T-box transcription factors are critical developmental regulators in all multi-cellular organisms, and altered T-box factor activity is associated with a variety of human congenital diseases and cancers. Despite the biological significance of T-box factors, their mechanism of action is not well understood. Here we examine whether SUMOylation affects the function of the C. elegans Tbx2 sub-family T-box factor TBX-2. We have previously shown that TBX-2 interacts with the E2 SUMO-conjugating enzyme UBC-9, and that loss of TBX-2 or UBC-9 produces identical defects in ABa-derived pharyngeal muscle development. We now show that TBX-2 is SUMOylated in mammalian cell assays, and that both UBC-9 interaction and SUMOylation depends on two SUMO consensus sites located in the T-box DNA binding domain and near the TBX-2 C-terminus, respectively. In co-transfection assays, a TBX-2:GAL4 fusion protein represses expression of a 5xGal4:tk:luciferase construct. However, this activity does not require SUMOylation, indicating SUMO is not generally required for TBX-2 repressor activity. In C. elegans, reducing SUMOylation enhances the phenotype of a temperature-sensitive tbx-2 mutant and results in ectopic expression of a gene normally repressed by TBX-2, demonstrating that SUMOylation is important for TBX-2 function in vivo. Finally, we show mammalian orthologs of TBX-2, Tbx2, and Tbx3, can also be SUMOylated, suggesting SUMOylation may be a conserved mechanism controlling T-box factor activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-013-1336-y) contains supplementary material, which is available to authorized users.

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T-box transcription factors are important regulators of development in all animals, and altered expression of T-box factors has been identified in an increasing number of diseases and cancers. Despite these important roles, the mechanism of T-box factor activity is not well understood. We have previously shown that the C. elegans Tbx2 subfamily member TBX-2 functions as a transcriptional repressor to specify ABa-derived pharyngeal muscle, and that this function depends on SUMOylation. Here we show that TBX-2 function also depends on interaction with the Groucho-family corepressor UNC-37. TBX-2 interacts with UNC-37 in yeast two-hybrid assays via a highly conserved engrailed homology 1 (eh1) motif located near the TBX-2 C-terminus. Reducing unc-37 phenocopies tbx-2 mutants, resulting in a specific loss of anterior ABa-derived pharyngeal muscles and derepression of the tbx-2 promoter. Moreover, double mutants containing hypomorphic alleles of unc-37 and tbx-2 exhibit enhanced phenotypes, providing strong genetic evidence that unc-37 and tbx-2 share common functions in vivo. To test whether interaction with UNC-37 is necessary for TBX-2 activity, we developed a transgene rescue assay using a tbx-2 containing fosmid and found that mutating the tbx-2 eh1 motif reduced rescue of a tbx-2 null mutant. These results indicate that TBX-2 function in vivo depends on interaction with UNC-37. As many T-box factors contain eh1 motifs, we suggest that interaction with Groucho-family corepressors is a common mechanism contributing to their activity.

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