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Single-cell RNA sequencing and total RNA sequencing data, respectively, evaluating the expression of selective genes from C57BL/6 mouse-derived small intestinal organoids and whole ileal tissue from naïve Il22Ra1fl/fl;Defa6-cre+/- mice

ABSTRACT: All intestinal epithelial lineages express the IL-22 receptor; however, it is not known whether IL-22-dependent regulation of small intestinal host defense is dependent on ISCs or a specific epithelial lineages. To examine differences in gene expression among small intestinal enterocytes, single cell RNA sequencing was performed on primary small intestinal organoids derived from naïve C57BL/6 mice. Our scRNA-seq results demonstrate that Paneth cells express the highest level of Il22ra1 when compared to ISCs as well as enterocyte, goblet, tuft, and endocrine lineages. Additionally, total RNA sequencing (RNA-seq) of the terminal ileum of naïve Il22Ra1fl/fl;Defa6-cre+ (Paneth cell-specific IL22Ra1 knockout) and littermate cre- mice was performed. Our sequencing data revealed reduced expression of Paneth cell-specific Lyz1, Mmp7, and select alpha-defensin antimicrobial peptides but increased expression of IL-22 inducible Reg3g and serum amyloid genes (Saa1 and Saa3) in the terminal ileum of naïve Il22Ra1fl/fl;Defa6-cre+ mice

ORGANISM(S): Mus musculus

PROVIDER: GSE159423 | GEO | 2020/10/14

REPOSITORIES: GEO

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Project description:Background & Aims: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions. Methods: We generated mice with intestinal epithelial-specific disruption of Cul4b (pVillin-cre; Cul4bfn/Y), inducible disruption of Cul4b (Lgr5-creERT2; Cul4bfn/Y, CAG-creERT2; Cul4bfn/Y) and their control (Cul4bfn/Y). Intestinal tissues were analyzed by histology, immunofluorescence, RNA sequencing and mass spectrum. Intestinal organoids deprived from mice with pVillin-Cre; Cul4bfn/Y, Lgr5-Cre; Cul4bfn/Y, Tg-Cul4b and their controls were used in assays to measure intestinal self-renewal, proliferation and differentiation. Wnt signaling and intestinal markers were analyzed by immunofluorescence and immunoblot assays. Differential proteins upon Cul4b ablation or Cul4b-interacting proteins were identified by mass spectrometry. Results: Cul4b specifically located at ISCs zone. Block of Cul4b impaired intestinal homeostasis maintenance by reduced self-renewal and proliferation. Transcriptome analysis revealed that Cul4b-null intestine lose ISC characterization and showed disturbed ISC niche. Mechanistically, reactivated Wnt pathway could recover intestinal dysfunction of Cul4b knockout mice. Analysis of differential total and ubiquitylated proteins uncovered the novel targeting substrate of Cullin-Ring ubiquitin ligase 4b (CRL4b), immunity-related GTPase family M member 1 (Irgm1) in intestine. Decreased Irgm1 rescued abnormally interferon signaling, overemphasized autophagy and downstream phosphate proteins in Cul4b knockout mice. Conclusion: We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

Dataset Information

Single-cell RNA sequencing and total RNA sequencing data, respectively, evaluating the expression of selective genes from C57BL/6 mouse-derived small intestinal organoids and whole ileal tissue from naïve Il22Ra1fl/fl;Defa6-cre+/- mice

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