Fig 2: Mbd3 deletion causes severe innate colitis while Batf deletion promotes colitis resolution.a ILC3s isolated from indicated mice were used for ELISA assay to analyze IL-17 production. TKO, circKcnt2−/−Batf−/−Rag1−/− mice. n = 3 independent samples. b Cell numbers of IL-17+ ILC3s were analyzed in indicated mice after DSS treatment. n = 5 for each group. c Analysis of body weight changes of indicated mice after DSS treatment. n = 5 for each group. d H&E staining of colons from indicated mice treated with DSS. Scale bar, 100 μm. e Colitis scores of indicated mice as in d (**P = 0.0011). f ILC3s (3 × 104 cells per mouse) isolated from Mbd3−/−, circKcnt2−/−, circKcnt2−/−Batf−/− (DKO), or WT control mice were transplanted into Rag1−/−Il2rg−/− mice. Two weeks later, mice were administrated with DSS and anti-IL-17 or isotype IgG. Colons were isolated and analyzed by H&E staining. Scale bar, 100 μm. g Colitis scores of indicated mice as in f (*P = 0.0219; ***P = 0.0006). n = 5 independent samples. *P < 0.05 and **P < 0.01. Data are shown as means ± SD. Data were analyzed by an unpaired Student’s t-test and representative of at least three independent experiments. Source data are provided as a Source Data file.

Fig 3: CircKcnt2 recruits the NuRD complex onto Batf promoter to suppress its expression.a Analysis of Mbd3 enrichment on Batf promoter by ChIP assay. ILC3s was isolated from DSS-treated WT mice. n = 3 independent samples. b Enrichment analysis of NuRD complex component proteins on Batf promoter in circKcnt2+/+ and circKcnt2−/− ILC3s after DSS treatment. n = 3 independent samples. c ChIP assay for enrichment of NuRD complex component proteins on Batf promoter in circKcnt2WT and circKcnt2Mut ILC3s from DSS-treated mice. n = 3 independent samples. d FISH assay for analysis of co-localization between Mbd3 and Batf promoter in circKcnt2+/+ and circKcnt2−/− ILC3s from DSS-treated mice. Green, Batf promoter; red, Mbd3; blue, circKcnt2. Scale bar, 5 μm. e LPLs isolated from DSS-treated circKcnt2+/+ and circKcnt2−/− mice were used for ChIP assay with anti-Mbd3. Eluents were subjected to sucrose gradient ultracentrifugation, followed by western blotting and PCR analysis. f LPLs were isolated from DSS-treated circKcnt2WT and circKcnt2Mut mice and used for CHIRP assay using biotin-labeled circKcnt2 probes. g ChIP assay for enrichment analysis of H3K27ac on Batf promoter in Mbd3−/−, circKcnt2−/−Mbd3−/−, or WT control ILC3s. n = 3 independent samples. h DNaseI accessibility assay for Batf promoter accessibility in Mbd3−/−, circKcnt2−/−Mbd3−/−, or WT control ILC3s. n = 3 independent samples. i ILC3s from DSS-treated Mbd3−/−, circKcnt2−/−Mbd3−/−, or WT control mice were subjected to nuclear run-on assay, followed by RT-PCR analysis of Batf. n = 3 independent samples. j, k Analysis of Batf expression by qRT-PCR and western blotting in Mbd3−/−, circKcnt2−/−Mbd3−/−, or WT control ILC3s. n = 3 independent samples. Data were analyzed by an unpaired Student’s t-test and shown as means ± SD. Data are representative of at least three independent experiments. Source data are provided as a Source Data file.

Fig 4: CircKcnt2 associates with Mbd3.a LPL cells from DSS-treated WT mice were lysed and incubated with biotin-labeled and linearized circKcnt2 transcripts, anti-sense, or Sepharose 4B beads control. Precipitants were resolved by SDS-PAGE, followed by silver staining. Indicated bands were identified via mass spectrometry. b LPL lysates were incubated with circKcnt2 probes or controls, followed by western blotting using indicated antibodies. c Electrophoretic mobility shift assay (EMSA) using biotin-labeled circKcnt2 and recombinant Mbd3 with or without anti-Mbd3. d EMSA assay using linearized circKcnt2 RNAs and Flag tagged NuRD complex component proteins. e RNA pulldown assay was performed using linearized and biotin-labeled circKcnt2 RNAs or anti-sense through LPL cell lysates. f CircKcnt2 was co-localized with Mbd3 in ILC3s by immunofluorescence staining. Scale bar, 5 μm. g RNA immunoprecipitation (RIP) assay was conducted using anti-Mbd3 or IgG through LPL lysates from DSS-treated mice (***P = 0.0001). n = 3 independent samples. h RNA pulldown assay was performed using WT circKcnt2 and its different mutations. i EMSA assay using WT circKcnt2 or HR5 mutation. ***P < 0.001. Data were analyzed by an unpaired Student’s t-test and shown as means ± SD. Data are representative of at least three independent experiments. Source data are provided as a Source Data file.