Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent upregulation, dependent on ERK signaling and inhibitable by stromal cell contact.Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor ␤ (CBF␤), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBF␤ up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBF␤, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1-mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and IntroductionDespite divergent phenotypes, megakaryocytic and erythroid lineages originate from a common bipotent progenitor, known variously as the blast-forming unit erythroid/megakaryocyte (BFU-E/MK) or the megakaryocyte erythroid progenitor (MEP). [1][2][3] As further evidence of a developmental link, human erythroblasts at relatively late stages of development retain the potential for megakaryocytic transdifferentiation. 4 The molecular basis for this developmental relationship appears to reside in the extensive sharing of lineage-restricted transcription factors. Many transcription factors initially identified as critical in erythroid development have been found through gene knock-out experiments to be important in megakaryocytic development. [5][6][7] GATA-1 is the prototypic erythro-megakaryocytic transcription factor, cooperating with its cofactor FOG-1 to serve essential roles in erythroid and megakaryocytic differentiation. 8 Enforced GATA-1 expression in myeloid cell lines promotes erythroid, megakaryocytic, or combined differentiation, depending on the cell type. [9][10][11] Knock-out of either the GATA-1 or the FOG-1 gene results in midgestation embryonic lethality because of severe anemia associated with abnormal or absent megakaryopoiesis. 7,12 Lineageselective knock-down in mice of GATA-1 expression in megakaryocytes causes increased megakaryocyte proliferation coupled with impaired maturation. 6,13 Knock-in mice with compound GATA-1 and GATA-2 mutations, causing the loss of FOG-1 binding, display a complete absence of megakaryopoiesis, a phenocopy of FOG-1 null mice. 8 Human hereditary mutations in the amino terminal...

It has been proposed that inhibitors of an oncogene's effects on multipotent hematopoietic progenitor cell differentiation may change the properties of the leukemic stem cells and complement the clinical use of cytotoxic drugs. Using zebrafish, we developed a robust in vivo hematopoietic differentiation assay that reflects the activity of the oncogene AML1-ETO. Screening for modifiers of AML1-ETO-mediated hematopoietic dysregulation uncovered unexpected roles of COX-2 and β-catenin-dependent pathways in AML1-ETO function. This approach may open doors for developing therapeutics targeting oncogene function within leukemic stem cells.

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Human acute myeloid leukemias with the t(8;21) translocation express the AML1-ETO fusion protein in the hematopoietic stem cell compartment and show impairment in erythroid differentiation. This clinical finding is reproduced in multiple murine and cell culture model systems in which AML1-ETO specifically interferes with erythroid maturation. Using purified normal human early hematopoietic progenitor cells, we find that AML1-ETO impedes the earliest discernable steps of erythroid lineage commitment. Correspondingly, GATA-1, a central transcriptional regulator of erythroid differentiation, undergoes repression by AML1-ETO in a nonconventional histone deacetylase-independent manner. In particular, GATA-1 acetylation by its transcriptional coactivator, p300/ CBP, a critical regulatory step in programming erythroid development, is efficiently blocked by AML1-ETO. Fusion of a heterologous E1A coactivator recruitment module to GATA-1 overrides the inhibitory effects of AML1-ETO on GATA-1 acetylation and transactivation. Furthermore, the E1A-GATA-1 fusion, but not wild-type GATA-1, rescues erythroid lineage commitment in primary human progenitors expressing AML1-ETO. These results ascribe a novel repressive mechanism to AML1-ETO, blockade of GATA-1 acetylation, which correlates with its inhibitory effects on primary erythroid lineage commitment. (Cancer Res 2006; 66(6): 2990-6)

Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.