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. Author manuscript; available in PMC: 2009 Nov 10.
Published in final edited form as: J Cell Biochem. 2009 Jun 1;107(3):377–382. doi: 10.1002/jcb.22142

Megakaryocytic Programming by a Transciptional Regulatory Loop: A Circle Connecting RUNX1, GATA-1, and P-TEFb

Adam N Goldfarb 1
PMCID: PMC2775554  NIHMSID: NIHMS136778  PMID: 19350569

Abstract

Transcription factors originally identified as drivers of erythroid differentiation subsequently became linked to megakaryopoiesis, reflecting the shared parentage of red cells and platelets. The divergent development of megakaryocytic and erythroid progenitors relies on signaling pathways that impose lineage-specific transcriptional programs on non-lineage-restricted protein complexes. One such signaling pathway involves RUNX1, a transcription factor upregulated in megakaryocytes and downregulated in erythroid cells. In this pathway, RUNX1 engages the erythro-megakaryocytic master regulator GATA-1 in a megakaryocytic transcriptional complex whose activity is highly dependent on the P-TEFb kinase complex. The implications of this pathway for normal and pathological megakaryopoiesis are discussed.

At a phenotypic level megakaryocytes and erythroblasts are polar opposites, but several studies have proven that they have a common origin in the bipotent MEP progenitor cell [Goldfarb, 2007]. Consistent with their shared heritage, striking similarities have been found in the transcriptional regulators programming each of these lineages. Transcription factors originally associated with highly specialized red cell functions, i.e. regulation of hemoglobin synthesis, were subsequently found to participate in normal megakaryocytic development. In fact, the majority of erythroid and megakaryocytic promoters have been found to recruit a common array of factors that includes GATA-1, FOG-1, SCL/tal, LMO2, Ldb-1, and NF-E2 [Goldfarb, 2007]. Thus divergence in erythroid and megakaryocytic transcriptional programs involves tight and complex regulation superimposed on highly overlapping transcriptional complexes. One pivotal element in this lineage divergence consists of RUNX1 and its cofactor CBFβ, which undergo upregulation early in megakaryocytic differentiation and downregulation early in erythroid differentiation [Elagib et al., 2003; Kundu et al., 2002; Lorsbach et al., 2004]. This article will discuss how the interaction of RUNX1 with GATA-1 provides a critical signal in the establishment and maintenance of the megakaryocytic transcriptional program. A model is presented in which the P-TEFb kinase complex, originally associated with transciptional elongation, functions as a critical integrating element in this developmental signaling pathway.

RUNX1 AND GATA-1 IN MEGAKARYOPOIESIS: LESSONS FROM HUMAN DISEASE

Human diseases have provided important information on the roles of RUNX1 and GATA-1 in megakaryopoiesis. Human germline mutations of RUNX1 and GATA-1 have both been identified in hereditary thrombocytopenia. In the case of RUNX1, familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML) manifests as an autosomal dominant thrombocytopenia with impaired platelet function, associated with mutations that predominantly impair the DNA binding function of the runt domain [Michaud et al., 2002; Song et al., 1999]. Marrows of patients with FPD/AML show increased numbers of small hypolobulated megakaryocytes [Heller et al., 2005] and impaired megakaryocytic colony formation [Song et al., 1999]. FPD/AML platelets express markedly diminished levels of the c-Mpl receptor [Heller et al., 2005] and show defects in α-granule contents [Sun et al., 2004]. In the case of GATA-1, X-linked thrombocytopenia (XLT) presents with moderate to severe thrombocytopenia, impaired platelet function, and variable degrees of concomitant anemia, associated with missense mutations affecting the GATA N-finger [Mehaffey et al., 2001; Nichols et al., 2000]. Marrows from these patients show a moderate to marked increase in megakaryocytes which may be small and hypolobulated [Mehaffey et al., 2001; Nichols et al., 2000]. Paradoxically, frequent erythroblasts in these patients show enlargement with multinucleation [Nichols et al., 2000]. EM on XLT marrows has revealed abnormal megakaryocytic ultrastructure, with diminished granulation and demarcation membranes [Nichols et al., 2000]. A single kindred has recently been described with a germline mutation in exon 2 of GATA-1, associated with exclusive expression of a short isoform, GATA-1s, which lacks amino acids 1-83; the clinical features in this kindred included X-linked macrocytic anemia and leukopenia (XL anemia/leukopenia); platelets were normal in number but abnormal in function [Hollanda et al., 2006]. The marrows displayed hypocellularity with increased megakaryocytes that were small and hypolobulated, as well as erythroblasts with enlargement and multinucleation. EM on the platelets revealed diminished granulation and a round shape. As discussed below, acquired somatic exon 2 mutations causing GATA-1s expression are a hallmark of megakaryocytic proliferative disorders in Down syndrome [Gurbuxani et al., 2004]. However, none of the 8 patients with germline GATA-1 exon 2 mutation has shown development of a proliferative disorder.

