ALBERT

Beschreibung: Abstract 159 The anemias of chronic disease (ACD) are a common complication of malignancy, inflammation and kidney disorders. In ACD, there is dysregulation of iron homeostasis, decreased proliferation of erythroid progenitors, diminished production of erythropoietin (EPO), and shortened lifespan of RBC. Multiple pathophysiologic mechanisms have been implicated in the development of ACD, including elevated production of hepcidin and inflammatory cytokines, IFNγ, TRAIL, Interleukins-1β, 6, 10, 15, & TNFα. These cytokines are thought to directly inhibit erythroid differentiation through unknown mechanisms. The current study addressed the hypothesis that inhibition of erythropoiesis in ACD may arise through synergistic effects of iron deprivation and specific inflammatory cytokines. To identify relevant cytokines, candidate factors were applied to primary human erythroid progenitors in iron replete and restricted cultures. Peripheral blood human CD34+ progenitors from healthy donors underwent standard prestimulation for 72 hours, followed by culture in unilineage erythroid medium (4.5 U/ml EPO + 25ng/ml SCF) for 4-5 days under iron replete (100% transferrin saturation) or iron restricted (15% transferrin saturation) conditions. Candidate cytokines were screened for effects on viability, proliferation, and differentiation using cell counting and flow cytometric analysis of the erythroid cell surface marker GPA and the megakaryocytic antigen CD41a. Contrary to previous reports, the majority of cytokines (TRAIL & Interleukins-1β, 6, 10, 15) showed no effects on erythroid proliferation or differentiation under iron replete or restricted conditions. By contrast, both IFNγ and TNFα displayed potent inhibitory effects under iron restricted conditions but only weak effects in iron replete cultures. Typically, iron restriction alone reduced the proportion of GPA+ cells by 50%, whereas IFNγ or TNFα combined with iron restriction caused a 90% reduction. While both cytokines cooperated with iron restriction in blocking upregulation of GPA and promoting cell death, each cytokine also had distinctive effects on morphology and differentiation. IFNγ enhanced megakaryocytic development, while TNFα retained cells as immature, CD34+ progenitors. The synergistic inhibition of erythroid differentiation with iron restriction and TNFα was confirmed in vivo using a murine model of dietary iron deprivation coupled with continuous infusion of low-dose TNFα. Regarding the mechanism for this synergy, we have previously shown that erythroid iron deprivation leads to inactivation of the aconitase enzymes, which normally convert citrate to isocitrate, and that provision of exogenous isocitrate abrogates the erythroid inhibition associated with iron deprivation. Accordingly, participation of this pathway was assessed in the more potent erythroid inhibition associated with IFNγ or TNFα plus iron deprivation. Strikingly, isocitrate administration not only abrogated effects due to iron deprivation but also those due to the inflammatory cytokines, leading to complete rescue of erythroid differentiation. To address the underlying basis for erythroid cross-talk of iron and cytokine signaling, we screened pathways implicated in iron metabolism and inflammation. Two relevant pathways identified were Jun kinase (JNK) and calmodulin-associated kinase II (CAMKII), important in TNFα and IFNγ signaling, respectively. In particular, TNFα and iron deprivation synergized in the activation of JNK, and IFNγ and iron deprivation synergized in activating CAMKII. In both cases, isocitrate partially restored the activation to basal levels. As an important negative control, iron deprivation did not affect IFNγ activation of STAT1 phosphorylation, indicating that its effects were not due to upregulation of receptor expression or function. Altogether, these data suggest that among the various cytokines implicated in ACD, only IFNγ and TNFα synergize with iron deprivation in the inhibition of erythropoiesis. These actions occur through cross-talk between intracellular signaling pathways, specifically pathways involving aconitase and cytokine-activated kinases. The connection of aconitase/metabolism with inflammation is novel and has implications for clinical treatment of ACD, as well as for new understanding of erythroid and inflammatory signaling. