Báo cáo Y học: Transcriptional regulation of erythropoiesis Fine tuning of combinatorial multi-domain elements ppt

REVIEW ARTICLE Transcriptional regulation of erythropoiesis Fine tuning of combinatorial multi-domain elements Chava Perry 1,2 and Hermona Soreq 1 1 Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Israel; 2 Department of Hematology, The Tel Aviv Sourasky Medical Center, Tel Aviv and Tel Aviv University, Israel Haematopoiesis, the differentiation of haematopoietic stem cells and progenitors into various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Commitment of pluripotent haematopoietic stem cells to the erythroid lineage induces erythropoiesis, the production of red blood cells. This pro- cess involves a concerted progression through an erythroid burst forming unit (BFU-E), an erythroid colony forming unit (CFU-E), proerythroblast and an erythroblast. The terminally differentiated erythrocytes, in mammals, lose their nucleus yet function several more months. A well- coordinated cohort of transcription factors regulates the formation, survival, proliferation and differentiation of multipotent progenitor into the erythroid lineage. Here, we discuss broad-spectrum factors essential for self-renewal and/or differentiation of multipotent cells as well as specific factors required for proper erythroid development. These factors may operate solely or as part of transcriptional complexes, and exert activation or repression. Sequence comparisons reveal evolutionarily conserved modular com- position for these factors; X-ray crystallography demon- strates that they include multidomain elements (e.g. HLH or zinc finger motifs), consistent with their complex interactions with other proteins. Finally, transfections and genomic studies show that the timing of each factor’s expression during the hematopoietic process, the cell lineages affected and the existing combination of other factors determine the erythroid cell fate. Keywords: transcriptional regulation; hematopoiesis; ery- thropoiesis; DNA binding motifs; acetylcholinesterase. EMBRYONIC ERYTHROPOIESIS In vertebrates, embryonic hematopoiesis involves primitive and definitive steps [1–3] (Fig. 1). Primitive, large nucleated erythroblasts that synthesize embryonic globin forms arise in blood islands that emerge from extraembryonic mesoderm in the yolk sac, at murine embryonic day 7.5 (E7.5) or day 15–18 in humans [4,5]. Definite hematopoiesis is established in the fetal liver beginning at mouse E9.5; it is multilineage, generating well-defined erythrocytes that synthesize adult forms of globin and become enucleated, as well as myeloid, megakaryocyte and lymphoid cells [6]. It is generally believed to initiate in the aorto-gonad-mesonephros (AGM) region [7], though a recent study suggests that the yolk sac is the predominant source of both primitive and definitive hema- topoietic progenitors [4]. Hematopoietic progenitors migrate through the blood stream to seed the fetal liver. Late in fetal life, bone marrow assumes hematopoietic activity and becomes the predominant hematopoietic organ in postnatal life [4,8]. Both embryonic and adult erythropoiesis require broad spectrum as well as erythroid transcription factors. Figure 1 presents the plethora of these factors within the context of the hematopoietic process. BROAD SPECTRUM FACTORS Stem cell leukemia (SCL) Originally identified in a chromosomal translocation in T-cell acute lymphoblastic leukemia (ALL), the stem cell leukemia (SCL) gene on chromosome 1p32–33 encodes a basic helix-loop-helix (bHLH) transcription factor [9,10]. SCL binds E-box (CAGGTG) DNA elements as a heterodimer in complex with E12/E47, the bHLH alternat- ively spliced products of the E2A gene [11,12]. It also participates in a DNA-bound complex containing the transcription factors E12/E47, GATA-1, Ldb-1 and LMO2 [13]. SCL is detected in early hematopoietic progenitors and in more mature megakaryocytes, erythroid and mast cells as Correspondence to H. Soreq, Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, 91904, Israel. Fax: + 972 2 6520258, Tel.: + 972 2 6585109, E-mail: soreq@cc.huji.ac.il Abbreviations: ALL, acute lymphoblastic leukemia; AGM, aorto- gonad-mesonephros; BFU-E, erythroid burst forming unit; CFU-E, erythroid colony forming unit; ES, embryonic stem; SCL, Stem cell leukemia; Epo, erythropoietin; FOG, friend of GATA; EKLF, erythroid Kruppel-like factor; BKLF, basic Kruppel-like factor; AChE, acetylcholinesterase; LCR, locus control region. HRD, hematopoietic regulatory domain; CF and NF, C-terminal and the N-terminal zinc-fingers, respectively; HS, hypersensitive domains; Rb, retinoblastoma; Stat, signal transducer and activator of transcription; HERF1, hematopoietic RING finger 1. Definitions: embryonic age is written as Ex,wherex represents the number of days post-conception. (Received 14 March 2002, revised 2 May 2002, accepted 16 May 2002) Eur. J. Biochem. 