TIF1γ requires sumoylation to exert its repressive activity on TGFβ signaling

TIF1γ, a new regulator of TGFβ signaling, inhibits the Smad4-mediated TGFβ response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1γ participates in TGFβ signaling as a negative regulator of Smad4 during the TGFβ-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1γ is sumoylated and interacts with Ubc9, the only known SUMO-conjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1γ, the Smad interaction domain. We show that a sumoylation-defective TIF1γ mutant significantly reduces TIF1γ inhibition of Smad complexes and that of the Smad-mediated TGFβ transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1γ sumoylation is required to limit Smad4 binding on the PAI-1 TGFβ target gene promoter. Ectopic expression of TIF1γ in mammary epithelial cells inhibits TGFβ-induced EMT, an effect relieved by expression of non-sumoylated TIF1γ. Taken together, our results identify a new TGFβ regulatory layer, whereby sumoylation strengthens the TIF1γ repressive action on canonical TGFβ signaling.