Acquired, somatic mutations of RUNX1 and GATA-1 occur consistently in human leukemias. RUNX1 and CBFβ collectively represent the genetic loci most frequently targeted for chromosomal translocations in acute leukemia [Downing and Shannon, 2002]. In addition, point mutations within RUNX1 frequently occur in myelodysplasia and occasionally in acute myeloid leukemia [Harada et al., 2004]. In cases of acute megakaryoblastic leukemia, FISH analysis has detected allelic loss or translocation of the RUNX1 locus in 25% of cases (4/15) [Berger et al., 2006]. Mutations of GATA-1 exon 2, leading to GATA-1s expression, occur in virtually all cases of the Down syndrome megakaryocytic proliferative disorders [Gurbuxani et al., 2004]. These disorders include the transient myeloproliferative disorder of Down syndrome and acute megakaryoblastic leukemia (DS-AMKL). These GATA-1 mutations do not occur in remission marrows of Down syndrome patients and have generally not been found in non-Down syndrome megakaryocytic leukemias (non-DS-AMKL). Microarray gene expression profiling has been exploited to compare DS-AMKL cells with non-DS-AMKL cells from age-matched patients [Bourquin et al., 2006; Ge et al., 2006]. Two independent microarray studies have found markedly increased erythroid gene expression in DS megakaryoblasts, suggesting that GATA-1s is defective in repressing the erythroid program during megakaryocytic differentiation. Bourquin et al. also identified upregulation of GATA-2 and downregulation of RUNX1 as specific features of DS-AMKL [Bourquin et al., 2006]. Table 1 highlights the similarities of human diseases with GATA-1 and RUNX1 mutations.

Table 1.

Human diseases with either GATA-1 or RUNX1 mutations

Gene Transmission Disease Mutation Platelets Megakaryocytes
RUNX1 Autosomal dominant FPD/AML Loss of function, runt domain Decreased, defective aggregation Increased, small, hypolobulated, decreased granules
GATA-1 X-linked XLT Loss of function, N-finger Decreased, defective aggregation Increased, small, hypolobulated, decreased granules
GATA-1 X-linked XL anemia/leukopenia Loss of function, GATA-1s expression N1 number, defective aggregation, Increased, small, hypolobulated, decreased granules
RUNX1 Acquired Non-DS-AMKL Allelic loss (25% of cases)
GATA-1 Acquired DS-AMKL Loss of function, GATA-1s
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RUNX1 AND GATA-1 IN MEGAKARYOPOIESIS: LESSONS FROM MOUSE MODELS

Knockout mice have also provided critical information on the contributions of RUNX1 and GATA-1 to megakaryopoiesis. Inducible deletion of RUNX1 in adult mice, RUNX1Δ/Δ, causes a rapid and prolonged 5-fold drop in peripheral blood platelet counts, with minimal effects on red cell or neutrophil numbers [Growney et al., 2005; Ichikawa et al., 2004]. Marrows from RUNX1Δ/Δ mice show virtual absence of normal-appearing megakaryocytes but a striking increase in overall numbers of megakaryocytes, as assessed by acetylcholinesterase staining. RUNX1-deficient megakaryocytes are small and hypolobulated, showing a marked decrease in ploidy as compared with normal megakaryocytes [Growney et al., 2005; Ichikawa et al., 2004]. RUNX1Δ/Δ marrows yield a 3-fold increase in numbers of CFU-MK as well as a marked increase in individual colony size [Growney et al., 2005; Ichikawa et al., 2004]. EM has identified ultrastructural disruption consisting of poorly developed demarcation membranes [Ichikawa et al., 2004].

In vivo assessment of GATA-1 function in megakaryopoiesis has relied on the GATA-1Lo mouse strain (also known as ΔneoΔHS Gata-1) in which knockout of an upstream regulatory element has led to megakaryocyte-specific loss of GATA-1 expression [Shivdasani et al., 1997]. These mice show diminished platelet counts at 15% of normal, coupled with a striking increase in marrow megakaryocytes. GATA-1-deficient megakaryocytes are also small and hypolobulated, with diminished ploidy on flow cytometry [Vyas et al., 1999]. Ultrastructural abnormalities include poorly developed demarcation membranes and diminished granulation [Shivdasani et al., 1997]. In ex vivo culture, GATA-1 deficient megakaryocytes are hyperproliferative, forming extremely large colonies in methylcellulose [Kuhl et al., 2005]. Interestingly, knock in mice expressing only GATA-1s (GATA1ΔN strain) display no abnormalities as adults but do manifest a proliferative burst of megakaryocytic progenitors in fetal yolk sac and liver from E9.5-E16.5 [Li et al., 2005]. These fetal GATA-1s megakaryocytic progenitors also display a hyperproliferative phenotype in ex vivo cultures but retain a capability for maturation. Table 2 highlights similarities between RUNX1 and GATA-1 knockout strains.

Table 2.

