Doctor of Philosophy

dissertationCell differentiation requires precise genetic regulation by transcription factors, their associated coregulators, and histone modifying enzymes. Growth factor independence (GFI) family of transcriptional repressors are crucial regulators during hematopoiesis and play an important role in multilineage fate specification. GFI1 and GFI1B are highly homologous proteins sharing a nearly identical N-terminal Snail/Slug/GFI (SNAG) domain and a highly conserved concatemer of six C-terminal zinc fingers (ZnFs). For transcriptional repression, GFI family members both recognize the same DNA response element via ZnF 3-5 and both recruit the dominant effector lysine specific demethylase (LSD)1 via their SNAG domain. However, the complete group of GFI regulatory proteins and complex members required for LSD1-dependent repression remains unknown. To gain additional insights into LSD1-dependent or ZnF-interacting GFI partners, we utilized proteome-wide proximity labeling comparing the interacting partners of full-length, wild type GFI1B (GFI1B-Full) to: 1) GFI1B mutants incapable of binding LSD1, and 2) a naturally occurring splice variant of GFI1B (GFI1B-Short) lacking ZnFs 1 and 2. First, we identify LSD1-dependent GFI1B binding partners consisting of core (RCOR1, HDAC1, HDAC2, HMG20B and PHF21A) and putative (HMG20A, GSE1, ZMYM2 and ZNF217) components of the BRAF-histone deacetylase (HDAC) (BHC) complex. We show enforced expression of GFI1B in K562 erythroleukemia cells drives erythroid differentiation and is dependent on LSD1. We demonstrate that depletion of both HMG20A and HMG20B, or GSE1 block GFI1B-mediated erythroid differentiation in K562 cells, phenocopying impaired erythroid differentiation by LSD1 iv depletion or disruption of GFI1B-LSD1 binding. Second, we demonstrate a functional deficiency of GFI1B-Short by using GFI1B-mediated erythroid differentiation in K562 cells. We identify GFI1B binding partners dependent on ZnFs 1 and 2 by comparing GFI1B-Full interactors to GFI1B-Short interactors. Among proteins enriched in GFI1B-Full samples are E3 SUMO ligases and nuclear import proteins. We further demonstrate that GFI1B-Full is SUMOylated while GFI1B-Short is not-despite the fact that both splice variants contain the receptor lysine, K61. Finally, we identify five basic (arginine or lysine) amino acids in ZnFs 1 and 2 required for GFI1B cellular localization and GFI1B-Full-mediated erythroid differentiation in K562 cells. Given the dependency of LSD1 for both GFI proteins and high homology between their protein domains, our work adds mechanistic understanding of the BHC complex in GFI-mediated transcriptional repression and protein binding partners required for GFI protein processing within the cell