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: AML1-ETO (A/E) is the fusion product of a chromosomal translocation, t(8;21) frequently associated with FAB M2 acute myeloid leukemia (AML). The fusion combines the runt domain of the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. Clinical cases of AML with t(8;21) are distinguished by blockade in erythroid differentiation. In addition, enforced expression of A/E in primary human erythroid progenitors impairs differentiation. Existing paradigms postulate that A/E exerts its leukemogenic effects through recruitment to RUNX binding sites of cofactors such as corepressors, histone deacetylases (HDACs), and DNA methyltransferases (DNMTs), causing repression of RUNX target genes. However, this paradigm fails to explain effects of A/E on erythropoiesis as erythroid genes generally lack functional RUNX sites. We have published a physical and functional interplay between RUNX1 and the erythroid master regulator GATA-1 (Blood 101:4333). Furthermore, A/E physically interacted and functionally interfered with GATA-1. In the current studies we have examined domain and cofactor requirements for A/E inhibition both of GATA-1 function and of erythroid differentiation. Deletional mutagenesis of A/E demonstrated that the zinc finger (NH4) and runt domains were absolutely required for GATA-1 inhibition. Treatment with HDAC and DNMT inhibitors failed to affect A/E repression of GATA-1. RNAi knockdown of all known NH4 interactors, HDACs 1-3, N-CoR, SMRT-A, and SMRT-B also failed to affect A/E inhibition of GATA-1. Inducible expression of A/E in MEL cells caused downregulation of endogenous GATA-1 protein and mRNA, an effect dependent on induction of erythroid differentiation. A coexpressed GATA-1-GFP fusion showed downregulation with identical kinetics to endogenous GATA-1. Interestingly, proteasome-specific inhibitors effectively prevented the downregulation of endogenous GATA-1 and GATA-1-GFP caused by induction of A/E coupled with erythroid differentiation. Fluorescence microscopy showed a striking relocation of GATA-1-GFP from the nucleus to discrete, paranuclear bodies upon joint induction of A/E expression and erythroid differentiation. Our findings indicate that A/E inhibition of GATA-1 occurs through a previously undescribed mechanism that involves GATA-1 redistribution to novel cellular structures followed by proteasome-mediated degradation. These findings expand the paradigm of A/E leukemogenicity to include a non-transcriptional mechanism in which a growth inhibitor/tumor suppressor, GATA-1, is targeted by A/E for proteolytic degradation in a manner reminiscent of human papilloma virus E6 targeting of p53 for degradation in cervical carcinogenesis.

Beschreibung: Programming of megakaryocytic differentiation requires precise coordination of multiple signal transduction and transcription pathways. Previous in vivo and in vitro studies have implicated RUNX1 and GATA-1 as transcription factors that collaborate in the execution of this program. Analysis of the mechanism for the synergy of these two factors revealed induction of RUNX1 hyperphosphorylation by GATA-1 coexpression. A pharmacologic screen identified roscovitine as an inhibitor of the transcriptional cooperation, implicating a cyclin-dependent kinase (Cdk). A screen employing a panel of dominant-negative Cdk mutants identified Cdk9 as a critical component of the GATA-1-RUNX1 cooperation. In addition, HEXIM1, an endogenous Cdk9 inhibitor, similarly blocked transcriptional synergy. Furthermore, two kinase inhibitory compounds, DRB and flavopiridol, also blocked GATA-1-RUNX1 cooperation at concentrations specific for Cdk9 inhibition. Regarding the mechanism for GATA-1 induction of RUNX1 phosphorylation, coimmunoprecipitation experiments showed GATA-1 binding to both Cdk9 and cyclinT1. To examine the role of P-TEFb in primary megakaryocytic differentiation, human CD34+ cells in megakaryocytic cultures underwent treatment with 50 nM flavopiridol, a dose selective for Cdk9 inhibition. This treatment blocked megakaryocytic polyploidization while having no effect on the cell cycle properties of the non-megakaryocytic cells in the cultures. The treatment also impaired upregulation of