Mouse strains with either RUNX1 or GATA-1 mutations

Strain Overall phenotype Platelets Megakaryocytes
GATA-1 -/- Embryonic lethal E10.5-11.5 Not evaluable Not evaluable
RUNX1 -/- Embryonic lethal E11.5-12.5 Not evaluable Not evaluable
GATA-1Lo (ΔneoΔHS) Viable adult, shortened lifespan 6-fold decrease Small, hypolobulated, decreased ploidy, hyperproliferative, poorly formed demarcation membranes
RUNX1f/f ∷Mx1-CRE (pIpC induced deletion) Viable adult, shortened lifespan 5-fold decrease Small, hypolobulated, decreased ploidy, hyperprolferative, poorly formed demarcation membranes
GATA-1s (GATA1ΔN) Normal adult. Transient expansion of embryonic megakaryocyte progenitors. Normal Hyperproliferative fetal megakaryocytes
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OVERLAPPING TRANSCRIPTIONAL PROGRAMS OF GATA-1 AND RUNX1 IN MEGAKARYOPOIESIS

Gene expression profiling of normal and GATA-1 deficient murine megakaryocytes has identified several positive and negative target genes [Muntean and Crispino, 2005]. Retroviral restoration of GATA-1 in GATA-1Lo fetal liver megakaryocytes demonstrated rescued expression of several positive target genes including GPIIβ, GPIbα, and JAK2. However, retrovirally expressed GATA-1 failed to repress the negative target genes GATA-2 and Ets1. Gene expression profiles of RUNX1 deficient megakaryocytes have not been published, but in one study RUNX1-/- murine ES cells subjected to in vitro megakaryocytic cultures showed loss of expression of GPIbβ and GPIIIα [Liu et al., 2006a]. In addition, expression of a RUNX1 inhibitor, AML1-ETO, in murine Lin- marrow cells caused downregulation of JAK2 and GPIIβ, implicating these genes as positive targets of wild type RUNX1 [Liu et al., 2006b]. Mice bearing hypomorphic alleles of CBFβ, a critical cofactor for RUNX1, have shown markedly decreased platelet expression of CD41 (GPIIbIIIα) [Talebian et al., 2007]. In vitro studies have identified CD41 and GPIbα as direct RUNX1 targets based on chromatin immunoprecipitation (ChIP) and reporter assays [Xu et al., 2006; Zhao et al., 2008]. In vivo evidence has implicated C-MPL as a shared RUNX1/GATA-1 target gene in that platelets from FPD/AML patients show diminished Mpl protein and mRNA [Heller et al., 2005], and megakaryocytes from GATA-1Lo mice show decreased MPL transcript levels [Vyas et al., 1999]. Thus, deficiencies of RUNX1 and of GATA-1 produce similar phenotypes in megakaryocytes and appear to affect a common pool of target genes which include GPIIβ, GPIbα, JAK2, and C-MPL.

GATA-2 CANNOT COMPENSATE FOR GATA-1 IN MEGAKARYOPOIESIS

GATA-2 bears a high degree of homology to GATA-1 but has been associated with driving expansion of early multipotent hemaopoietic progenitors [Goldfarb, 2007]. The degree of functional redundancy between these two factors remains the subject of active investigation. Loss of GATA-1 expression in megakaryocytic and erythroid lineages leads to markedly increased GATA-2 mRNA levels, reflecting the role of GATA-1 as a critical repressor of the GATA-2 locus [Welch et al., 2004]. Primary purified GATA-1-deficient megakaryocytes, despite a five-fold increase in GATA-2 mRNA, manifest multiple abnormalities in growth control and differentiation [Muntean and Crispino, 2005]. One complicating issue is that GATA-2 protein is much less stable than GATA-1, raising questions about the relevance of mRNA levels [Minegishi et al., 2005]. A recent study addressed this issue by breeding a GATA-2 transgene onto a GATA-1 null background such that the protein levels and spatiotemporal patterns of GATA-2 expression closely recapitulated those of normal GATA-1 [Ferreira et al., 2007]. Unexpectedly, these mice, and similar strains with GATA-3 replacement of GATA-1, showed almost complete rescue of erythropoiesis. However, both the GATA-2 and GATA-3 replacement strains displayed significant thrombocytopenia, with 2-3-fold reductions in platelet counts. Thus, the most carefully controlled in vivo experiments to date indicate that the hematopoietic GATA factors are functionally redundant with regard to erythropoiesis, but that the GATA-1 protein exerts a unique function in megakaryopoiesis.

