Bone Abstracts

The Runt-domain transcription factors Runx2 and Runx3 are known to drive chondrocyte maturation from prehypertrophic to the terminal stage. The RUNX family proteins form dimers with CBFb, and bind to consensus sequences of 5′-PuACCPuCa-3′ upstream of target genes to activate or repress transcription.

To address the role of Runx3 transcription factor in zebrafish, we have isolated the different splice variants encoding distinct runx3 protein isoforms and their corresponding expression patterns were assessed in zebrafish tissues, during development and in bone-derived cells undergoing differentiation, by real-time RT-PCR.

To further understand the molecular mechanism affecting runx3 gene expression, we analyzed the activity of its two promoters, P1 and P2, that drive transcription of the different variants leading to distinct protein isoforms, in osseous and non-osseous cells, using a promoter-derived luciferase reporter system. We performed transient transfection assays with either the full promoters or serial deletion constructs. We observed a reduction in runx3 P1 promoter activity when the complete 5′-UTR was deleted (from −17 to −701 bp), and in P2-promoter activity when two regions (from −1232 to −663 and from −713 to −554 bp) were delected. These results indicate that the identified regions contain important transcription factors or enhancer binding sites for Runx3 transcritpion. To identify upstream regulators of the runx3-P1 and P2 promoters, we performed cotransfection assays in Hek293 cells with runx3-P1 or runx3-P2 luciferase constructs and several transcription factors expression vectors. Runx2 was identified as one regulator of runx3-P2 promoter activity. The runx2-responsive site on the runx3 promoter was identified by in silico analysis and is being confirmed by mutation analysis.

Collectively, our results identify the regions of runx3-P1 and P2 promoters important for runx3 basal transcription and provide a first basis to correlate expression of distinct Runx3 variants with specific transcription factors affecting P1 or P2 promoter activity.