CD41. Extending these findings to an in vivo model system, mice underwent treatment with daily low dose flavopiridol (5–7 mg/kg/day), a regimen previously shown to have no toxicity. Wild type C57BL/6 (wt BL/6) mice were compared with the ΔneoΔHS strain (GATA-1Lo) which has diminished GATA-1 expression in megakaryocytes. After only 1 week of treatment, the GATA-1Lo mice developed worsening thrombocytopenia associated with new-onset anemia, with several dying after 2 weeks of treatment. Flow cytometry on marrow from the treated GATA-1Lo mice revealed a marked expansion of abnormal megakaryocytes showing coexpression of CD71 plus CD41 and loss of polyploidization. Marrow and spleen histology showed extensive replacement by immature-appearing megakaryocytes with hypolobulated nuclei, as well as frequent pyknotic megakaryocytes. The control mice, flavopiridol treated wt BL/6 and saline treated GATA-1Lo, displayed none of these abnormalities. Additional experiments determined the flavopiridol effect on the GATA-1Lo mice to be completely reversible, with normalization of all parameters 2 weeks after ending treatment. In aggregate, these data implicate P-TEFb recruitment by GATA-1 in mediating cooperative activation of megakaryocytic promoters with RUNX1. This pathway may depend in part on the direct phosphorylation of RUNX1 by Cdk9. In mice, a synthetic lethal relationship between megakaryocytic GATA-1 deficiency and Cdk9 inhibition exists, manifesting as a fulminant but reversible megakaryocytic proliferative disorder reminiscent of the Down syndrome-associated megakaryocyte proliferations. A model is proposed in which P-TEFb, as a component of GATA-1-RUNX1 transcriptional complexes, plays an integral role in the specific programming of megakaryocytic differentiation, with particular importance in the unique cell cycle changes associated with this lineage.

Beschreibung: Erythropoietin (Epo) signaling drives normal erythropoiesis by promoting the survival, proliferation and maturation of committed erythroid progenitor cells. Epo acts at an early stage in erythroid development, prior to the initiation of hemoglobin synthesis. Under conditions of iron restriction, erythroid progenitors become refractory to Epo, resulting in hypoplastic anemia. The resulting iron restriction checkpoint protects iron stores from depletion by preventing Epo-driven erythroid expansion and inappropriate iron utilization for hemoglobin synthesis. In addition to diminished body stores, defects in iron uptake or intracellular trafficking can also activate this checkpoint and contribute to Epo-refractory anemias, e.g. sideroblastic anemias. Our previous work using primary human hematopoietic cultures had implicated aconitase enzymes, which interconvert citrate and isocitrate, as critical regulators of the erythroid iron-restriction checkpoint. In those studies, supplying cells with isocitrate had completely abrogated the block in erythroid development caused by iron restriction. In the current studies, we examine the mechanism for isocitrate rescue of erythropoiesis in iron deprived human progenitors and determine the in vivo effects of isocitrate administration in mice with iron deficiency anemia. Initial experiments addressed whether the activity of isocitrate was due to its catabolism to yield ATP and succinyl CoA, a precursor of heme. Several independent findings argued against such a metabolic mechanism. Firstly, erythroid progenitors showed no changes in cellular [ATP] with iron deprivation −/+ isocitrate. Secondly, a bioactive analog of alpha-ketoglutarate, TaKG, failed to rescue erythropoiesis under iron deprivation. Thirdly, isocitrate promoted erythroid differentiation in progenitors with blockade in mitochondrial biogenesis, induced by chloramphenicol. In these last studies, isocitrate reversed chloramphenicol inhibition of glycophorin A and globin expression; exogenous hemin by contrast reversed only the inhibition of globin expression. The combination of isocitrate and hemin, however, showed strong synergy in the rescue of growth and globin expression in cholaramphenicol treated progenitors. Subsequent experiments tested the hypothesis that isocitrate functions as a second messenger in erythroid development. Accordingly, iron deprived erythroid progenitors exposed to a range