Systematic evaluation of how Hand2 dimerization partners regulate DNA binding specificity and biological activity

Mia I. Crawford

Document Type

Article

Publication Date

2025

Keywords

JMG

Abstract

Our study aims to characterize how different dimerization partners alter Hand2 biological activity and binding site specificity during mandible development. To address these questions, we applied complementary experimental and computational approaches to assess Hand2 function as either an obligate homo- and heterodimer and modeled its 3D structure with different interactors. Twist1, Tcf12, and Tcf4 were identified using mass spectrometry as candidate Hand2 dimerization partners that modulate E-box binding specificity in the mandible. In this study, we used transient transfections in NIH-3T3 cells to verify nuclear localization and confirm protein production of synthetic Hand2-dimers. Thus far, our work has validated all obligate dimers can be imported into the nucleus and protein for monomeric Hand2 and dimeric Hand2-Hand2 can be detected via Western blot. Currently, studies are underway to perform Western blot for the other dimer pairs. We have shown successful transfection and expression in transfected NIH3T3 cell lines for Hand2-Hand2-V5 and Hand2-V5 constructs. Colocalization of Hand2-Hand2-V5 and Hand2-V5 were also verified with Hoechst stain, indicating nuclear colocalization of constructs.

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