RUNX-GATA-FOG INTERACTIONS: A PHYLOGENETICALLY CONSERVED PATHWAY

Transcriptional cooperativity of RUNX1 and GATA-1 in the activation of megakaryocytic promoters has been established by reporter assays using transient transfection of several mammalian cell lines [Elagib et al., 2003; Xu et al., 2006]. These studies have also demonstrated physical interaction of the two factors via their conserved DNA binding domains. Interestingly, DS-AMKL derived GATA-1 mutants fully retain physical interaction with RUNX1 but manifest diminished transcriptional cooperation [Xu et al., 2006]. Strong phylogenetic conservation of this pathway is evident in that Drosophila counterparts of RUNX1 and GATA-1, Lozenge (Lz) and Serpent (Sp), cooperate in programming fly hematopoietic development, promoting crystal cell lineage commitment [Fossett et al., 2003; Waltzer et al., 2003]. The Drosophila homolog of FOG, U-shaped, blocks transcriptional cooperation of Lz and Sp and crystal cell development, in a manner dependent on its recruitment by the Sp amino terminal zinc finger module [Fossett et al., 2003; Waltzer et al., 2003]. In a striking correspondence, human GATA-1 mutants (ΔNF) defective in FOG1 binding show marked enhancement of its transcriptional cooperativity with RUNX1 [Xu et al., 2006]. The mechanism for U-shaped/FOG interference with Lz-Sp/RUNX-GATA cooperation remains unknown but does not rely on FOG engagement of the NuRD corepressor complex as U-shaped lacks a NuRD recruitment domain [Hong et al., 2005].

P-TEFb KINASE COMPLEX MEDIATES RUNX-GATA COOPERATION AND MEGAKARYOCYTIC DEVELOPMENT

While analyzing transcriptional cooperation of RUNX1 and GATA-1, we found via multiple approaches an essential role for the kinase P-TEFb [Elagib et al., 2008]. In particular, pharmacologic P-TEFb inhibitors (roscovitine, flavopiridol, DRB), a dominant-negative kinase mutant, and the endogenous inhibitory protein HEXIM1 all interfered with the combined transcriptional activity of RUNX1 plus GATA-1 but did not affect the activity of each factor alone. Furthermore, induction of megakaryocytic differentiation tightly correlated with activation of P-TEFb, while inhibition of P-TEFb blocked megakaryocytic differentiation both in vitro and in vivo [Elagib et al., 2008].

The functional core of P-TEFb consists of the cyclin-dependent kinase Cdk9 in association with the regulatory subunit cyclin T. The majority of P-TEFb in cells resides within a large inactive complex containing HEXIM1, the small nuclear RNA (snRNA) 7SK, and numerous additional proteins and ribonucleoproteins [Price, 2008]. Activating signals release the Cdk9/cyclin T dimer from the large complex, permitting the latter to activate promoter-specific transcriptional elongation through phosphorylation of RNA polymerase II carboxy terminal domain serine 2 (RNAP II CTD S2), as well as the negative elongation factors DSIF and NELF [Peterlin and Price, 2006].

P-TEFb signaling specifically contributes to the developmental regulation of genes during cellular differentiation and is not required for maintenance of cell growth and survival. In fact, a functional shRNA screen in 21 different cell lines examining 85% of all human kinases has distinguished Cdk9 as one of the most dispensable factors in the entire kinome [Grueneberg et al., 2008]. In vivo knockdown of Cdk9, applying morpholino oligonucleotides to zebrafish embryos, specifically blocked hematopoietic development with no obvious effects on non-hematopoietic tissues [Meier et al., 2006]. In mammalian systems, P-TEFb activity specifically participates in myogenic, cardiac hypertrophic, and megakaryocytic differentiation programs [Elagib et al., 2008; Sano et al., 2002; Simone et al., 2002]. The contribution of P-TEFb to cellular differentiation may derive in part from its gene-specific enhancement of transcriptional elongation but may also arise from its direct phosphorylation of other nuclear factors [Simone et al., 2002].

Earlier studies have suggested that both GATA-1 and RUNX1 regulate transcription through functional and physical interactions with the P-TEFb kinase complex. RUNX1 has been shown negatively to regulate P-TEFb function through its sequestration of cyclin T1 into inactive chromatin loops [Jiang and Peterlin, 2008; Jiang et al., 2005]. In this manner, RUNX1 can retain target genes in an inactive but transcriptionally “poised” state. These target genes may then become rapidly activated through remodeling of chromatin loops and release of activated PTEFb/RNAP II [Jiang and Peterlin, 2008; Peterlin and Price, 2006]. By contrast, GATA-1 has been shown to activate target genes by promoting transcriptional elongation [Dore et al., 2008]. This function most likely arises from the ability of GATA-1 to physically interact with P-TEFb. Importantly, the formation of GATA-1-P-TEFb complexes is highly dynamic, with complex assembly occurring during megakaryocytic induction and disassembly occurring during erythroid differentiation [Elagib et al., 2008; Meier et al., 2006]. A related biochemical function of GATA-1 consists of its ability to induce remodeling of chromatin loops [Jing et al., 2008]. Thus, P-TEFb serves as a common cofactor for both RUNX1 and GATA-1, most likely acting to integrate signals provided by these two transcriptional factors during megakaryocytic differentiation.