of Epo levels (0.05–20 U/ml) underwent isocitrate treatment. Remarkably, isocitrate showed no rescue of iron deprived erythroid cultures with 0.05 U/ml Epo, a dose that still promotes erythroid differentiation in high iron cultures. Partial rescue was obtained with 0.2 U/ml Epo, and complete rescue with 〉 4.5 U/ml. Previous studies have suggested that Epo-mediated calcium signaling shows a strong dosage dependency, requiring relatively high doses of Epo to activate calcium influx. In human CD34+ progenitors exposed to 4.5 U/ml Epo for either 5 hours or 3 days, iron deprivation induced a drop in steady state intracellular calcium levels. Inclusion of isocitrate in the medium restored the intracellular calcium to the levels seen in the iron-replete cultures. Finally, the in vivo activity of isocitrate was assessed in a murine model of iron deficiency anemia. Strikingly, intraperitoneal injections of isocitrate (200 mg/kg/day for 5 days) significantly augmented the red cell counts in C57BL/6 weanlings subjected to a low iron diet: mean RBC of 9.3 ± 0.35 × 10e12 cells/liter for the isocitrate group compared to an average RBC of 7.4 ± 0.51 × 10e12 cells/liter for the saline control group (P=0.012, N = 6 for each group). Interestingly, this increase accompanied a parallel decrease in red cell mean corpuscular hemoglobin concentration. Taken together, our results suggest a role for isocitrate as a second messenger coupled to Epo signaling and potentially involved in intracellular calcium regulation. In vivo administration abrogates the iron restriction checkpoint on red cell production, as in ex vivo studies, but cannot drive hemoglobin synthesis in the face of limited iron stores. The ability of isocitrate to collaborate with hemin in overriding erythroid mitochondrial defects offers novel therapeutic avenues for sideroblastic anemias.

Beschreibung: 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 up-regulation, 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 β (CBFβ), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFβ up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFβ, 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 RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins αIIb and α2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).

Beschreibung: Infants with Down syndrome (DS) display a high incidence of a reversible megakaryoblastic proliferation known as transient myeloproliferative disorder (DS-TMD). The clinical features of DS-TMD include marrow and liver infiltration by abnormal megakaryoctic precursors. These cells show a high propensity for spontaneous death, leading to liver damage and occasionally tumor lysis syndromes. Rapid disease onset is followed by gradual spontaneous remission over weeks to months. 10–20% of patients experience disease recurrence, which manifests as irreversible acute megakaryoblastic leukemia (DS-AMKL). DS-TMD pathogenesis requires trisomy 21 combined with acquired mutations of GATA-1, a transcription factor that programs megakaryocytic and erythroid maturation. The mutant sGATA-1 consists of an 84 amino acid amino-terminal truncation with diminished transcriptional activation. Evolution of DS-TMD to DS-AMKL may involve acquisition of p53 mutations, according to a recent clinical study in which such mutations occurred in 0/7 DS-TMD and in 2/3 DS-AMKL cases. Our lab has recently developed a murine model for DS-TMD based on GATA-1 cross-talk with the P-TEFb kinase complex promoting megakaryocytic maturation (see Elagib et al., Blood, prepublication 2008). In this model, Flavopiridol inhibition of P-TEFb in GATA-1Lo mice, which have megakaryocytic deficiency of GATA-1, induces a rapid onset megakaryoblastic proliferative disorder with many features of DS-TMD: high rate of spontaneous cell death within megakaryoblasts, collateral damage to normal cells in involved tissues (marrow and spleen), defective megakaryoblastic polyploidization, aberrant coexpression of erythroid antigens on megakaryoblasts, reversibility of disease upon withdrawal of P-TEFb inhibitor, requirement for defective GATA-1 in megakaryocytes. To determine the influence of p53 signaling on disease phenotype, the GATA-1Lo mutation was bred onto a TP53−/− background, followed by in vivo P-TEFb inhibition with low-dose