Function of the C. elegans T-box factor TBX-2 depends on SUMOylation

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.

Function of the C. elegans T-box factor TBX-2 depends on interaction with the UNC-37/Groucho corepressor

“…Mutating two consensus residues in the TBX-2 eh1 motif eliminated interaction of the TBX-2 bait with the UNC-37 prey (Figure 1B; Supplemental Figure 1). In contrast, this mutation did not affect the interaction between TBX-2 and the E2 SUMO conjugating enzyme UBC-9, which binds a site near the eh1 motif (Huber et al, 2013), indicating that the mutant TBX-2 is expressed in yeast (Supplemental Figure 1). A similar mutation in MLS-1 eliminates interaction with UNC-37 (Miller and Okkema, 2011), and in subsequent experiments we refer to this mutation as TBX-2 FDV➔ADA .…”

“…We have previously used the hypomorphic mutant tbx-2(bx59) as a sensitized genetic background to ask if mutations in other genes affect TBX-2 activity in vivo (Huber et al, 2013; Milton et al, 2013). To test if UNC-37 affects TBX-2 activity, we compared the phenotype of the tbx-2(bx59) and unc-37(e262) single mutants to that of unc-37(e262); tbx-2(bx59) double mutants.…”

“…The following strains were used in this study: N2 wild type Bristol, DP38 unc-119(ed3) (Maduro and Pilgrim, 1995), OK0460 tbx-2(ok529)/dpy-17(e164) unc-32(e189) (Roy Chowdhuri et al, 2006), OK0507 cuIs21[ceh-22∷gfp] III (Roy Chowdhuri et al, 2006), OK0640 cuIs31[ceh-22∷gfp] X , CB262 unc-37(e262) I (Brenner, 1974), OK0660 tbx-2(bx59) III (Huber et al, 2013). …”

“…tbx-2(ok529) homozygotes were distinguished from heterozygotes by progeny tests in which rescued hermaphrodites were crossed into N2 males, and 100% of male progeny possessed the ok529 allele. To generate the synthetic lethal unc-37(e262); tbx-2(bx59) strain, individual animals were PCR genotyped for tbx-2(bx59) allele as previously described (Huber et al, 2013). …”

SUMOylation is essential for sex-specific assembly and function of the Caenorhabditis elegans dosage compensation complex on X chromosomes

“…SUMOylation is also important for pharyngeal muscle development in the embryo (33) and development of the reproductive system, including morphogenesis of the vulva (34)(35)(36). DCC SUMOylation is unique among these examples in representing SUMO modification of a protein complex.…”