269, 3607–3618 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02999.x well as in the mesencephalon, metencephalon, embryonic skeleton, endothelial cells and neurons [14–16]. Its expres- sion increases during erythroid differentiation, where it evokes enhanced proliferation and differentiation. SCL confers proliferation advantage while repressing differenti- ation in myeloid progenitors, and is absent from most mature myeloid and lymphoid cells [17]. SCL null mice die in utero at about E8.5, showing no evidence of blood formation. SCL null embryonic stem (ES) cells fail to give rise to any hematopoietic lineage, suggesting that SCL is crucial for primitive hematopoiesis/erythropoi- esis [18,19]. Clonogenic assays show failure in myelopoiesis, pointing at SCLs critical role in very early hematopoietic differentiation [11]. LIM-only protein 2 (LMO2) Also known as Rbtn2 and TTG2, LMO2 includes two cysteine-rich LIM domains homologous to the DNA binding domain of GATA transcription factors. Localized to chromosome 11p13, the LMO2 gene is involved in the 11;14 translocation of childhood T cell ALL [20]. Highest LMO2 expression levels are found in hematopoietic tissues [21]. LMO2 does not bind DNA by itself, but acts as a bridge between DNA-binding transcription factors such as SCL and GATA-1. Over half of the erythroid LMO2 protein associates with SCL [11,13]. LMO2 null mice die around E9 of severe anemia, with lack of any yolk sac hematopoiesis, identifying an essential role for LMO2 in early hematopoiesis (Table 1) [22,23]. However, LMO2 may also participate in the lineage-specific mechanisms that regulate erythropoiesis, as it takes part in an erythroid transcription-activation complex, together with SCL, E2A, GATA-1 and Lbd1. The complex recognizes an E box motif approximately one helix turn (10 bp) upstream from a GATA site. The GATA-1 gene itself includes sites promo- ting formation of this multimeric erythroid complex [13,24]. LMO2 on its own, like SCL and GATA-1, had little effect on developing Xenopus embryos. However, ectopic coexpression of LMO2, SCL1 and GATA-1 in Xenopus embryos enlarged the ventral blood islands at the expense of dorsal mesoderm (muscle and notochord) embryogenesis [25]. Ectopic expression of LMO2 in Xenopus pole explants treated with basic fibroblast growth factor (bFGF) resulted in erythroid differentiation and extensive globin gene expression. LMO2, SCL1 and GATA-1 overexpression in activin-treated Xenopus pole explants further increased the production of hemoglobinized cells. This suggests the Fig. 1. Embryonic erythropoiesis. Shown are developmental stages in primitive and definitive hematopoiesis up to erythroid commitment. 3608 C. Perry and H. Soreq (Eur. J. Biochem. 269) Ó FEBS 2002 Table 1. Effects of Hematopoietic/Erythroid transcription factors. Gene Motifs Phenotype of genomic disruption Lethality (mouse embryonic day) Overexpression DNA binding sequence Reference SCL bHLH Bloodless mice-absence of yolk sac E8.5 Myeloid proliferation; reduced differentiation response CAGGTG [19] hematopoiesis Erythroid proliferation and differentiation (E box) LMO2 LIM domain Bloodless mice-absence of yolk sac hematopoiesis E8.5–9 Erythroid differentiation and globin gene expression in Xenopus pole explants, not in whole embryo – [22] GATA-2 Zinc finger Decreased embryonic erythrocytes (primitive and definitive); poorly proliferating multipotent progenitors; absence of mast cells E10-11 Promotes proliferation and blocks erythroid differentiation in erythroid precursors T/AGATAA/G [16] c-Myb Helix- Normal primitive but E15 Inhibits erythroid differentiation TAACGG [11,95,96] turn-helix severely impaired Leucine- zipper definitive erythropoiesis region GATA-1 Zinc finger Ablated embryonic erythropoiesis