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Cul4b promotes CD4 T cell expansion by aiding the repair of damaged DNA

ABSTRACT: The capacity for T cells to become activated and clonally expand during pathogen invasion is pivotal for protective immunity. Our understanding of how T cell receptor (TCR) signaling prepares cells for this rapid expansion remains limited. We have identified an E3 ubiquitin ligase, Cul4b, that regulates this process. Cul4b levels and activity increase following TCR stimulation. Disruption of Cul4b resulted in impaired proliferation and survival of activated T cells. Additionally, Cul4b-deficient CD4 T cells accumulated DNA damage. In T cells, Cul4b preferentially associated with the substrate receptor DCAF1, and Cul4b and DCAF1 were found to interact with proteins that promote the sensing or repair of damaged DNA. While Cul4b-deficient CD4 T cells showed evidence of DNA damage sensing, downstream phosphorylation of SMC1A did not occur. These findings reveal an essential role for Cul4b in promoting the repair of damaged DNA to allow survival and expansion of activated T cells.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hossein Fazelinia

LAB HEAD: Steven H. Seeholzer

PROVIDER: PXD017699 | Pride | 2021-01-04

REPOSITORIES: Pride

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			Action	DRS
	q170917_PO1228rp_KO1_01.raw	Raw
	q170917_PO1228rp_KO1_02.raw	Raw
	q170917_PO1228rp_KO1_03.raw	Raw
	q170917_PO1228rp_KO1_04.raw	Raw
	q170917_PO1228rp_KO1_05.raw	Raw

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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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Cul4b promotes CD4 T cell expansion by aiding the repair of damaged DNA

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