Flavopiridol (5 mg/kg/day for 9 days). The resultant megakaryoblastic disorder in the GATA-1Lo::TP53−/− compound mutants showed several features distinct from findings in GATA-1Lo::TP53+/+ mice. In the peripheral blood, the GATA-1Lo::TP53−/− mice showed no significant decline in platelet counts: 2/5 mice had decreases, each 50%) in platelet counts with P-TEFb inhibition. On necropsy, the GATA-1Lo::TP53−/− mice showed splenomegaly of ~2-fold, while GATA-1Lo::TP53+/+ mice showed splenic shrinkage. Light microscopy revealed extensive splenic infiltration by sheets of megakaryoblasts with minimal evidence of cell death in GATA-1Lo::TP53−/− mice. Marrows from these mice also showed infiltration by megakaryoblasts, but with relative preservation of tissue architecture and bystander cellular elements. Flow cytometry on these marrows confirmed the presence of an abnornal population of megakaryocytic cells with erythroid antigen coexpression and highlighted the lack of intramedullary cell death, distinct from the extensive cell death seen in the GATA-1Lo::TP53+/+ marrows. Another histologic feature unique to the GATA-1Lo::TP53−/− mice consisted of hepatic infiltration by megakaryoblasts, without evidence of hepatocellular damage. Withdrawal of Flavopiridol for 14 days lead to clearance of megakaryoblasts from all involved organs, as seen with GATA-1Lo::TP53+/+ mice. Thus, p53 clearly modulates the phenotype of the megakaryoblastic disease seen in GATA-1Lo mice undergoing P-TEFb inhibition. In the absence of p53 signaling, this disease shows more extensive proliferation, as indicated by the splenomegaly and liver infiltration, combined with markedly decreased cell death. This decrease in cell death is accompanied by a decrease in collateral damage of bystander cells/tissues and by an ability to maintain platelet counts at pre-treatment levels. These findings provide in vivo validation that P-TEFb inhibition can activate p53 (see Gomes et al., Genes Dev., 20:601, 2006) and suggest that the cell death, tissue damage, and spontaneous regression seen in human DS-TMD could be p53-driven. Loss of p53 function may promote transformation to irreversible leukemia, but P-TEFb under such circumstances retains the potential to induce disease regression.

Beschreibung: Hematopoietic transitions that accompany fetal development, for example erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors show impaired execution of the morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects also contribute to inferior platelet recovery after cord blood stem cell transplantation and to inefficient platelet production by megakaryocytes (Mk) derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism known to drive adult Mk morphogenesis. A central feature of this pathway is known to involve sustained high amplitude activation of the P-TEFb kinase (Cdk9/cyclin T1). This cascade is initiated by downregulation of core components of the repressive complex of P-TEFb: the 7SK snRNP consisting of the 7SK small nuclear RNA (7SK) and its stabilizing factors MePCE and LARP7. The resulting high amplitude activation of P-TEFb drives multiple features of Mk differentiation: induction of cytoskeletal morphogenetic factors (ACTN1, FLNA, MKL1, HIC5), silencing of erythroid genes, promotion of histone H2B K120 monoubiquitiniation (H2BUb1), and phosphorylation of Spt5 at T806 (pSpt5 T806). Critical, rate-limiting steps triggering this pathway comprise MePCE proteolysis by calpain 2 and downregulation of LARP7, both resulting in destabilization of 7SK. In comparison to Mk derived from adult peripheral blood stem cells (adult Mk), Mk derived from umbilical cord blood stem cells (neonatal Mk) showed evidence of decreased P-TEFb activation with decreases in: 1) the expression of the cytoskeletal morphogenetic factors, 2) global H2BUb1, and 3) pSpt5 T806. In addition, neonatal Mk retained expression of the erythroid marker glycophorin A (GPA). Surprisingly, neonatal Mk failed to downregulate 7SK despite the downregulation of its stabilizers MePCE and LARP7, suggesting the existence of alternative 7SK stabilizing factor/s unique to neonatal Mk. Our screening identified the oncofetal RNA-binding protein IGF2BP3 to be expressed in neonatal but not adult Mk, and ectopic expression of IGF2BP3 in adult Mk