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...

Human red cell differentiation requires the action of erythropoietin on committed progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in hypoplastic anemia. To address the basis for iron regulation of erythropoiesis, we established primary hematopoietic cultures with transferrin saturation levels that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identified as a critical regulatory element the aconitases, multifunctional iron-sulfur cluster proteins that metabolize citrate to isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or megakaryocytic progenitors. An active site aconitase inhibitor, fluorocitrate, blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid iron restriction response in vitro and reversed anemia progression in irondeprived mice. The mechanism for aconitase regulation of erythropoiesis most probably involves both production of metabolic intermediates and modulation of erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase C␣/␤, or possibly protein kinase C␦, whose activities were regulated by iron, isocitrate, and erythropoietin. IntroductionRed cell production results from erythropoietin (Epo)-driven survival, proliferation, and maturation of committed bone marrow progenitor cells. This process critically depends on cellular uptake of adequate bioavailable iron, provided in the form of diferric transferrin. Compromise in iron uptake or intracellular trafficking results in iron-restricted erythropoiesis, characterized by diminished marrow responsiveness to Epo. 1 Epo acts during an early phase of erythroid development, between late (erythroid burst-forming unit) and early pronormoblast stages, before the initiation of hemoglobin synthesis. [1][2][3] Therefore, iron restriction serves as a checkpoint to restrain Epo-driven progenitor expansion in the face of limited iron stores. It has been documented in zebrafish, in which defects in intracellular iron utilization block early erythroid differentiation, 4 and in mammals, in which dietary iron deficiency impairs the transition from erythroid colony-forming unit to pronormoblast. 5 In the clinical setting, iron-restricted erythropoiesis underlies many of the anemias that are refractory to Epo treatment (eg, anemias of chronic renal disease and inflammation). 1 The lineage-and stage-selective nature of the erythroid iron restriction response has suggested involvement of a specialized signaling pathway distinct from the iron depletion response that occurs in a wide variety of cell types treated with chelators. Supporting this notion, chelator-induced iron depletion in MCF-7 cells causes cell-cycle arrest in G 1 phase followed by apoptosis, 6 neither of which is seen in iron-restricted erythropoiesis in vivo. 2,6,7 Thus, to identify mechanisms applicable to ...

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.

The transcription factor GATA-1 participates in programming the differentiation of multiple hematopoietic lineages. In megakaryopoiesis, loss of GATA-1 function produces complex developmental abnormalities and underlies the pathogenesis of megakaryocytic leukemia in Down syndrome. Its distinct functions in megakaryocyte and erythroid maturation remain incompletely understood. In this study, we identified functional and physical interaction of GATA-1 with components of the positive transcriptional elongation factor P-TEFb, a complex containing cyclin T1 and the cyclindependent kinase 9 (Cdk9). Megakaryocytic induction was associated with dynamic changes in endogenous P-TEFb composition, including recruitment of GATA-1 and dissociation of HEXIM1, a Cdk9 inhibitor. shRNA knockdowns and pharmacologic inhibition both confirmed contribution of Cdk9 activity to megakaryocytic differentiation. In mice with megakaryocytic GATA-1 deficiency, Cdk9 inhibition produced a fulminant but reversible megakaryoblastic disorder reminiscent of the transient myeloproliferative disorder of Down syndrome. P-TEFb has previously been implicated in promoting elongation of paused RNA polymerase II and in programming hypertrophic differentiation of cardiomyocytes. Our results offer evidence for P-TEFb cross-talk with GATA-1 in megakaryocytic differentiation, a program with parallels to cardiomyocyte hypertrophy. IntroductionMegakaryocytes and erythroblasts develop from bipotential megakaryocyte-erythroid progenitors (MEPs) under the influence of multiple critical transcription factors. Some of these factors, such as GATA-1, GATA-2, FOG1, and SCL, promote both lineages, whereas others, such as RUNX1 and EKLF, promote only megakaryocyte or erythroid development, respectively. 1,2 Many of these key factors serve dual functions in development, activating lineage-appropriate genes while simultaneously repressing lineageinappropriate genes. 2 Numerous in vitro and in vivo studies have emphasized the centrality of GATA-1 in megakaryocyte development. Virtually all megakaryocytic promoters contain functionally important GATA binding sites. 3 Mice with diminished GATA-1 in megakaryocytes display thrombocytopenia and aberrant megakaryocytic maturation. 4 In particular, the GATA-1-deficient megakaryocytes show defects in growth regulation, polyploidization, proplatelet formation, granule biogenesis, and surface antigen expression. [5][6][7] Interestingly, these mice also display MEP abnormalities, implicating GATA-1 in development of a properly primed bipotent progenitor. 8,9 In humans, hereditary and acquired GATA-1 mutations have both been associated with defective megakaryopoiesis. Familial X-linked thrombocytopenia results from missense mutations affecting the N-terminal zinc finger of GATA-1, a domain involved in recruitment of the cofactor FOG1. [10][11][12][13][14] Acquired mutations of GATA-1 occur in the setting of the 2 Down syndrome (DS)-associated megakaryoblastic proliferative disorders, transient myeloproliferative disorder (DS-TMD) and...