Abstract
Intestinal stem cells (ISCs) are maintained by stemness signaling for precise modulation of self-renewal and differentiation under homeostasis. However, the way in which intestinal immune cells regulate the self-renewal of ISCs remains elusive. Here we found that mouse and human Lgr5+ ISCs showed high expression of the immune cell–associated circular RNA circPan3 (originating from the Pan3 gene transcript). Deletion of circPan3 in Lgr5+ ISCs impaired their self-renewal capacity and the regeneration of gut epithelium in a manner dependent on immune cells. circPan3 bound mRNA encoding the cytokine IL-13 receptor subunit IL-13Rα1 (Il13ra1) in ISCs to increase its stability, which led to the expression of IL-13Rα1 in ISCs. IL-13 produced by group 2 innate lymphoid cells in the crypt niche engaged IL-13Rα1 on crypt ISCs and activated signaling mediated by IL-13‒IL-13R, which in turn initiated expression of the transcription factor Foxp1. Foxp1 is associated with β-catenin in rendering its nuclear translocation, which caused activation of the β-catenin pathway and the maintenance of Lgr5+ ISCs.
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Data availability
Microarray data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE123051, GSE123083 and GSE123353. Full scans of all blots and gels are shown in Supplementary Fig. 8. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank Y. Xu and Y. Teng for technical support. We thank J. Li (Cnkingbio Company) for technical support. This work was supported by the National Natural Science Foundation of China (grants 91640203, 31530093, 31771638, 81672897, 81672956, 81472413, 81572433, 81772646 and 31601189), Strategic Priority Research Programs of the Chinese Academy of Sciences (XDB19030203 and XDA12020219) and Beijing Natural Science Foundation (grant 7181006); and a Postdoctoral Innovative Talent Support Program to P.Z.
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P.Z. designed and performed the experiments, analyzed the data and wrote the paper. X.Z. and D.F. generated genome-modified mice. J.W., L.H. and T.L. performed the experiments and analyzed the data. Y.W., B.L., B.Y., L.S., J. W., L.Y., X.Q., Y.D. and C.L. performed some of the experiments. L.H., W.R. and X.W. provided human samples. Y.T. initiated and analyzed the data. Z.F. initiated the study and organized, designed and wrote the paper.
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Supplementary Figure 1 Validation of upregulated circRNAs in ISCs.
a, Validation of circRNA expression levels in Lgr5-GFP+ ISCs and Lgr5-GFP– non-ISCs through real-time PCR. CircRNA expression levels were normalized to those of non-ISCs. n = 6 mice. b, Complementary DNA (cDNA) and genomic DNA (gDNA) were used as templates to amplify circRNAs in Lgr5-GFP+ ISCs with divergent and convergent primers. Due to large introns in gDNAs, different sets of divergent and convergent primers were used in the lower panel. c, Validation of circRNAs in Lgr5-GFP+ ISCs. Genomic compositions of circRNAs were depicted (upper panel). PCR products generated from Lgr5-GFP+ ISCs using divergent primers were sequenced (lower panel). d, Total RNAs from Lgr5-GFP+ ISCs were treated with or without 3 U/μg RNase R for 1 h, followed by RNA extraction and real-time PCR analysis. n = 3 independent experiments. e, Lgr5-GFP+ ISCs were treated with 2 μg/ml actinomycin D, followed by RNA extraction and real-time PCR analysis of circRNAs. n = 3 independent experiments. f, real-time PCR detection for knockdown efficiency of indicated circRNAs in Lgr5-GFP+ ISCs. n = 3 independent experiments. In all panels, data are shown as the mean ± s.d. ** P < 0.01; *** P < 0.001, by one-tailed Student’s t-test.
Supplementary Figure 2 Characterization of circPan3 and generation of circPan3RFP reporter mice.
a, Schematic representation of mouse circPan3. Black arrowheads denote primers for linear Pan3 detection and gray arrowheads denote primers for circPan3. b, Diagram of 21 cricRNAs derived from Pan3 transcript. CircRNAs were named by their lengths. c, Expression levels of the indicated circRNAs were examined by real-time PCR. The used primers were listed in Supplementary Table 3. n = 4 mice. d, Nuclear and cytoplasmic fractionation of Lgr5-GFP+ ISC lysates with real-time PCR analysis. n = 4 independent experiments. e, Western blot to detect the peptide-coding potential of circPan3. f, Scheme for generation of circPan3RFP mice. g, circPan3RFP mice were identified by agarose gel electrophoresis. h, CircPan3 and Pan3 expression levels in 2-month-old mouse tissues were analyzed by real-time PCR. n = 4 independent experiments. i, Indicated tissues were used for circPan3 examination by Northern blot. 18 S rRNA was detected as a loading control. j, CircPan3RFPLgr5GFP mice were generated by crossing CircPan3RFP with Lgr5GFP mice, and intestinal tissues were analyzed by confocal microscopy. k, Indicated intestinal sections from Lgr5GFP mice were used for circPan3 in situ hybridization. l, Schematic representation of human circPAN3. Black arrowheads denote primers for linear PAN3 detection and gray arrowheads denote primers for circPAN3. m, Northern blot for circPAN3 detection in human intestine tissues. 18 S rRNA was used as a loading control.
