Proteomic analysis identifies transcriptional cofactors and homeobox transcription factors as TBX18 binding proteins

The TBX18 transcription factor is a crucial developmental regulator of several organ systems in mice, and loss of its transcriptional repression activity causes dilative nephropathies in humans. The molecular complexes with which TBX18 regulates transcription are poorly understood prompting us to use an unbiased proteomic approach to search for protein interaction partners. Using overexpressed dual tagged TBX18 as bait, we identified by tandem purification and subsequent LC-MS analysis TBX18 binding proteins in 293 cells. Clustering of functional annotations of the identified proteins revealed a highly significant enrichment of transcriptional cofactors and homeobox transcription factors. Using nuclear recruitment assays as well as GST pull-downs, we validated CBFB, GAR1, IKZF2, NCOA5, SBNO2 and CHD7 binding to the T-box of TBX18 in vitro. From these transcriptional cofactors, CBFB, CHD7 and IKZF2 enhanced the transcriptional repression of TBX18, while NCOA5 and SBNO2 dose-dependently relieved it. All tested homeobox transcription factors interacted with the T-box of TBX18 in pull-down assays, with members of the Pbx and Prrx subfamilies showing coexpression with Tbx18 in the developing ureter of the mouse. In summary, we identified and characterized new TBX18 binding partners that may influence the transcriptional activity of TBX18 in vivo.