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The transcription factor ThPOK suppresses Runx3 and imposes CD4+ lineage fate by inducing the SOCS suppressors of cytokine signaling

Abstract

Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4+ lineage fate and Runx3 promoting the CD8+ lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8+ lineage fate, nor is it understood why ThPOK itself imposes the CD4+ lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4+ lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4+ lineage fate in the thymus.

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Figure 1: ThPOK induces the expression of genes encoding members of the SOCS family.
Figure 2: ThPOK is a transcriptional activator of SOCS-encoding genes.
Figure 3: Imposition of the CD4+ lineage fate by ThPOK requires SOCS1.
Figure 4: ThPOK-Tg Socs1−/− thymocytes that adopt the CD8+ cytotoxic T cell fate have minimal expression of any SOCS-encoding gene.
Figure 5: SOCS1 can replace ThPOK function during commitment to the CD4+ lineage in the thymus.
Figure 6: Transgenically expressed SOCS1 can replace ThPOK in the generation of CD4+ T cells.
Figure 7: ThPOK-independent and ThPOK-dependent generation of CD4+ T cells.

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Acknowledgements

We thank D. Singer and R. Hodes for critical review of this manuscript; R. Bosselut (National Cancer Institute) for Thpok−/− and ThPOK-Tg (C8) mice; J. Ihle (St. Jude Children's Research Hospital) for mice with germline deficiency in Socs1 and Ifng; D. Kappes (Fox Chase Cancer Center) for Thpokhd/hd mice; T. Kay (St. Vincent's Institute of Medical Research) for human CD4–Socs1 reporter mice; and M. Kubo (RIKEN) for SOCS1-Tg mice. Supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research.

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Authors

Contributions

M.A.L., M.Y.K. and A.T.W. designed and did experiments, and analyzed data; L.F. generated reagents; A.S. designed the study, analyzed data and wrote the manuscript; and J.-H.P. designed and did experiments, analyzed data, provided overall direction and wrote the manuscript.

Corresponding author

Correspondence to Jung-Hyun Park.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Induction of genes encoding SOCS proteins byThPOK in CD4+ T cells.

a) Purified CD4+ LN T cells from WT and ThPOK-transgenic mice (lines L1, C8, and L6) were assessed for Socs gene mRNA expression by qRT-PCR. Results are mean ± SE of three independent experiments. b) ThPOK induction of SOCS1 expression is cell intrinsic. TCRb+CD4SP thymocytes of B6 and ThPOK-Tg origin from B6+ThPOK-Tg -> Rag2-/- mixed bone marrow chimeras were compared by qRT-PCR for ThPOK and Socs1 mRNA expression. Mean ± SE of CD4SP cells from three mixed bone marrow chimeric mice. *, P <.05; ***; P <.001.

Supplementary Figure 2 ThPOK-binding sites in promoters of genes encoding SOCS proteins.

Identification of potential ThPOK binding sites in the Socs1 and Cish gene promoter regions. The 5′ upstream regions of mouse Socs1 and Cish genes were screened for evolutionary conserved sequences using rVista 2.0. Two potential ThPOK binding sites were identified in the promoter regions of both Socs1 (top, middle) and Cish (bottom) as indicated in red boxes.

Supplementary Figure 3 IFN-γ deficiency does not impair Th-POK's ability to impose the CD4+ lineage fate on developing thymocytes.

Thymocyte profiles from the indicated mouse strains. Data are representative of three independent experiments.

Supplementary Figure 4 Genes encoding SOCS proteins mediate the antagonism between ThPOK and Runx3.

In intermediate (INT) thymocytes, persistent TCR signaling induces expression of ThPOK (top) while cessation of TCR signaling followed by cytokine signaling induces Runx3 (bottom). ThPOK expression is directly suppressed by Runx3. Runx3 expression, on the other hand, is antagonized by ThPOK via its regulation of Socs gene expression.

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Luckey, M., Kimura, M., Waickman, A. et al. The transcription factor ThPOK suppresses Runx3 and imposes CD4+ lineage fate by inducing the SOCS suppressors of cytokine signaling. Nat Immunol 15, 638–645 (2014). https://doi.org/10.1038/ni.2917

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