Abstract LB-357: Notch and PIAS3 differentially regulate sumoylation of GFI1 to modulate transcriptional repression.

Abstract

Abstract The zinc-finger (ZF) transcriptional repressor, Growth factor independence (GFI)-1, is a master regulator of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. Gfi1 is necessary for T-lymphopoiesis following Notch activation, while Notch-driven T-cell leukemia/lymphoma requires Gfi1 to maintain a malignant phenotype. Granulopoiesis also requires Gfi1, and Gfi1 mutations cause severe congenital neutropenia. GFI1 shares near invariant SNAG and ZF domains with its paralog, GFI1B, at their N- and C-termini, respectively. These conserved regions confer comparable DNA binding and transcriptional repression abilities to both proteins, yet Gfi1b specifies erythroid and megakaryocytic fates and is often abundantly expressed in myeloid leukemias. A non-homologous linker separates SNAG and ZF domains in GFI proteins. We hypothesized Notch dependence on GFI1 in normal and malignant T-lymphopoiesis arises from interactions and post-translational modifications occurring uniquely within its linker region. We show the GFI1 linker contains a type-I sumoylation consensus motif centered on K239 that is absolutely conserved among mammalian GFI1 proteins but absent from GFI1B. GFI1 is sumoylated on K239, and arginine substitution (K239R) abolishes modification by ubiquitin family proteins. We confirm GFI1 binds the E3 sumo ligase, PIAS3, and extend this observation to mammalian cells. The binding interface includes the PIAS3 RING domain and a subdomain within the GFI1 linker that contains the sumoylation consensus motif. Transcriptional repression by GFI1 is impaired by PIAS3 co-expression. GFI1-K239R resists this effect of PIAS3, and its half-life is prolonged 3-fold over wild type GFI1. These data suggested GFI1 sumoylation and turnover limit its functions in transcriptional control, and that responses dependent upon GFI1 might alter sumoylation to reinforce GFI1 functions. To explore this notion, we leveraged the dependence of Notch on GFI1. We show that Notch intracellular domain (NICD) expression blocks GFI1 sumoylation and reverses the inhibitory effect of PIAS3 on GFI1-mediated transcriptional repression. Moreover, NICD binds the GFI1 linker and competes with PIAS3 for GFI1 binding. These results define an oppositional relationship between NICD and PIAS3 in GFI1 sumoylation and transcription, and intimate a post-translational mechanism by which Notch signaling supports GFI1 functions in normal and malignant T-lymphopoiesis. Citation Format: Daniel Andrade, Jason Singer, Diana Bareyan, David McClellan, Helena Lucente, Mahesh Chandrasekharan, Michael E. Engel. Notch and PIAS3 differentially regulate sumoylation of GFI1 to modulate transcriptional repression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-357. doi:10.1158/1538-7445.AM2013-LB-357