A MODEL FOR INTEGRATIVE DEVELOPMENTAL PROGRAMMING: RUNX1 BOOKMARKING

Megakaryocytic lineage commitment from bipotent MEP cells requires the precisely coordinated activation of megakaryocytic genes in a temporally appropriate sequence, all while erythroid genes are actively maintained in a repressed state. One of the earliest events distinguishing megakaryocytic and erythroid lineages consists of differential RUNX1 expression [Elagib et al., 2003; Lorsbach et al., 2004]. However, RUNX1 alone does not suffice for activation of megakaryocytic genes and in fact initially acts to repress C-MPL prior to the onset of megakaryocytic differentiation [Satoh et al., 2008]. We postulate that as an initial step in megakaryocytic differentiation, RUNX1 is recuited to megakaryocytic promoters, assembling inactive P-TEFb and paused RNAPII, a process consisting of “bookmarking for future reading”. As megakaryocytic differentiation proceeds, GATA-1 expression replaces that of its more primitive counterpart GATA-2 [Muntean and Crispino, 2005]. This “GATA switch” is proposed to promote remodeling of P-TEFb into an active complex, releasing RNAPII for transcription of megakaryocytic target genes. Two critical target genes known to be highly responsive to PTEFb activity are RUNX1 itself and HEXIM1 [Elagib et al., 2008; He et al., 2006; Meier et al., 2006]. Emergence of a positive loop for RUNX1 expression is postulated to reinforce megakaryocytic lineage commitment, while HEXIM1 upregulation acts in a contrary manner to buffer P-TEFb activity.

We further postulate a central role for the GATA-RUNX-P-TEFb pathway in maintaining erythroid genes in a repressed state. Initial evidence for this notion derived from experiments over a decade ago by Hensold and colleagues in which DRB, a specific P-TEFb inhibitor, induced erythroid differentiation of MEL cells, including upregulated transcription of β-GLOBIN and BAND 3, very paradoxical given the role of P-TEFb in transcriptional elongation! Subsequent experiments have documented a correlation between chemical induction of HEXIM1 expression and erythroid lineage commitment in MEL cels [He et al., 2006; Turano et al., 2006]. Megakaryoblasts in DS-AMKL display aberrant coexpression of several erythroid markers including CD71, suggesting a defect in this repressive pathway potentially arising from a failure of the mutant GATA-1s to properly engage P-TEFb [Bourquin et al., 2006; Ge et al., 2006]. In vivo P-TEFb blockade by flavopiridol administration induces a megakaryoproliferative disorder in GATA-1Lo mice characterized by the expansion of CD71+ megakaryoblasts, again implicating GATA-1 cross-talk with P-TEFb in repression of the erythroid program during megakaryopoiesis [Elagib et al., 2008].

A model is shown in Figure 1 depicting how the RUNX1-GATA-1-P-TEFb signaling axis may function in programming megakaryocytic lineage commitment . Key remaining questions include: 1) what factors mediate the remodeling of P-TEFb from an inactive to an active complex during megakaryocytic development? 2) what are the relevant downstream targets of P-TEFb? 3) how do RUNX1 and GATA-1 exert their influence on P-TEFb? 4) how do signals that negatively regulate megakaryocytic development, e.g. cAMP signaling [Freson et al., 2008], interact with the RUNX1-GATA-1-P-TEFb signaling axis?

Figure 1. A hypothetical model of RUNX1 (R1), GATA-1 (G1), and P-TEFb interactions during megakaryocytic differentiation.

Figure 1

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In the bipotent megakaryocyte erythroid progenitor (MEP), key target genes are “bookmarked for future reading” via RUNX1 (R1) recruitment of inactive P-TEFb, as well as RNA polymerase II (RNAPII). The key target genes include megakaryocytic genes (Mk), as well as RUNX1 (R1) and HEXIM1 (H1). The positive and negative signals regulating P-TEFb remodeling remain unknown but are somehow coupled to the GATA switch in which GATA-1 (G1) replaces GATA-2, and possibly to the conversion of RUNX1 from a repressor to activator. Once activated, P-TEFb promotes transcriptional elongation through phosphorylation of RNAPII and the negative regulators of elongation, DSIF and NELF. However, it is likely that P-TEFb also phosphorylates other factors, possibly even those involved in its own remodeling. Additional players include FOG1 (F1), which negatively regulates RUNX1-GATA-1 cooperation, and p300, which can serve as coactivator for GATA-1 and RUNX1.

ACKNOWLEDGMENTS

This work was supported by the National Cancer Institute (CA 100057). The author also wishes to thank Kamal Elagib, Lorrie Delehanty, and Ivailo Mihaylov for their many experimental and intellectual contributions to this work.

