Exploring Twist1 and its Mutants' Regulation of Bone Formation and Cancer Metastasis

Twist1, the focus of this study, is a key transcription factor that functions as a master regulator of cancer metastasis and early embryonic development. Runx2 (OSF2), another important gene in this study, is a major transcription factor involved in bone differentiation.  Developmental mutations in Twist1 can cause craniosynostosis due to early onset of Runx2 expression, which results in early coronal sutures in the skull.  Twist1 has been noted to reduce Runx2 protein level, especially when it forms a complex with another transcription factor called Snail (Twist-Snail complex). This complex can only form when the WR domain of Twist1 interacts with Snail. Taking all this into account, the proposed hypothesis is that if Twist1 and Runx2 have a functional relationship, then different mutants of Twist1 might have different regulatory capabilities on Runx2. The chosen mutants are Twist1 deltaWR, Twist1 L131P,  Twist1 I156V, Twist1 L83P, Twist1 Y185A, Twist1 A186T, Twist1 S188A, Twist1 S188D, Twist1 S188L and Twist1 S188P.  These Twist1 mutants were used along with the wild type. The procedure was to transform E.coli with the Twist1 wild type and mutant DNAs, do midi preparation for the plasmids, culture COS-1 cells, transfect them with the plasmid DNA, prepare the cell lysate, run functional assays (beta galactosidase, protein estimation, and luciferase), and perform western blotting. The results that were observed supports the hypothesis that different Twist1 mutants had different effects on the level of Runx2 protein. Direct applications for this study could be the development of therapeutics for developmental abnormalities and different cancers.