Isoform Runx2-II is sufficient for the development of calvaria, clavicles, and limb bones in the absence of Runx2-I.

Runx2 is an essential transcription factor for osteoblast differentiation and chondrocyte maturation. Heterozygous mutations in RUNX2 cause cleidocranial dysplasia, characterized by an open fontanel, hypoplastic clavicles, and short stature. Runx2 is transcribed from two promoters, P1 and P2, generating Runx2-II and Runx2-I, respectively. However, the function of Runx2-I remains unclear. To clarify its function, we generated Runx2-I-specific knockout mice by systematically deleting the P2 region while retaining the downstream sequence from the branch site for intron 1 splicing. As the 2.5-kb and 24-kb deletions did not disrupt P2, the deletion was extended to 76 kb (Int1del/+ mice). Because this intron contains Supt3, whose homozygous deletion was lethal on embryonic day 9.5-10.5, we crossed Int1del/+ mice with Runx2+/- mice. Cap analysis of gene expression showed that Runx2-I mRNA was absent in Int1del/+Runx2+/- mice. In newborn calvaria and 6-week-old femurs, real-time reverse transcription-polymerase chain reaction analysis showed that Runx2-II mRNA in Int1del/+Runx2+/- mice was upregulated to wild-type levels, whereas the total Runx2 mRNA was comparable to Runx2+/- mice. At E16.5 and the newborn stage, skeletal preparation and histological and micro-CT analyses showed that the impaired development of cranial bones, clavicles, and limb bones in Runx2+/- mice was rescued in Int1del/+Runx2+/- mice, whereas that of the ribs, sternum, and vertebrae was not. In 6-week-old females, reduced femoral cortical thickness in Runx2+/- mice was restored in Int1del/+Runx2+/- mice. Neither Runx2-I nor Runx2-II overexpression exhibited negative autoregulation of endogenous expression. Depending on the cell type examined, their overexpression or Runx2 knockdown differentially affected P1 and P2 reporter activities, which were also influenced by an osteoblast-specific enhancer. These findings indicate that wild-type levels of Runx2-II can substitute for Runx2-I in calvaria, clavicle, and limb bone development. Upregulation of Runx2-II in Int1del/+Runx2+/- mice cannot be explained by negative autoregulation, offering a hypothetical model of reciprocal P1/P2 regulation.