Molecular identification of transcription factor Runx1 variants in grass carp (Ctenopharyngodon idella) and their responses to immune stimuli

The Runt-related transcription factor (Runx) family consists of three members, Runx1, Runx2 and Runx3 in mammals, which are involved in various biological processes. Recent studies have demonstrated that Runx1 plays critical roles in the immunity of higher vertebrates. In fish, zebrafish and fugu Runx family members have been identified, and their chromosome location, promoter usage and expression patterns have been elucidated. However, their expression profiles in immune responses are still unknown. In this study, we identified grass carp five Runx1 (gcRunx1) variants (v1-5) possibly generated through alternative promoter usage and alternative splicing. The gcRunx1 v1-3 encodes the proteins possessing intact structural characteristics of Runx family, but the putative proteins of gcRunx1 v4-5 lack a transactivation domain, an inhibitory domain and a C-terminal pentapeptide motif (VWRPY). Tissue distribution assays revealed that gcRunx1 was preferentially expressed in some immune-related tissues including thymus and spleen, indicating its potential roles in teleost immunity. The changes of gcRunx1 expression to various immune stimuli was examined in periphery blood lymphocytes, showing that gcRunx1 v1-3 mRNA levels were increased after LPS, poly I:C and PHA treatment, whereas gcRunx1 v4-5 mRNA expression were stimulated only by LPS and PHA. Furthermore, in vivo studies confirmed that bacterial challenge enhanced gcRunx1 mRNA levels. In particular, in vitro and in vivo studies revealed that gcRunx1 v4-5 mRNA expression was induced with a delayed kinetics compared with that of gcRunx1 v1-3. These findings not only provide the evidence for the involvement of gcRunx1 in immune response, but also reveal the inducible expression diversity of fish Runx1 splicing variants, thereby facilitating further elucidating the role of Runx1 in piscine immunity.