AbstractsBiology & Animal Science

The transcription factor c-Myb is an important regulator involved in proliferation, differentiation, and apoptosis of hematopoietic cells. Its activity is regulated through interactions with co-repressors and co-activators. Some of these interactions are in turn directed by several post-translational modifications, including SUMOylation. SUMO (Small Ubiquitin-related Modifier) is covalently conjugated to lysine in the consensus motif ΨKxE, and two sites; K503 and K527, located in c-Myb c-terminal regulatory domain (CRD) are described to be SUMO-modified [1]. c-Mybs transactivation potential is severely reduced, even after a small increase in the level of SUMOylation. Functional effects resulting from SUMOylation are, however, challenging to analyze due to the low level of SUMO-modified c-Myb in the cell. SUMOylation is a highly dynamic and reversible process, where SUMO is attached to the target protein in three steps, involving three different enzymes, including the E2 conjugating enzyme Ubc9. SUMO is deconjugated from substrates in a fourth step by SUMO specific proteases SENPs, hence, making the reaction reversible. Tools available to study c-Myb SUMOylation, including SUMO fusion constructs and ectopic expression of SUMO pathway components, are either static or globally acting. This makes them unsuitable for studies of the modification in a specific, dynamic, and time-controlled manner. We have therefore, inspired by the USDDS method (Ubc9/substrate dimerization dependent SUMOylation) [2], designed a system to induce c-Myb SUMOylation and deSUMOylation by bringing c-Myb together with Ubc9 or SENP1 in vivo. We have taken advantage of the heterodimerization system from ARIAD. The system is based on a rapamycin derivate (AP21967), which binds the heterodimerization domains FKBP and FRB with high affinity. Hence, any two proteins fused to the domains can be brought together in vivo by addition of the dimerizer. A system for inducible SUMOylation of c-Myb was established by fusing c-Myb and Ubc9 to FKBP and FRB, respectively. The two proteins were by addition of AP21967 brought together in vivo, allowing SUMO modification to take place. A corresponding system for deSUMOylation was established by fusing SENP1 to the FRB domain. After having established and validated them, both systems were applied to address functional effects of SUMOylation and deSUMOylation in an effector-reporter assay. Finally, we addressed the systems’ biological relevance by dynamically study SUMO-dependent phosphorylation and SUMO-dependent protein interactions. Inducible dimerization may prove to be an important tool to unravel the dynamic role SUMO has in regulating c-Myb, and may also be a valuable tool for the study of posttranslational modifications in general. [1] Dahle, O., et al., 2003 [2] Zimnik, S., et al., 2009