Sequence-Specific DNA Binding by the αNAC Coactivator Is Required for Potentiation of c-Jun-Dependent Transcription of the Osteocalcin Gene

Abstract

Since the c-Jun coactivator αNAC was initially identified in a differential screen for genes expressed in differentiated osteoblasts, we examined whether the osteocalcin gene, a specific marker of terminal osteoblastic differentiation, could be a natural target for the coactivating function of αNAC. We had also previously shown that αNAC can specifically bind DNA in vitro, but it remained unclear whether the DNA-binding function of αNAC is expressed in vivo or if it is required for coactivation. We have identified an αNAC binding site within the murine osteocalcin gene proximal promoter region and demonstrated that recombinant αNAC or αNAC from ROS17/2.8 nuclear extracts can specifically bind this element. Using transient transfection assays, we have shown that αNAC specifically potentiated the c-Jun-dependent transcription of the osteocalcin promoter and that this activity specifically required the DNA-binding domain of αNAC. Chromatin immunoprecipitation confirmed that αNAC occupies its binding site on the osteocalcin promoter in living osteoblastic cells expressing osteocalcin. Inhibition of the expression of endogenous αNAC in osteoblastic cells by use of RNA interference provoked a decrease in osteocalcin gene transcription. Our results show that the osteocalcin gene is a target for the αNAC coactivating function, and we propose that αNAC is specifically targeted to the osteocalcin promoter through its DNA-binding activity as a means to achieve increased specificity in gene transcription.

ACKNOWLEDGMENTS

We thank Gérard Karsenty (Baylor College of Medicine) and Shoukat Dedhar (University of British Columbia) for providing expression vectors and reporter plasmids. We are indebted to Renny T. Franceschi (University of Michigan) for his generous gift of the MC3T3-E1 subclone stably transfected with the osteocalcin-luciferase reporter and for tips on the ChIP assay. We are grateful to McGill University and the Genome Quebec Innovation Centre for allowing repeated usage of the real-time PCR instrument. Mark Lepik and Guylaine Bédard prepared the figures.

This work was supported by the Shriners of North America.