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, domainswapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbf␤, 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.

A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with MYC

“…Our ®nding that full-length Cbfa1 forms complexes with Cbfb in vivo is in apparent con¯ict with previous studies which indicated that inhibitory sequences must be deleted from the full-length protein for it to interact with Cbfb Thirunavukkarasu et al, 1998). However, as these conclusions were based on in vitro interaction of recombinant proteins (Thirunavukkarasu et al, 1998) or transfection of 3T3 ®broblasts , it is possible that essential binding cofactors were absent or limiting in each case.…”

“…However, as these conclusions were based on in vitro interaction of recombinant proteins (Thirunavukkarasu et al, 1998) or transfection of 3T3 ®broblasts , it is possible that essential binding cofactors were absent or limiting in each case. In support of this interpretation, it has recently been shown that interaction of Cbfa2 (AML1/PEBPB) with Ets1 causes mutual activation of their DNA binding potential by inactivation of inhibitory domains in each protein (Kim et al, 1999).…”

MINT, the Msx2 Interacting Nuclear Matrix Target, Enhances Runx2-dependent Activation of the Osteocalcin Fibroblast Growth Factor Response Element

“…However, Runx2 (1-392) still robustly responds to FGF2 using the OCFRE as a reporter. Overall Runx2 and FGF responsiveness is lost in mutant Runx2 (1-312), indicating that critical TAF domains for Runx2 activity lie between amino acids 312 and 392, a region encompassing the AD3 initially identified by Karsenty and colleagues (30). Thus, Runx2 provides the major transactivation function in the osteoblast nuclear protein complexes assembled by the OCFRE.…”

Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis

“…1B). This region is a transactivation domain in mammals (Thirunavukkarusu et al, 1998), but it is not found in any orthologs outside of the mammalian RUNX2.…”