conferred neonatal phenotypic features including reduction in size, increased proliferation and leaky erythroid antigen expression. Immunoprecipitation and glycerol gradient studies indicated the participation of IGF2BP3 in the 7SK snRNP complex. Mining of endogenous IGF2BP3 iCLIP data indicated that 7SK is one of the top direct targets of IGF2BP3 and further mapped binding to the 7SK fourth hairpin, a critical stability determinant. In loss of function studies, the knockdown of IGF2BP3 in neonatal Mk resulted in destabilization of 7SK and upregulation of the Mk morphogenetic cytoskeletal factors, as well as increased levels of H2BUb1, pSpt5 T806, and hyperphosphorylated RNA Pol II. Phenotypically, the knockdown of IGF2BP3 in neonatal Mk elicited adult features including increased size, enhanced polyploidization, reduced proliferation and silencing of erythroid antigen expression. Collectively, these findings suggest that the block in P-TEFb activation in neonatal Mk results from ontogentic stage-specific expression of IGF2BP3 which prevents the 7SK destabilization normally associated with adult megakaryocytic P-TEFb activation. We also identified a pharmacologic approach to inhibit IGF2BP3 expression, through inhibition of bromodomain and extra- terminal (BET) proteins, which reproducibly promoted adult features in neonatal Mk including enlargement, inhibition of erythroid antigen expression, upregulation of morphogenetic cytoskeletal factors, and increased platelet formation in vitro. Enforced expression of IGF2BP3 in neonatal Mk significantly blunted the effects of BET inhibitors indicating the specificity of their action in downregulating IGF2BP3. These results identify IGF2BP3 as a human ontogenic masterswitch that restricts megakaryocyte development through modulating a lineage-specific P-TEFb activation mechanism, revealing new strategies toward enhancing platelet production. Disclosures No relevant conflicts of interest to declare.

Beschreibung: RUNX1 and GATA-1 both play essential roles in the transcriptional programming of normal mammalian megakaryocytic development, deficiencies of either factor having similar phenotypic consequences. We have previously characterized physical and functional interactions between these two factors, and others have confirmed analogous cooperations in Drosophila and Danio homologs. We now present data on molecular mechanisms for the cooperation of these two factors in the transcriptional activation of the megakaryocytic aIIb integrin promoter. In these studies, GATA-2 also physically interacted with RUNX1 but failed to cooperate in transcriptional activation. In fact, increasing amounts of GATA-2 repressed the functional interplay between GATA-1 and RUNX1. Through generation of GATA-2/GATA-1 chimeras, we identified a conserved subdomain within the GATA-1 amino terminus that was both necessary and sufficient for transcriptional cooperation with RUNX1. Coexpression of wild type GATA-1 or of cooperating GATA-2/GATA-1 chimeras, but not of GATA-2 or of non-cooperating chimeras, induced a mobility shift in wild type RUNX1. Using immunoprecipitation followed by immunoblot with a panel of phosphospecific antibodies, we found GATA-1 to induce RUNX1 phosphorylation at recognition sites for cyclin-dependent kinases (cdks). Treatment of cells with roscovitine, a specific cdk inhibitor, blocked the transcriptional cooperation of GATA-1 with RUNX1 and eliminated the RUNX1 mobility shift caused by GATA-1 coexpression. Mutagenesis of RUNX1 identified a cluster of serine/threonine-proline (S/TP) sites collectively required for the transcriptional augmentation and mobility shift induced by GATA-1. In addition, intact DNA binding by RUNX1 was required for cooperation with GATA-1. These results provide a new paradigm for cooperation of interacting transcription factors, in which one partner recruits a kinase leading to phosphorylation and activation of the other partner. Furthermore, these results provide a biochemical basis for the previously inexplicable functional differences between GATA-1, which promotes megakaryocytic maturation, and GATA-2 which promotes proliferation without maturation.

Beschreibung: 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 cyclin-dependent 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.