DIFFERENTIAL FUNCTION AND REGULATION OF RUNX2 ISOFORMS

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DESCRIPTION (provided by applicant): The complex Cbfa1/Runx2 gene gives rise to two isoforms, Runx2-II and Runx2-I, respectively derived from the distal "bone-related" (P1) and proximal (P2) promoters. Except for differences in their 5' untranslated regions (5'UTRs) and N-termini, the Type II and I Cbfa1 gene products are identical. The molecular mechanisms whereby these isoforms impart differential function to developmental pathways involving osteogenesis, chondrogenesis, and odontogenesis remain to be defined, but are likely derived from distinct N-terminal functional domains and possibly differential expression imparted by the ability of their 5'UTRs to modulate translational efficiency. Distinct function of the Runx2-II II N-terminal isoform is suggested by its later expression during bone development, but prior transgenic studies that deleted both isoforms were not designed to evaluate their separate function, in addition, the Runx2 gene generates multiple mRNAs with 5'UTRs having complex secondary structures that regulate translation in vitro through internal ribosome entry site (IRES) initiation of protein synthesis. The overall goals of these investigations are to establish the distinct function of Runx2-II and Runx2-I isoforms and to confirm that IRES-dependent translation control plays an important role in regulating its expression and tissue specific functions in vivo. In aim 1, we will determine the separate function of the "bone-related" isoform by completing the characterization of mice selectively deficient in the Runx2-II isoform. In addition, we will create a selective Runx2-I null mouse that leaves the expression and function of the Runx2-II isoform intact. In aim 2, we will investigate the ability of the 5'UTRs of Type I and Il Cbfa1 to regulate translation efficiency through cap-and IRES-dependent mechanisms in vitro and in transgenic mice. These studies will define the unique functions of the N-terminal domains, and determine if translational regulation contributes to the tissue-specific expression of Runx2-I and Runx2-II isoforms.