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- Molecular structure and functional analysis of runx3 in zebrafishPublication . Rodrigues, Brigite Sandra Nunes Simões; Cancela, Leonor; Kelsh, Robert; Conceição, NatérciaIn mammals, runt-related family of transcription factors is encoded by the three distinct genes, RUNX1, RUNX2 and RUNX3 that share an evolutionarily conserved 128 amino acid Runt domain, which is responsible for the dual function of DNA binding and hetero-dimerization with the co-factor CBFβ. Gene ablation and gain of function experiments established all three proteins as key regulators of lineage-specific gene expression in major developmental pathways. Mutations in RUNX genes have been frequently associated with human diseases. Despite many studies to unveil mechanisms of RUNX3 action, available data is insufficient to fully understand its physiological role, particularly the importance of each isoform. We cloned for the first time a variety of transcript variants for both zebrafish runx3 and cbfβ genes, identified their temporal expression by qPCR and localized runx3 sites of expression by in situ hybridization in adult tissues and during early embryonic development. As runx3-P1 and runx3-P2 transcripts were found to be differentially expressed, we used a promoter in silico comparative approach for both P1 and P2 runx3 gene promoter regions and in vitro and in vivo promoter analysis to identify regulatory regions and conserved transcription factor binding sites, allowing us to select, from an extensive list of putative transcription binding sites, the best candidates to regulate runx3 promoter regions. Furthermore, through in vitro analysis, we have examined the possible cross- and auto-regulation of runx3 promoters by Runx2 and Runx3 isoforms, respectively. In conclusion, we have used computational and molecular approaches to improve our understanding of the complexity of Runx and Cbfβ variants and their implication for function, using zebrafish as a model. Altogether, our structural and functional data provide further support to the assumption that the expression of runx3 variants is tightly regulated, leading to a highly specific spatial-temporal expression pattern.