Identification of a New pebp2αA2 Isoform From Zebrafish runx2 Capable of Inducing Osteocalcin Gene Expression In Vitro

The zebrafish runx2b transcription factor is an ortholog of RUNX2 and is highly conserved at the structural level. The runx2b pebp2αA2 isoform induces osteocalcin gene expression by binding to a specific region of the promoter and seems to have been selectively conserved in the teleost lineage. Introduction: RUNX2 (also known as CBFA1/Osf2/AML3/PEBP2αA) is a transcription factor essential for bone formation in mammals, as well as for osteoblast and chondrocyte differentiation, through regulation of expression of several bone‐ and cartilage‐related genes. Since its discovery, Runx2 has been the subject of intense studies, mainly focused in unveiling regulatory targets of this transcription factor in high vertebrates. However, no single study has been published addressing the role of Runx2 in bone metabolism of low vertebrates. While analyzing the zebrafish (Danio rerio) runx2 gene, we identified the presence of two orthologs of RUNX2, which we named runx2a and runx2b and cloned a pebp2αA‐like transcript of the runx2b gene, which we named pebp2αA2. Materials and Methods: Zebrafish runx2b gene and cDNA were isolated by RT‐PCR and sequence data mining. The 3D structure of runx2b runt domain was modeled using mouse Runx1 runt as template. The regulatory effect of pebp2αA2 on osteocalcin expression was analyzed by transient co‐transfection experiments using a luciferase reporter gene. Phylogenetic analysis of available Runx sequences was performed with TREE_PUZZLE 5.2. and MrBayes. Results and Conclusions: We showed that the runx2b gene structure is highly conserved between mammals and fish. Zebrafish runx2b has two promoter regions separated by a large intron. Sequence analysis suggested that the runx2b gene encodes three distinct isoforms, by a combination of alternative splicing and differential promoter activation, as described for the human gene. We have cloned a pebp2αA‐like transcript of the runx2b gene, which we named pebp2αA2, and showed its high degree of sequence similarity with the mammalian pebp2αA. The cloned zebrafish osteocalcin promoter was found to contain three putative runx2‐binding elements, and one of them, located at −221 from the ATG, was capable of mediating pebp2αA2 transactivation. In addition, cross‐species transactivation was also confirmed because the mouse Cbfa1 was able to induce the zebrafish osteocalcin promoter, whereas the zebrafish pebp2αA2 activated the murine osteocalcin promoter. These results are consistent with the high