An SCF-FBXW7 Ubiquitin Ligase Mediated Feedback Loop Facilitates GATA Factor Switching and Reinforces Commitment to Terminal Erythroid Maturation

Abstract

The GATA family transcription factors GATA2 and GATA1 play reciprocal roles during terminal erythroid and megakaryocytic maturation. GATA2 is expressed early in hematopoiesis and is required for early progenitor cell proliferation and survival. It must be down regulated in order for terminal maturation to occur. In contrast, GATA1 increases in expression during erythropoiesis and megakaryopoiesis, and is required for terminal maturation of these lineages. During this process, GATA1 displaces GATA2 at a large number of chromatin loci, typically leading to repression of early progenitor genes and activation of terminal maturation genes. This “GATA factor switch” is facilitated by the considerably shorter half-life of GATA2 (~30 min) compared to GATA1 (>4-6 hours). GATA2’s short half-life is mediated by ubiquitin-proteosomal degradation mechanisms. Yet the ubiquitin complex(es) responsible for GATA2-specific clearance has not been identified. In this study, we show that the Skip1-Cullin-Fbox (SCF) substrate recognition factor Fbxw7 is involved in GATA2 ubiquitin-mediated degradation. Proteomic and co-immunoprecipitation experiments show physical association of Fbxw7 with GATA2, but not GATA1. CRISPR/Cas9 deletion of Fbxw7 in G1ER4 cells results in elevated GATA2 steady-state protein levels, a prolonged GATA2 half-life, delayed GATA2 clearance upon estradiol induction, and impaired erythroid terminal maturation. Importantly, Fbxw7 mRNA and protein levels normally increase during terminal erythroid maturation and this occurs in a GATA1 dependent manner. We identified a key cis-regulatory element upstream of the Fbxw7 gene that is occupied by GATA2 during early erythropoiesis, but becomes bound by GATA1/TAL1 during late erythroid maturation in induced G1-ER4 cells and human CD34+ in vitro differentiated erythroblasts. This is accompanied by the acquisition of active enhancer histone marks at this site. Deletion of this regulatory element in G1ER4 cells blocks the GATA1 dependent increase in Fbxw7 mRNA transcript levels during erythroid cell maturation. Lastly, we identified a family with congenital hypoplastic anemia (lacking mutations in all known Diamond Blackfan Anemia genes) who harbor a germline Fbxw7missense mutation. Collectively, these findings identify GATA2 as a novel Fbxw7 substrate and uncover an ubiquitin-mediated post-transcriptional GATA factor feedback loop that reinforces GATA factor switching and commitment to terminal erythroid maturation. DisclosuresCantor:Amgen: Membership on an entity’s Board of Directors or advisory committees.