Grant sponsor: National Cancer Institute; Grant number: CA100057

REFERENCES

  1. Berger R, Busson M, Dastugue N, Radford-Weiss I, Michaux L, Hagemeijer A, Quilichini B, Benattar L, Bernard O, Romana SP. Acute megakaryoblastic leukemia and loss of the RUNX1 gene. Cancer Gen Cytogen. 2006;164:71–73. doi: 10.1016/j.cancergencyto.2005.05.002. [DOI] [PubMed] [Google Scholar]
  2. Bourquin J-P, Subramanian A, Langebrake C, Reinhardt D, Bernard O, Ballerini P, Baruchel A, Cave H, Dastugue N, Hasle H, Kaspers GL, Lessard M, Michaux L, Vyas P, van Wering E, Zwaan CM, Golub TR, Orkin SH. Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. Proc Natl Acad Sci USA. 2006;103:3339–3344. doi: 10.1073/pnas.0511150103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dore LC, Amigo JD, dos Santos CO, Zhang Z, Gai X, Tobias JW, Yu D, Klein AM, Dorman C, Wu W, Hardison RC, Paw BH, Weiss MJ. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA. 2008;105:3333–3338. doi: 10.1073/pnas.0712312105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Downing JR, Shannon KM. Acute leukemia: a pediatric perspective. Cancer Cell. 2002;2:437–445. doi: 10.1016/s1535-6108(02)00211-8. [DOI] [PubMed] [Google Scholar]
  5. Elagib KE, Mihaylov IS, Delehanty LL, Bullock GC, Ouma KD, Caronia JF, Gonias SL, Goldfarb AN. Cross-talk of GATA-1 and P-TEFb in megakaryocyte differentiation. Blood. 2008;112:4884–4894. doi: 10.1182/blood-2008-03-145722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elagib KE, Racke FK, Mogass M, Khetawat R, Delehanty LL, Goldfarb AN. RUNX-1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation. Blood. 2003;101:4333–4341. doi: 10.1182/blood-2002-09-2708. [DOI] [PubMed] [Google Scholar]
  7. Ferreira R, Wai A, Shimizu R, Gillemans N, Rottier R, von Lindern M, Ohneda K, Grosveld F, Yamamoto M, Philipsen S. Dynamic regulation of GATA factor levels is more important than their identity. Blood. 2007;109:5481–5490. doi: 10.1182/blood-2006-11-060491. [DOI] [PubMed] [Google Scholar]
  8. Fossett N, Hyman K, Gajewski K, Orkin SH, Schulz RA. Combinatorial interactions of Serpent, Lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis. Proc Natl Acad Sci USA. 2003;100:11451–11456. doi: 10.1073/pnas.1635050100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Freson K, Peeters K, De Vos R, Wittevrongel C, Thys C, Hoylaerts MF, Vermylen J, Van Geet C. PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications. Blood. 2008;111:1885–1893. doi: 10.1182/blood-2007-06-098558. [DOI] [PubMed] [Google Scholar]
  10. Ge Y, Dombkowski AA, LaFiura KM, Tatman D, Yedidi RS, Stout ML, Buck SA, Massey G, Becton DL, Weinstein HJ, Ravindranath Y, Matherly LH, Taub JW. Differential gene expression, GATA1 target genes, and the chemotherapy sensitivity of Down syndrome megakaryocytic leukemia. Blood. 2006;107:1570–1581. doi: 10.1182/blood-2005-06-2219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldfarb AN. Transcriptional control of megakaryocyte development. Oncogene. 2007;26:6795–6802. doi: 10.1038/sj.onc.1210762. [DOI] [PubMed] [Google Scholar]
  12. Growney JD, Shigematsu H, Li Z, Lee BH, Adelsperger J, Rowan R, Curley DP, Kutok JL, Akashi K, Williams IR, Speck NA, Gilliland DG. Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype. Blood. 2005;106:494–504. doi: 10.1182/blood-2004-08-3280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grueneberg DA, Degot S, Pearlberg J, Li W, Davies JE, Baldwin A, Endege W, Doench J, Sawyer J, Hu Y, Boyce F, Xian J, Munger K, Harlow E. Kinase requirements in human cells: I. Comparing kinase requirements across various cell types. Proc Natl Acad Sci USA. 2008;105:16472–16477. doi: 10.1073/pnas.0808019105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gurbuxani S, Vyas P, Crispino JD. Recent insights into the mechanisms of myeloid leukemogenesis in Down syndrome. Blood. 2004;103:399–406. doi: 10.1182/blood-2003-05-1556. [DOI] [PubMed] [Google Scholar]
  15. Harada H, Harada Y, Niimi H, Kyo T, Kimura A, Inaba T. High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. Blood. 2004;103:2316–2324. doi: 10.1182/blood-2003-09-3074. [DOI] [PubMed] [Google Scholar]
  16. He N, Pezda AC, Zhou Q. Modulation of a P-TEFb functional equilibrium for the global control of cell growth and differentiation. Mol Cell Biol. 2006;26:7068–7076. doi: 10.1128/MCB.00778-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heller PG, Glembotsky AC, Gandhi MJ, Cummings CL, Pirola CJ, Marta RF, Kornblihtt LI, Drachman JG, Molinas FC. Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation. Blood. 2005;105:4664–4670. doi: 10.1182/blood-2005-01-0050. [DOI] [PubMed] [Google Scholar]
