tukey’s test adjusted for multiple comparisons (c). Data are representative of three independent experiments (a, c).

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