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Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop

Oncogene volume 29, pages 2807–2816 (2010)Cite this article

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Abstract

Spi-1/PU.1 oncogene is downregulated as proerythroblasts undergo terminal differentiation. Insertion of the Friend virus upstream of the Spi-1/PU.1 locus leads to the constitutive upregulation of Spi-1/PU.1, and a subsequent block in the differentiation of the affected erythroblasts. We have shown that sustained overexpression of Spi-1/PU.1 also inhibits the erythroid splicing of protein 4.1R exon 16, irrespective of chemical induction of differentiation. Here, we show a positive feedback loop that couples constitutive phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling to high expression of Spi-1/PU.1 in Friend erythroleukemia cells. Inhibition of PI3K/AKT results in Spi-1/PU.1 downregulation in a stepwise manner and induces cell differentiation. Chromatin immunoprecipitation assays further supported the positive autoregulatory effect of Spi-1/PU.1. Mutational analysis indicated that Ser41, but not Ser148, is necessary for Spi-1/PU.1-mediated repression of hemoglobin expression, whereas both Ser residues are required for Spi-1/PU.1 inhibition of the erythroid splicing event. We further show that inhibition of the erythroid transcriptional and splicing events are strictly dependent on distinct Spi-1/PU.1 phosphorylation modifications rather than Spi-1/PU.1 expression level per se. Our data further support the fact that Spi-1/PU.1 inhibits 4.1R erythroid splicing through two different pathways, and bring new insights into the extracellular signal impact triggered by erythropoietin on late erythroid regulatory program, including pre-mRNA splicing.

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Abbreviations

ChIP:

chromatin immunoprecipitation

DMSO:

dimethylsulfoxide

Epo:

erythropoietin

MEL:

mouse erythroleukemia

SFFV:

spleen focus-forming virus

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Acknowledgements

We thank Dr I Merida (Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Madrid, Spain) for pEFbosEGFP_C1 plasmid, Dr F Moreau-Gachelin (Inserm U830, Institut Curie, Paris, France) for anti-Spi-1/PU.1 antibody, G Giraud for helping with the ChIP assay and Dr G Mouchiroud and Dr F Morlé (CGMC, Villeurbanne, France) for sharing reagents, including anti-p85 antibody. This work was supported by grants from the ‘Ligue contre le Cancer, Comité de la Loire’. Authors were supported by the INSERM, the ‘Ligue contre le Cancer, Comité de la Loire’, the ‘Ligue contre le Cancer, Comité du Doubs’

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Authors and Affiliations

  1. mRNA Metabolism in Normal and Pathological Cells,

    O Breig, O Théoleyre, A Douablin & F Baklouti

  2. CGMC, CNRS, Université Lyon 1, Villeurbanne, France

    O Breig, O Théoleyre, A Douablin & F Baklouti

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  1. O Breig
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Correspondence to F Baklouti.

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Breig, O., Théoleyre, O., Douablin, A. et al. Subtle distinct regulations of late erythroid molecular events by PI3K/AKT-mediated activation of Spi-1/PU.1 oncogene autoregulation loop. Oncogene 29, 2807–2816 (2010). https://doi.org/10.1038/onc.2010.29

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