T‐cell receptor signaling induces proximal Runx1 transactivation via a calcineurin–NFAT pathway

Abstract

Runx1 transcription factor is a key player in the development and function of T cells.Runx1 transcripts consist of two closely related isoforms (proximal and distal Runx1) whose expressions are regulated by different promoters. Which Runx1 isoform is expressed appears to be tightly regulated. The regulatory mechanism for differential transcription is, however, not fully understood. In this study, we investigated the regulation of the proximal Runx1 promoter in T cells. We showed that proximal Runx1 was expressed at a low level in naïve T cells from C57BL/6 mice, but its expression was remarkably induced upon T-cell activation. In the promoter of proximal Runx1, a highly conserved region was identified which spans from −412 to the transcription start site and harbors a NFAT binding site. In a luciferase reporter assay, this region was found to be responsive to T-cell activation through Lck and calcineurin pathways. Mutagenesis studies and chromatin immunoprecipitation assay indicated that the NFAT site was essential for NFAT binding and transactivation of the proximal Runx1 promoter. Furthermore, TCR signalinginduced expression of proximal Runx1 was blocked by treatment of cells with cyclosporin A. Together, these results demonstrate that the calcineurin-NFAT pathway regulates proximal Runx1 transcription upon TCR stimulation. Keywords: Cellular activation r Naive cells r T cells r TCR r Transcription factorsAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionIn T cells, engagement of the T-cell receptor (TCR) with the peptide-major histocompatibility complex on antigen-presenting Correspondence: Dr. Won Fen Wong e-mail: wonfen@um.edu.my cells recruits various molecules to the contact site, triggers a signaling cascade, induces cytokine expression, and eventually determines cell fate: differentiation, proliferation, anergy, or apoptosis [1]. TCR activation causes phosphorylation of Lck kinase whose signal is transmitted through several downstream signaling pathways to transcription factors in the nucleus. The calcineurin pathway is one of such pathways. Calcineurin dephosphorylates cytoplasmic nuclear factor of activated T-cell (NFAT) transcription C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 894-904 Leukocyte signaling 895 factor, which is then translocated into the nucleus [1] resulting in the expression of various genes including interleukin-2 (IL-2) [2]. The Runx1 transcription factor is one of the key players in T-cell functions. Runx1 is abundantly expressed in thymus and spleen [3] and plays important roles in multiple stages of T-cell development as well as the differentiation of T helper (Th) cell subsets through the interplay with other transcription factors [4]. In thymus, Runx1 cooperates with LEF-1, Ets, or c-myb to form an enhanceosome complex that activates TCRs and MHC class I gene expression [5,6]. Interestingly, on one hand, Runx1 deficiency impairs transition of double ...

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