  18. Hollanda LM, Lima CSP, Cunha AF, Albuquerque DM, Vassallo J, Ozelo MC, Joazeiro PP, Saad STO, Costa FF. An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis. Nat Genet. 2006;38:807–812. doi: 10.1038/ng1825. [DOI] [PubMed] [Google Scholar]
  19. Hong W, Nakazawa M, Chen Y-Y, Kori R, Vakoc CR, Rakowski C, Blobel GA. FOG-1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA-1. EMBO J. 2005;24:2367–2378. doi: 10.1038/sj.emboj.7600703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ichikawa M, Asai T, Saito T, Yamamoto G, Seo S, Yamazaki I, Yamagata T, Mitani K, Chiba S, Hirai H, Ogawa S, Kurokawa M. AML1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis. Nature Med. 2004;10:299–304. doi: 10.1038/nm997. [DOI] [PubMed] [Google Scholar]
  21. Jiang H, Peterlin BM. Differential chromatin looping regulates CD4 expression in immature thymocytes. Mol Cell Biol. 2008;28:907–912. doi: 10.1128/MCB.00909-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jiang H, Zhang F, Kurosu T, Peterlin BM. Runx1 binds positive transcription elongation factor b and represses transcriptional elongation by RNA polymerase II: possible mechanism of CD4 silencing. Mol Cell Biol. 2005;25:10675–10683. doi: 10.1128/MCB.25.24.10675-10683.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jing H, Vakoc CR, Ying L, Mandat S, Wang H, Zheng X, Blobel GA. Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus. Mol Cell. 2008;29:232–242. doi: 10.1016/j.molcel.2007.11.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuhl C, Atzberger A, Iborra F, Nieswandt B, Porcher C, Vyas P. GATA1-mediated megakaryocyte differentiation and growth control can be uncoupled and mapped to different domains in GATA1. Mol Cell Biol. 2005;25:8592–8606. doi: 10.1128/MCB.25.19.8592-8606.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kundu M, Chen A, Anderson S, Kirby M, Xu L, Castilla LH, Bodine D, Liu PP. Role of Cbfb in hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene Cbfb-MYH11. Blood. 2002;100:2449–2456. doi: 10.1182/blood-2002-04-1064. [DOI] [PubMed] [Google Scholar]
  26. Li Z, Godinho FJ, Klusmann J-H, Garriga-Canut M, Yu C, Orkin SH. Developmental stage-selective effect of somatically mutated leukemogenic transcription factor GATA1. Nat Genet. 2005;37:613–619. doi: 10.1038/ng1566. [DOI] [PubMed] [Google Scholar]
  27. Liu H, Carlsson L, Grundstrom T. Identification of an N-terminal transactivation domain of Runx1 that separates molecular function from global differentiation function. J Biol Chem. 2006a;281:25659–25669. doi: 10.1074/jbc.M603249200. [DOI] [PubMed] [Google Scholar]
  28. Liu Y, Cheney MD, Gaudet JJ, Chruszcz M, Lukasik SM, Sugiyama D, Lary J, Cole J, Dauter Z, Minor W, Speck NA, Bushweller JH. The tetramer structure of the Nervy homology two domain, NHR2, is critical for AML1/ETO's activity. Cancer Cell. 2006b;9:249–260. doi: 10.1016/j.ccr.2006.03.012. [DOI] [PubMed] [Google Scholar]
  29. Lorsbach RB, Moore J, Ang SO, Sun W, Lenny N, Downing JR. Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression. Blood. 2004;103:2522–2529. doi: 10.1182/blood-2003-07-2439. [DOI] [PubMed] [Google Scholar]
  30. Mehaffey MG, Newton AL, Gandhi MJ, Crossley M, Drachman JG. X-linked thrombocytopenia caused by a novel mutation of GATA-1. Blood. 2001;98:2681–2688. doi: 10.1182/blood.v98.9.2681. [DOI] [PubMed] [Google Scholar]
  31. Meier N, Krpic S, Rodriguez P, Strouboulis J, Monti M, Krijgsveld J, Gering M, Patient R, Hostert A, Grosveld F. Novel binding partners of Ldb1 are required for haematopoietic development. Dev. 2006;133:4913–4924. doi: 10.1242/dev.02656. [DOI] [PubMed] [Google Scholar]
  32. Michaud J, Wu F, Osato M, Cottles GM, Yanagida M, Asou N, Shigesada K, Ito Y, Benson KF, Raskind WH, Rossier C, Antonarakis SE, Israels S, McNicol A, Weiss H, Horwitz M, Scott HS. In vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis. Blood. 2002;99:1364–1372. doi: 10.1182/blood.v99.4.1364. [DOI] [PubMed] [Google Scholar]
  33. Minegishi N, Suzuki N, Kawatani Y, Shimizu R, Yamamoto M. Rapid turnover of GATA-2 via ubiquitin-proteasome protein degradation pathway. Genes Cells. 2005;10:693–704. doi: 10.1111/j.1365-2443.2005.00864.x. [DOI] [PubMed] [Google Scholar]
