Abstract
EZH2 plays an essential role at the β-selection checkpoint of T lymphopoiesis by regulating histone H3 lysine 27 trimethylation (H3K27me3) via its canonical mode of action. Increasing data suggest that EZH2 could also regulate other cellular functions, such as cytoskeletal reorganization, via its noncanonical pathway. Consequently, we investigated whether the EZH2 noncanonical pathway could be involved in early T-cell maturation, which requires cell polarization. We observed that EZH2 localization is tightly regulated during the early stages of T-cell development and that EZH2 relocalizes in the nucleus of double-negative thymocytes enduring TCRβ recombination and β-selection processes. Furthermore, we observed that EZH2 and EED, but not Suz12, colocalize with the microtubule organization center (MTOC), which might prevent its inappropriate polarization in double negative cells. In accordance with these results, we evidenced the existence of direct or indirect interaction between EED and α-tubulin. Taken together, these results suggest that the EZH2 noncanonical pathway, in association with EED, is involved in the early stages of T-cell maturation.
Highlights
During the DN2 to DN3 transition, TCRβ gene segments rearrange to produce a TCRβ chain that associates with the pTCRα chain and CD3 to form a pre-TCR-CD3 complex
Since EZH2 has a noncanonical cytoplasmic function in mature T cells[3], we examined the variation in EZH2 subcellular localization during T lymphopoiesis
EZH2 is essential for the differentiation of lymphocyte lineages, as its deletion induces T-cell differentiation arrest at the β-selection c heckpoint[3,5] and B-cell differentiation arrest at the pre-BCR selection s tep[2], while its homolog EZH1 is only essential for B-cells d evelopment[21]
Summary
During the DN2 to DN3 transition, TCRβ gene segments rearrange to produce a TCRβ chain that associates with the pTCRα chain and CD3 to form a pre-TCR-CD3 complex (pTCR-CD3) The functionality of this pTCRCD3 complex is tested through β-selection occurring at the DN3 stage, allowing the elimination of T-cells expressing nonfunctional TCRβ. While no correlation was found between EZH2 expression and canonical activity, we established that EZH2 subcellular localization was tightly orchestrated during the early stages of T-cell differentiation. We observed that EZH2 and EED localized in the cytoplasm, where they could act through a noncanonical pathway. Co-immunoprecipitation experiments suggest an interaction between EED (and potentially EZH2) and the tubulin network These results lead us to propose a novel link between the EZH2/EED noncanonical pathway and MTOC polarization during early T lymphopoiesis
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