Supplementary Figure 3 CircPan3 knockout impairs the stemness of Lgr5-GFP+ ISCs.
a, Confirmation of intronic sequences responsible for circPan3 formation. b, Minigene assay for circPan3 detection in circPan3 silenced Lgr5-GFP+ ISCs infected with lentiviruses carrying the indicated constructs. Black arrowhead denotes circPan3. c, Scheme for circPan3 knockout mice. Intronic sequences (#2) were deleted through CRISPR/Cas9 approach. d, circPan3−/− mice were identified by agarose gel electrophoresis. e, circPan3−/− in Lgr5-GFP+ ISCs was detected by PCR. Black arrowheads denote primers for linear Pan3 detection and gray arrowheads denote primers for circPan3. f, g, CircPan3 and Pan3 expression levels in circPan3−/− mice were detected by Northern blot (f) and Western blot (g). h, Expression levels of the indicated circRNAs in circPan3−/− and circPan3+/+ Lgr5-GFP+ ISCs were examined by real-time PCR. n = 4 independent experiments. i, Pan3 expression levels in Lgr5GFPcircPan3−/− mice were detected by Western blot. j, FACS analysis of Lgr5-GFP+ ISCs in small intestines of Lgr5GFPcircPan3+/+ and Lgr5GFPcircPan3−/− mice. Numbers indicated the percent of Lgr5-GFP+ ISCs in the gate, and were shown as means ± SD. n = 6 for each group. k, mRNA expression levels of the indicated ISC markers were examined by real-time PCR. n = 5 for each group. l, Western blot for Pan3 detection in LRlacZcircPan3+/+ and LRlacZcircPan3–/– mice. m, Representative β-galactosidase images from LRlacZcircPan3+/+ and LRlacZcircPan3–/– intestines (n = 6 per group). n, Schematic representation of intestinal regeneration. o, Scheme for generation of circPan3CTG mice. ATG codon of circPan3 peptide was mutated to CTG. p, PCR products from circPan3CTG mice were used for DNA sequencing to confirm the mutation of ATG. q, Lgr5GFPcircPan3CTG/CTG mice were used for Lgr5-GFP+ ISCs observation by confocal microscopy. Scale bars, 50 μm. n = 200 fields pooled across 6 mice were used for Lgr5-GFP+ ISC detection, and data are shown as mean ± s.d. ns, not significant, by one-tailed Student’s t-test. r, Pan3 expression in indicated mice was detected by Western blot. Data represent at least three independent experiments.
Supplementary Figure 4 Pan3 knockout does not affect intestinal structure or ISC self-renewal maintenance.
a, b, Establishment of Pan3 knockout mice. Pan3 knockout mice were generated through CRISPR/Cas9 approach, and confirmed by DNA sequencing (a). Pan3 knockout efficiency was confirmed by Western blot (b). c, circPan3 expression in Pan3 knockout mice via real-time PCR. n = 4 independent experiments. d, Representative H&E images from Pan3 knockout intestine from 2-month-old Pan3+/+ and Pan3–/– mice were sacrificed and intestines were analyzed with H&E staining. n = 200 fields pooled across four mice. e, ISC staining for ISC in intestines from Pan3+/+ and Pan3–/– mice. n = 200 fields pooled across four mice were observed for cell number calculation. f, Organoid formation of Pan3+/+ and Pan3–/– Lgr5-GFP+ ISCs. n = 6 independent experiments. g, Organoid formation of Pan3 overexpressing Lgr5-GFP+ ISCs established using lentivirus. n = 6 independent experiments. h, i, poly(A) tail was detected in circPan3 knockout and overexpressing Lgr5-GFP+ ISCs. For h, WT, Pan3–/– and circPan3–/–Lgr5-GFP+ ISCs were used for poly(A) detection. For i, circPan3-overexpressing Lgr5-GFP+ ISCs were used for poly(A) detection. In all panels, data are shown as the mean ± s.d. ns, not significant, by one-tailed Student’s t-test. For all representative images, at least three independent experiments were performed with similar results.