  34. Muntean AG, Crispino JD. Differential requirements for the activation domain and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development. Blood. 2005;106:1223–1231. doi: 10.1182/blood-2005-02-0551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nichols KE, Crispino JD, Poncz M, White JG, Orkin SH, Maris JM, Weiss MJ. Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA-1. Nature Genet. 2000;24:266–270. doi: 10.1038/73480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peterlin BM, Price DH. Controlling the elongation phase of transcription with P-TEFb. Mol Cell. 2006;23:297–305. doi: 10.1016/j.molcel.2006.06.014. [DOI] [PubMed] [Google Scholar]
  37. Price DH. Poised polymerases: on your mark…get set…go! Mol Cell. 2008;30:7–10. doi: 10.1016/j.molcel.2008.03.001. [DOI] [PubMed] [Google Scholar]
  38. Sano M, Abdellatif M, Oh H, Xie M, Bagella L, Giordano A, Michael LH, DeMayo FJ, Schneider MD. Activation and function of cyclin T-Cdk9 (positive transcription elongation factor-b) in cardiac muscle-cell hypertrophy. Nat Med. 2002;8:1310–1317. doi: 10.1038/nm778. [DOI] [PubMed] [Google Scholar]
  39. Satoh Y, Matsumura I, Tanaka H, Ezoe S, Fukushima K, Tokunaga M, Yasumi M, Shibayama H, Mizuki M, Era T, Okuda T, Kanakura Y. AML1/RUNX1 works as a negative regulator of c-Mpl in hematopoietic stem cells. J Biol Chem. 2008;283:30045–30056. doi: 10.1074/jbc.M804768200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocytic growth and platelet development. EMBO J. 1997;16:3965–3973. doi: 10.1093/emboj/16.13.3965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Simone C, Stiegler P, Bagella L, Pucci B, Bellan C, De Falco G, De Luca A, Guanti G, Puri PL, Giordano A. Activation of MyoD-dependent transcription by cdk9/cyclin T2. Oncogene. 2002;21:4137–4148. doi: 10.1038/sj.onc.1205493. [DOI] [PubMed] [Google Scholar]
  42. Song W-J, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D, Ratajczak J, Resende IC, Haworth C, Hock R, Loh M, Felix C, Roy D-C, Busque L, Kurnit D, Willman C, Gewirtz AM, Speck NA, Bushweller JH, Li FP, Gardiner K, Poncz M, Maris JM, Gilliland DG. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukemia. Nature Genet. 1999;23:166–175. doi: 10.1038/13793. [DOI] [PubMed] [Google Scholar]
  43. Sun L, Mao G, Rao AK. Association of CBFA2 mutation with decreased platelet PKC-theta and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation. Blood. 2004;103:948–954. doi: 10.1182/blood-2003-07-2299. [DOI] [PubMed] [Google Scholar]
  44. Talebian L, Li Z, Guo Y, Gaudet JJ, Speck ME, Sugiyama D, Kaur P, Pear WS, Maillard I, Speck NA. T-lymphoid, megakaryocyte, and granulocyte development are sensitive to decreases in CBFb dosage. Blood. 2007;109:11–21. doi: 10.1182/blood-2006-05-021188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Turano M, Napolitano G, Dulac C, Majello B, Bensaude O, Lania L. Increased HEXIM1 expression during erythroleukemia and neuroblastoma cell differentiation. J Cell Physiol. 2006;206:603–610. doi: 10.1002/jcp.20502. [DOI] [PubMed] [Google Scholar]
  46. Vyas P, Ault K, Jackson CW, Orkin SH, Shivdasani RA. Consequences of GATA-1 deficiency in megakaryocytes and platelets. Blood. 1999;93:2867–2875. [PubMed] [Google Scholar]
  47. Waltzer L, Ferjoux G, Bataille L, Haenlin M. Cooperation between the GATA and RUNX factors Serpent and Lozenge during Drosophila hematopoiesis. EMBO J. 2003;22:6516–6525. doi: 10.1093/emboj/cdg622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Welch JJ, Watts JA, Vakoc CR, Yao Y, Wang H, Hardison RC, Blobel GA, Chodosh LA, Weiss MJ. Global regulation of erythroid gene expression by transcription factor GATA-1. Blood. 2004;104:3136–3147. doi: 10.1182/blood-2004-04-1603. [DOI] [PubMed] [Google Scholar]
  49. Xu G, Kanezaki R, Toki T, Watanabe S, Takahashi Y, Terui K, Kitabayashi I, Ito E. Physical association of the patient-specific GATA1 mutants with RUNX1 in acute megakaryoblastic leukemia accompanying Down syndrome. Leukemia. 2006;20:1002–1008. doi: 10.1038/sj.leu.2404223. [DOI] [PubMed] [Google Scholar]
  50. Zhao X, Jankovic V, Gural A, Huang G, Pardanani A, Menendez S, Zhang J, Dunne R, Xiao A, Erdjument-Bromage H, Allis CD, Tempst P, Nimer SD. Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity. Genes Dev. 2008;22:640–653. doi: 10.1101/gad.1632608. [DOI] [PMC free article] [PubMed] [Google Scholar]

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