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Sox4 regulates proliferation, differentiation, and DNA methylation patterns in the intestinal epithelium

ABSTRACT: Intestinal stem cells (ISCs) are responsible for maintaining the physiological function of the intestinal epithelium through the production of differentiated absorptive and secretory cellular lineages. In addition to producing specialized functional cells, ISCs must also drive constant proliferation in order to maintain the especially high rate of cellular turnover in the intestinal epithelium, which undergoes near total renewal every 5-7 days. Understanding the transcriptional mechanisms that control how ISCs and transit-amplifying progenitors (TAs) balance differentiation and proliferation in the intestine may provide valuable insight into common pathologies, such as inflammatory bowel disease and cancer. Here, we show that the Sry¬¬-box containing transcription factor, Sox4, is involved in the regulation of proliferation in the ISC/TA zone, the expression of ISC-associated genes, and the differentiation of enteroendocrine (EE) cells. Interestingly, we also observed a significant reduction in the expression of the methylcytosine dioxygenase, Tet1, in Sox4-deficient intestines. We find that Tet1, which initiates derepression of target genes via DNA demethylation, is specifically upregulated in ISC populations in wild-type animals. Additionally, Sox4-deficient intestines showed a significant reduction in levels of 5-hydroxymethylcytosine, the catalytic byproduct of TET protein activity, in the ISC/TA zone. Together, our data demonstrate that Sox4 regulates differentiation and proliferation in the intestinal epithelium, and suggests that it may influence these processes through induction of Tet1 and subsequent derepression of target genes through epigenetic mechanisms.

ORGANISM(S): Mus musculus

PROVIDER: GSE52675 | GEO | 2014/12/31

SECONDARY ACCESSION(S): PRJNA230560

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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Sox4 regulates proliferation, differentiation, and DNA methylation patterns in the intestinal epithelium

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