Two Domains Unique to Osteoblast-Specific Transcription Factor Osf2/Cbfa1 Contribute to Its Transactivation Function and Its Inability To Heterodimerize with Cbfβ

ABSTRACT

Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfβ, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

ACKNOWLEDGMENTS

K.T. thanks Patricia Ducy for pointing out the NLS, for providing various reagents, and for suggestions throughout the course of the study. We thank Yoshiaki Ito (Kyoto University, Kyoto, Japan) for providing the Cbfβ cDNA clone, Marilyn Szentirmay for the pSG424 and GAL4VP16 expression vectors, and Jennifer Philhower for the pGAL4SVluc reporter construct. Thanks are due to Brendan Lee and Thorsten Schinke for critical reading of the manuscript and to members of the Karsenty lab for helpful suggestions.

S.S. is a scholar of the Fonds de la Recherche en Sante du Quebec and a Killam Scholar of the Montreal Neurological Institute. This work was supported by a grant from the Medical Research Council of Canada, PG11473, to S.S. and by grants from the National Institutes of Health, DE11290 and HD97006, and a Basic Science Award of the March of Dimes Foundation to G.K.