Abstract
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.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.T
VKKE is located in a region that is not highly conserved among T-box factors, high scoring small ubiquitin-like modifier peptide (SUMO) consensus sites are found near the C-terminus of TBX-2 proteins from C. elegans, C. briggsae, and C. remanei, suggesting this site may be functionally conserved (Fig. 1b)
We mutated each of these two sites in C. elegans TBX-2 to all alanines either in single mutants (LKIE→AAAA or VKKE→AAAA) or in a double mutant (LKIE/VKKE→AAAA) and tested whether these mutants affected the ability of a TBX-2 bait to interact with UBC-9 prey
Summary
Electronic supplementary material The online version of this article (doi:10.1007/s00018-013-1336-y) contains supplementary material, which is available to authorized users. Over-expression of the Tbx2-subfamily genes TBX2 and TBX3 is found in a number of human cancers [8] Despite their developmental and clinical importance, relatively little is known about the mechanism by which T-box factors function. We first used the two-hybrid assay to map interaction sites between TBX-2 and UBC-9 and found two SUMO consensus sites in TBX-2 that mediate interaction with UBC-9 One of these sites is located near the TBX-2 C-terminus, while the other is located in a highly conserved region of the T-box DNA binding domain. We examined TBX-2 transcriptional activity and found that in mammalian cells a TBX-2-GAL4 DNAbinding domain (GAL4-DBD) fusion protein represses expression of a GAL4-responsive reporter, but surprisingly this repression did not require SUMOylation. We suggest SUMOylation is a common mechanism regulating activity of T-box transcription factors
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