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Epigenetic changes from ATAC-seq of intestine stem cells from mice with graft-versus-host disease

ABSTRACT: Intestinal stem cells (ISC) encounter inflammatory insults in immune mediated gastro-intestinal (GI) diseases. It remains unknown whether, and how, they adapt, and if the adaptation leaves scars on the ISCs that affects their subsequent regeneration capacity. We investigated the consequences of inflammation on Lgr5+ISCs in well-defined clinically relevant models of gastro-intestinal acute graft-versus-host disease (GI GVHD). Utilizing single cell transcriptomics, organoid, metabolic, epigenomic and in vivo models we found that Lgr5+ISCs undergo metabolic changes that lead to accumulation of succinate, which reprograms its epigenome. We performed transposase-accessible chromatin sequencing (ATAC-seq) in sorted Lgr5+ISCs harvested from the BALB/c→B6-GFP-Lgr5 model of GVHD.

ORGANISM(S): Mus musculus

PROVIDER: GSE223814 | GEO | 2024/09/11

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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Epigenetic changes from ATAC-seq of intestine stem cells from mice with graft-versus-host disease

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