Notch Alters Sumoylation To Govern GFI1 Protein Stability and Support Its Transcriptional Repression Function

Abstract

The zinc-finger (ZF) transcriptional repressors, Growth factor independence (GFI)-1 and -1B are master regulators of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. GFI family members share near invariant SNAG and ZF domains at their N- and C-termini, respectively. Their SNAG domains recruit Lysine Specific Demethylase (LSD)-1, a dominant effector of transcriptional repression by GFI proteins, while ZFs 3-5 of both GFI1 and GFI1B recognize a common consensus element in target promoters. A non-homologous linker separates the SNAG and ZF domains. Despite near identical DNA binding and co-factor recruitment characteristics, GFI family proteins direct distinct hematopoietic outcomes. Granulopoiesis requires Gfi1, and Gfi1 mutations cause severe congenital neutropenia (SCN) type II. Gfi1 is also necessary for T-lymphopoiesis and maintenance of the malignant phenotype in lymphoid leukemias and lymphomas. In contrast, Gfi1b specifies erythroid and megakaryocytic fates and is often abundantly expressed in myeloid leukemias. These distinct outcomes cannot be explained by patterns of expression alone, as Gfi1 and Gfi1B transcripts partially overlap during hematopoiesis. These findings suggest a need to distinguish between GFI family proteins as they integrate developmental signals to coordinate alternate hematopoietic fates. How this occurs is not understood. We hypothesized that functional differences between GFI1 and GFI1B may arise from interactions and post-translational modifications occurring uniquely within their linker regions. 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. K239 is required for robust GFI1 SUMOylation, as arginine substitution (K239R) significantly impairs formation of high molecular weight, SUMO-conjugated GFI1 derivatives. Coincidentally, K239R substitution also significantly impairs LSD1 recruitment by the GFI1 SNAG domain. The SUMOylation site in the GFI1 linker is contained within the binding site for PIAS3, an E3 SUMO ligase. PIAS3 co-expression antagonizes transcriptional repression by GFI1, but not GFI1B. The GFI1-K239R derivative represses transcription comparable to wild type GFI1, but resists the inhibitory effects of PIAS3. Moreover, GFI1-K239R preferentially co-purifies with chromatin in fractionated cells, displaying a prolonged half-life relative to wild type GFI1. These data suggest that GFI1 SUMOylation occurs on chromatin, facilitates LSD1 recruitment and serves as a prelude to GFI1 degradation. We surmised that the Notch1 intracellular domain (N1-ICD), which requires GFI1 for both normal and malignant T-lymphopoiesis, might alter GFI1 SUMOylation to reinforce its functions as a transcriptional regulator. We show that N1-ICD interacts directly with the GFI1 linker via the same motif that binds PIAS3. N1-ICD and PIAS3 compete for GFI1 binding and display an oppositional relationship in GFI1-mediated transcriptional repression. N1-ICD reinforces GFI1 as a transcriptional repressor, blocks GFI1 conjugation by ubiquitin family proteins and prevents GFI1 degradation. Our findings suggest a post-transcriptional mechanism by which Notch signaling endorses GFI1 as a transcriptional regulator and intimate GFI1 binding partners like LSD1 as candidate biological response modifiers in Notch-driven malignancies. Disclosures:No relevant conflicts of interest to declare.