STRUCTURE-FUNCTION ANALYSIS OF MSX2-MEDIATED TRANSCRIPTIONAL SUPPRESSION

Osteocalcin (OC) is a calcium binding protein expressed in mature oste oblasts undergoing mineralization. The OC gene has been identified as a target for transcriptional suppression by Msx2, a homeodomain transc ription factor that controls ossification in calvarial bone of the dev eloping skull. We have initiated systematic structure-function analyse s of Msx2, using OC promoter suppression (luciferase reporter) in MC3T 3-E1 calvarial osteoblasts as an assay. Msx2 variants were epitope ('' FLAG'')-tagged for monitoring Msx2 protein expression by Western blot analysis. Functional analyses of N- and C-terminally truncated molecul es identify Msx2 residues 97-208 as the core suppressor domain. Intern al deletion analyses indicate that suppressor function is dependent up on structural features encoded by residues 132-148-upstream of the hom eodomain and overlapping the homeodomain N-terminal extension-but not upon residues in the three homeodomain helices. Mutations that enhance DNA binding activity do not proportionally enhance Msx2 suppressor fu nction; moreover, a Msx2 point mutant Msx2(T147A) that completely lack s DNA binding activity is indistinguishable from wild-type Msx2! in it s ability to suppress the OC promoter, demonstrating that direct inter action with DNA is not required for Msx2 suppressor function. This sug gests that Msx2 suppresses transcription via protein-protein interacti ons with components of the basal transcriptional machinery, either alo ne or in concert with co-regulators. Using interaction ''Far Western'' blotting assays, we systematically tested for protein-protein interac tions between Msx2 and components of the basal transcriptional machine ry known to mediate transcriptional activation (TBP, TFIIB, and TFIIF) . Msx2 binds both components of TFIIF (RAP74, RAP30), but not TFIIB or TBP. Msx2(55-208) encompasses core suppressor domain residues and bin ds TFIIF; in this context, deletion of the seventeen amino acid residu es 132-148 that are required for core suppressor function abrogates in teractions with TFIIF components. Go-expression of RAP74 in MC3T3-E1 c ells partially reverses (>50%) suppression of OC promoter activity by Msx2, while co-expression of TFIIB or RAP30 has no effect. Thus the co re suppressor domain of Msx2 participates in functionally important in teractions with RAP74 that regulate OC promoter activity in calvarial osteoblasts.