Supplementary Figure 5 circPan3 interacts with Il13ra1 mRNA in Lgr5-GFP+ ISCs.
a, The indicated downregulated genes in circPan3−/− Lgr5-GFP+ ISCs were confirmed by real-time PCR. n = 3 PCR primers were listed in Supplementary Table 3. b, The indicated downregulated genes were depleted in Lgr5-GFP+ ISCs, followed by reassociation organoid formation assays. Organoid formation ratios were calculated. n = 6 independent experiments. c, d, Lgr5-GFP+ ISC cells were isolated from indicated mice, and IL-13Rα1 protein expression on surface and in total was detected by FACS (c) and Western blot (d), respectively. e, Double FISH assays were performed for circPan3 (red) and Il13ra1 mRNA (green) co-localization. f, RNA pulldown assay by incubation of Biotin-labeled circPan3 and Lgr5-GFP+ ISC lysates. Eluates were collected for RNA extraction, and Il13ra1 was detected by real-time PCR. Gapdh served as a negative control. n = 4 independent experiments. g, Prediction of stem-loop structures of circPan3. Predictions were based on minimum free energy (MFE) and partition function. Color scales denote confidence of predictions for each base with shades of red indicating strong confidence (http://rna.tbi.univie.ac.at/). HR, hairpin region. h, Lgr5-GFP+ ISCs carrying indicated circPan3 mutant were used for detection of circPan3-Il13ra1 mRNA interaction, and Il13ra1 enrichment was detected by real-time PCR. n = 3 independent experiments. i, Il13ra1 mRNA was labeled with Biotin via in vitro transcription, incubated with indicated circPan3 transcript, and subjected into native PAGE for RNA mobility shift assays. j, circPan3-Il13ra1 mRNA interaction detection in Lgr5-GFP+ ISCs carrying WT/mutant. Eluates were examined for circPan3 enrichment by real-time PCR. n = 3 independent experiments. k, Biotin-labeled Il13ra1 mRNA was incubated with indicated circPan3 mutations, followed by RNA mobility shift assays. l, Diagram of Il13ra1 mRNA was shown in upper panel and matching sequences and positions were shown in lower panel. In all panels, data are shown as the mean ± s.d. *** P < 0.001, by one-tailed Student’s t-test. For all representative images, at least three independent experiments were performed with similar results.
Supplementary Figure 6 circPan3 promotes Il13ra1 mRNA stability through competitively binding Il13ra1 mRNA with Ksrp protein.
a, MS-MS profiles of Ksrp, corresponding peptide sequences are listed on the top of the corresponding graphs. b, c, Il13ra1 mRNA levels in Ksrp+/+ and Ksrp−/− Lgr5-GFP+ ISCs by real-time PCR (b) and Western blot (c). For b, n = 5 independent experiments. d, Ksrp overexpressing Lgr5-GFP+ ISCs were established and incubated in organoid medium. After actinomycin D treatment, Lgr5-GFP+ ISCs were obtained for detection of Il13ra1 mRNA by real-time PCR. For mRNA curve, Il13ra1 mRNA levels were normalized to its expression of 0 min. n = 3 independent experiments. e, f, circPan3 expressing Lgr5-GFP+ ISCs were used for Il13ra1-Ksrp detection by Il13ra1 mRNA pulldown (e) and Ksrp RNA immunoprecipitation (f). For f, n = 6 independent experiments. g, h, circPan3 mutant transcript (circPan3-Mut) was added into Lgr5-GFP+ ISC lysates, followed by detection of Il13ra1 mRNA pulldown (g) and Ksrp RNA immunoprecipitation (h). For h, n = 6 independent experiments. i, Ksrp+/+ and Ksrp−/− Lgr5-GFP+ ISCs were used for circPan3 overexpression, and Il13ra1 mRNA expression was detected by real-time PCR. n = 5 independent experiments. j, Il13ra1 mRNA stability in indicated Lgr5-GFP+ ISCs was detected by real-time PCR as in (d). n = 3 independent experiments. k, Schematic representation for generation of Il13ra1flox mice. Two loxPs were inserted into Il13ra1 allele flanking at the exon 2 of Il13ra1 gene. l, Il13ra1 knockout was tested by agarose gel electrophoresis. m, Lgr5GFPIl13ra1f/f mice were intraperitoneally injected with 2 μg TAM. Then Il13ra1 deletion was confirmed by Western blot. n, Representative images of LRlacZIl13ra1f/f and LRlacZ intestines (n = 6 per group). Scale bars, 100 μm. o, Scheme for generation of Il13 loxp-Stop-loxp (Il13lsl) mice. p, Il13lsl/lsl mice were identified by agarose gel electrophoresis. q, Il13 deletion was detected by Western blot. Samples were entire small intestines of Il13lsl/lsl mice. r, Il13lsl/lsl mice were crossed with LRlacZ mice, and Lgr5-GFP+ ISCs was detected by lacZ staining. n = 8 mice were observed for each group. In all panels, data are shown as the mean ± s.d. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant, by one-tailed Student’s t-test. For all representative images and blots, at least three independent experiments were performed with similar results.
