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RNA-binding Mex3a promotes epithelial stemness and represses Klf4-dependent differentiation upon intestinal homeostasis, regeneration and cancer

ABSTRACT: The precise control of the self-renewal and differentiation of intestinal stem cells (ISCs) is essential for intestinal homeostasis, efficient regeneration and prevention of tumorigenesis. However, the underlying molecular mechanisms remain only partially understood, especially in regeneration and cancer. Here, it is demonstrated that RNA-binding protein Mex3a is specifically expressed in ISCs and promotes their stemness and proliferative status. Its depletion results in compromised ISCs, impaired intestinal regeneration and suppressed oncogenic transformation. Mechanistically, upregulation of Mex3a in ISCs at homeostasis, in postirradiation regenerative foci and in colorectal cancer model is regulated by the upstream transcription factor E2f3. In turn, Mex3a activates Wnt signaling by directly suppressing pro-differentiation transcription factor Klf4 at the mRNA level. Furthermore, expression of E2F3 and MEX3A significantly increases in human radiation enteritis and colorectal carcinoma (CRC), accompanied by KLF4 downregulation and Wnt signaling hyperactivation. These findings indicate that the E2f3-Mex3a-Klf4-Wnt as the critical molecular switch between ISC self-renewal and differentiation. It can represent a putative therapeutic target for CRC and regeneration-related gut diseases.

ORGANISM(S): Mus musculus

PROVIDER: GSE179493 | GEO | 2021/07/09

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.

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RNA-binding Mex3a promotes epithelial stemness and represses Klf4-dependent differentiation upon intestinal homeostasis, regeneration and cancer

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