Search

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGCΔ) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity. [ABSTRACT FROM AUTHOR]

GATA1 and NF-E2 p45 are two important regulators of megakaryopoiesis. Whereas GATA1 is known to regulate the p45 gene, details of the GATA1 contribution to the spatiotemporal expression of the p45 gene remain to be elucidated. To clarify the relationship between GATA1 and p45, we performed genetic complementation rescue analysis of p45 function in megakaryocytes utilizing the hematopoietic regulatory domain of the Gata1 gene (G1HRD). We established transgenic mouse lines expressing p45 under G1HRD regulation and crossed the mice with p45-null mice. Compound mutant mice displayed normal platelet counts and no sign of hemorrhage, indicating that G1HRD has the ability to express p45 in a spatiotemporally correct manner. However, deletion of 38 amino acids from the N-terminal region of p45 abrogated the p45 rescue function, suggesting the presence of an essential transactivation activity in the region. We then crossed the G1HRD-p45 transgenic mice with megakaryocyte-specific Gata1 gene knockdown (Gata1ΔneoΔHS) mice. The G1HRD-p45 transgene was insufficient for complete rescue of the Gata1ΔneoΔHS megakaryocytes, suggesting that GATA1 or other factors regulated by GATA1 are required to cooperate with p45 for normal megakaryopoiesis. This study thus provides a unique in vivo validation of the hierarchical relationship between GATA1 and p45 in megakaryocytes. [ABSTRACT FROM AUTHOR]