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ATAC-seq analyses of developmental EpCAM+ intestinal epithelial cells

ABSTRACT: To identify potential cell signaling pathways regulating the proliferative transition of Bmi1+ ISCs from highly proliferative to slow cycling, and also to identify influence for lineage differentiation, we performed bulk ATAC-Seq analyses of FACS-isolated EpCAM+ intestinal cells across developmental time points. Using differential chromatin accessibility analysis we identified enrichment of multiple pathways in the E12.5 population that progressively decreased at E17.5. We analyzed the dataset using identification of motif discovery within this dataset, we found enhanced utilization of transcription factors in pathways including the Hippo signaling pathway (Tead1, Tead2 and Tead4), a known pathway that regulates organ development and size. When we evaluated enrichment motifs for canonical Wnt-responsive loci, we identified occupancy peaking at E14.5, again consistent with transition of proliferative dependency on Bmi1+ to Lgr5+ ISCs. Global motif enrichment for Wnt-related targets was assessed using ChromVAR, which displayed a similar trend, with the canonical Wnt pathway signature peaking at E14.5 and E15.5 (e.g. Cdx2, Tcf4).

ORGANISM(S): Mus musculus

PROVIDER: GSE251949 | GEO | 2025/05/01

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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ATAC-seq analyses of developmental EpCAM+ intestinal epithelial cells

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