Supplementary Figure 7 IL-13 produced by ILC2s maintains Lgr5-GFP+ ISCs.
a, Lgr5GFP mice were stained with anti-CD3 and anti-CD4 antibodies for Th cells. b, ILC2s and ILC3s enriched from CD45.1 mice were transplanted into Lgr5GFP;Rag1−/− mice, and CD45.1 was examined in intestinal tissues of CD45.2 recipient mice 3 days later. c. Cell numbers of ILC1s, ILC2s and ILC3s in intestine tissues of PlzfCreRosa26lsl-DTR mice after DT treatment. d, ILC1, ILC2 and ILC3 cells were transferred into DT treated PlzfCreRosa26lsl-DTR mice, and Lgr5-GFP+ ISCs were stained with Lgr5 FISH. e, 200 μg anti-CD90, anti-CD25 or control IgG was injected (i.p.) into mice and ILC2 numbers were tested by FACS 3 days later. n = 8 mice were observed for each group. f, C57BL/6 mice were treated with the indicated antibodies to deplete ILCs, plus treatment with IL-13 and anti-IL-13 antibody, followed by Lgr5 detection via in situ hybridization. n = 200 fields pooled across 6 mice were used for cell number detection. g, Lgr5-GFP+ ISCs were mixed with activated ILC2s at 1:1, followed by organoid formation. Scale bars, 200 μm. n = 4 independent experiments. h, 1 × 104 YFP labeled circPan3 or Il13ra1 knockout Lgr5-GFP+ ISCs (LRYFP) were mixed with lacZ labeled control Lgr5-GFP+ ISCs (LRlacZ), and organoids were collected and analyzed by FACS two weeks later. i, Phosphorylation of Stat6 in the indicated ISCs was examined with Western blot. j, Western Blot for knockout efficiency in Stat6+/+ and Stat6−/− Lgr5-GFP+ ISCs. k, Gene set enrichment analysis (GSEA) for Wnt/β-catenin activation. n = 65 Wnt/β-catenin target genes were used for GSEA. l, β-catenin subcellular location in Stat6 blockade intestine tissues was examined by immunohistochemistry. n = 200 fields pooled across 6 mice were used for β-catenin observation. m, Nuclear and cytoplasmic separation was performed using Stat6+/+ and Stat6−/− crypts, followed by Western blot for β-catenin subcellular location. n, Stat6 blockade and Lgr5-GFP+ ISCs were collected for Foxp1 mRNA examination by real-time PCR. n = 6 mice were used for each group. o, p, Representative organoids from of Foxp1−/− and Stat6−/− Lgr5-GFP+ ISCs were shown in o, and Lgr5-GFP+ ISCs in organoids were examined by FACS (p). DKO, double knockout. n = 3 mice were used for each group. q, The indicated Lgr5-GFP+ ISCs were used for β-catenin nuclear translocation. n = 20 fields were used for β-catenin observation. r, Foxp1−/− and recused crypts were collected and expression levels of the indicated Wnt/β-catenin target genes were examined by real-time PCR. n = 3 independent experiments. s, β-catenin nuclear translocation was observed in the indicated Lgr5-GFP+ ISCs. n = 20 fields were used for β-catenin observation. t, β-catenin nuclear translocation in circPan3, Il13 and Il13ra1 knockout mice. n = 200 fields pooled across 6 mice were used forβ-catenin observation. In all panels, data are shown as the mean ± s.d. ** P < 0.01, *** P < 0.001, ns, not significant, by one-tailed Student’s t-test. For all representative images and blots, at least three independent experiments were performed with similar results.
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Supplementary Figures 1-7, Supplementary Tables 1-3 and Supplementary Dataset 1
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Zhu, P., Zhu, X., Wu, J. et al. IL-13 secreted by ILC2s promotes the self-renewal of intestinal stem cells through circular RNA circPan3. Nat Immunol 20, 183–194 (2019). https://doi.org/10.1038/s41590-018-0297-6
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DOI: https://doi.org/10.1038/s41590-018-0297-6
