Functional characterization of the mouse Tbx22 protein

Objective: Mutations in the TBX22 gene cause X-linked cleft palate and ankyloglosia in humans. The objective of this project was to examine the functional domains and subcellualr localization of the orthologous mouse Tbx22 protein. Methods: Full-length and 5'-truncated Tbx22 cDNAs were subcloned by using RT-PCR and fused inframe with EGFP to investigate subcellular localization by using cell transfection studies. In addition, a polyclonal antibody made against the carboxyl terminal peptide of the human TBX22 protein was tested for cross reactivity to mouse Tbx22 by using western and immunofluoresenct staining protocol. Results: Both EGFP-tagged full-length human and mouse Tbx22 proteins were localized in the nuclei of transfected NIH3T3 cells. Nuclear localization of the the full-length Tbx22 proteins in the transfected cells was also detected with the human TBX22 polyclonal antibody following methanol fixation. Deletion of the N-terminal region containing the putative nuclear localization signals abolished nuclear import of the EGFP-Tbx22 fusion protein. These data suggest that targeted deletion of the first three exons of the Tbx22 gene in mice would disrupt its in vivo function. Although the human TBX22 antibody worked for immunofluorescent staining of methanol fixed transfected NIH3T3 cells, it did not detect a specific signal in paraformaldehyde-fixed, paraffin-sectioned mouse embryonic craniofacial tissues. Since this polyclonal antibody is currently the only one recognizing a region of the TBX22 protein conserved in human and mice, new antibody reagents will need to be developed to fully characterize the in vivo function of Tbx22 in the mouse model. Conclusion: Tbx22 is a nuclear protein, consistent with its predicted role as a transcription factor. The N-terminal putative nuclear localization signal residues are essential for Tbx22 nucelar localization.