Two Distinct Transactivation Domains of GATA1 Contribute Megakaryopoiesis,

Abstract 3384

Transcription factor GATA1 orchestrates erythroid/megakaryocytic cell differentiation by coordinately regulating the expression of multiple genes encoding proteins responsible for the proliferation, differentiation and survival of progenitors. Mice lacking GATA1 expression die in utero due to insufficient primitive erythropoiesis. GATA1 retains three functional domains; N-terminus (NT) transactivation domain (TAD) and N-terminus and C-terminus zinc finger domains that are both critical for DNA-binding and interaction with co-factors. We previously showed that each domain of GATA1 confers distinguishable properties to GATA1 during primitive and definitive hematopoiesis in vivo. Especially, GATA1 mutant lacking NT domain (GATA1s) is sufficient to rescue the GATA1-deficient mice from embryonic lethality, but the rescued mice suffered from anemia and thrombocytopenia. Showing very good agreement with this finding, inherited mutation yielding GATA1s was found in a family with dyserythropoiesis and dysmegakaryopoiesis. Whereas previous biochemical studies argued that NT domain was a unique TAD in GATA1, these findings suggest that alternative TAD domain is vital for the GATA1 activity in vivo. In this study, we focus on the C-terminus (CT) region of GATA1. The transactivation activity of GATA1 lacking the CT region (G1-DCT) was significantly reduced to approximately 30% of the wild-type GATA1, which was similar to the GATA1s or NT deletion level. Practically no transactivation activity was found in the GATA1 lacking both NT and CT domains. We also examined transactivation potential of the NT and CT fused with GAL4-DBD. CT-GAL4-DBD fusion protein showed Luciferase reporter activity over 20-fold higher than the GAL4-DBD. Furthermore, retrovirally transduced G1-DCT failed to control the hyperproliferation of megakaryocyte progenitors lacking endogenous GATA1, similar to the salient feature reported for GATA1s. These results thus indicate that the coordinated function of CT- and NT-TADs of GATA1 is required in the megakaryopoiesis. GATA1s mutation was found in majority cases of Down syndrome patients with transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Since CT domain is rich in Ser, Thr and Pro residues, while the NT domain is an acidic transactivation domain rich in Glu, we surmise that GATA1 regulates target gene expression by utilizing two distinct transactivation domains. The imbalance in gene expression in megakaryocytic progenitors, which is caused by GATA1s loosing the function of NT-TAD, may be the key aspect for the understanding of the onset of TMD and AMKL.