Transcriptional Regulation of Thrombopoiesis.

Over the past two decades, a number of key transcription factors have been identified that play essential roles in megakaryocyte development. These include GATA-1, GATA-2, Friend of GATA-1 (FOG-1), Runx-1, Cbf-β, Fli-1, GABPα, TEL, NF-E2 p45, Gfi-1b, and SCL/TAL. Importantly, mutations in genes encoding several of these have been linked to human disorders of thrombopoieisis. Germline GATA-1 mutations that disrupt binding to FOG-1 cause X-linked macrothrombocytopenia and dyserythropoietic anemia. Acquired GATA-1 mutations that lead to exclusive production of a short isoform (GATA-1s) play initiating roles in Down Syndrome Transient Myeloproliferative Disorder (DS-TMD) and subsequent Acute Megakaryoblastic Leukemia (DS-AMKL). Haploinsufficiency of Runx-1 causes Familial Platelet Disorder with Propensity to Develop AML (FPD/ AML). Heterozygous loss of the Fli-1 gene leads to the macrothrombocytopenia seen in Jacobsen’s (Paris-Trousseau) syndrome. Important outstanding questions include:

1. how these transcription factors act together to control megakaryocyte terminal maturation;

2. how they differentially act as activators or repressors depending on gene context;

3. how they intersect with cell signaling pathways;

4. how they may coordinate terminal megakaryocyte maturation with spatial location within the bone marrow;

5. how they may control cell fate decisions of bipotential erythroid/megakaryocytic progenitor cells; and

6. whether additional key transcription factors exist.

Application of proteomic approaches involving multi-protein complex purification has provided novel insights into some of these questions. We have isolated GATA-1 containing complexes from megakaryocytic cells and identified the Krüppel-type zinc finger transcription factor ZBP-89 as a novel regulator of megakaryocyte and erythroid development. Knockdown of ZBP-89 expression in zebrafish embryos and mice results in blocked early megakaryopoiesis and definitive erythropoiesis, phenocopying aspects of GATA-1- and FOG-1-deficient animals. We have also found that the focal adhesion component Kindlin-3 co-localizes to the nucleus and interacts with FOG-1, suggesting a possible link between integrin signaling and megakaryocyte transcriptional control. Runx-1 multi-protein complex purifications have led to the identification of Fli-1 as a direct binding partner. This interaction results in synergistic transcriptional activation of megakaryocyte-specific genes. Interestingly, the interaction between Runx-1 and Fli-1 occurs preferentially in cells that are differentiating, even though both proteins are expressed abundantly in undifferentiated megakaryoblastic cells. This binding event correlates with assembly of a large complex containing Runx-1/ Fli-1/GATA-1/FOG-1 based on gel filtration chromatography experiments. These factors may, therefore, act as a megakaryocyte-specific enhancesome. Key future directions are aimed at elucidating the molecular mechanisms that regulate these protein-protein interactions and how cell signaling pathways may modulate them.