due to E11.5 Promotes megakaryocytic differentiation in an early T/AGATAA/G [28,36] blocked maturation at proerythroblasts myeloid cell line (with apoptosis); arrested megakaryocyte development (with hyperproliferation) FOG Zinc finger Blocked erythroid maturation at E12.5 Inhibits red cell formation and maturation in whole – [42] proerythroblasts; ablated megakaryocytopoiesis Xenopus embryos (mFOG2); represses GATA-1- induced activity (FOG1) EKLF Zinc finger Severe anemia; b-globin deficiency E16 Earlier switch from fetal to adult type globin; enhanced differentiation and hemoglobinization, reduced proliferation (in EKLF null cells) CACC GC rich [56] BKLF Zinc finger Myeloproliferative disorder – – CACC [51] Stat5 – Transient anemia due to fetal liver – – TTCC(A > T)GGAA [77,97] erythroid progenitors apoptosis at E13.5; mild anemia, exacerbated by stress, at adult life PU.1 Winged helix-turn-helix Blocked erythropoiesis (relieved by GATA-1) [74] Fli-1 Winged helix- turn-helix Inhibited erythroid differentiation, impaired ability to respond to specific erythroid inducers and reduced levels of GATA-1 [69] Ó FEBS 2002 Transcriptional regulation of erythropoiesis (Eur. J. Biochem. 269) 3609 formation of synergistic multiprotein complexes that pro- mote red cell formation and differentiation during embryo- genesis, in addition to SCL and LMO2s crucial role in early hematopoiesis [25]. A pentameric complex of LMO2, SCL, E2A, Lbd1 and pRb was shown to repress gene expression in erythroblasts [26], likely counteracting tran- scriptional activation to limit erythroid differentiation [25]. Figure 2 presents a scheme of the erythroid transcription- activation complex along promoters of erythropoietically active genes. GATA-2 All members of the GATA family of transcription factors contain two homologous zinc-finger domains and bind to the DNA GATA-consensus sequence (T/AGATAA/G), present in regulatory elements of many erythroid genes [e.g. globins, band 3, EKLF, FOG, erythropoietin receptor (EpoR) and heme biosynthetic enzymes] [27,28]. GATA-2, a member of the GATA family, is expressed in hematopoietic and ES cells and endothelial cells. Its forced expression in erythroid precursors promotes proliferation and blocks erythroid differentiation [11]. Expression of GATA-2 precedes that of another family member, GATA-1, and must decrease as GATA-1 expression increases to enable erythroid differentiation. GATA-2 null mice are embryonic lethal, due to severe anemia during the early phase of yolk sac hematopoiesis (E10–11) [16]. The most pronounced decrease occurs in the frequency of primitive and definitive erythroid and mast cell colonies, differenti- ating from GATA-2 null ES cells. Multipotential progen- itors arising from GATA-2 null ES cells proliferate poorly and undergo excessive apoptosis [11,16], suggesting that GATA-2 is essential for appropriate expansion and survival of early hematopoietic cells, at the expense of differenti- ation. The proto-oncogene c-Myb c-Myb is abundantly expressed in immature hematopoietic cells of erythroid, myeloid and lymphoid lineages but decreases as they differentiate. Moreover, its forced expres- sion inhibits erythroid differentiation [11]. c-Myb is required for early definitive cellular expansion and, like GATA-2, it needs to be downregulated to allow terminal differentiation [11,29]. c-Myb null mice exhibit normal primitive but severely impaired definitive hematopoiesis, resulting in death at E15. Mature circulating definitive erythrocytes as well as other lineage progenitors are decreased, while megakaryocytes, granulocytes and monocytes appear to be normal. The v-Myb gene, transduced by avian myeloblastosis virus (AMV), is an oncogene that specifically blocks terminal differentiation in macrophage precursors, activates their self-renewal capacities and determines the commitment of progenitors to macrophages while suppressing develop- ment of other lineages [30,31]. This specificity, distinct from the multilineage effects of c-Myb, likely reflects the loss of some c-Myb functions due to deletions and point mutations. The macrophage precursor-restricted activity of v-Myb resides in its leucine-zipper region (LZR), mutation of which enables v-Myb to affect uncommitted progenitors, support- ing development of erythroid cells, granulocytes and megakaryocytes [32]. v-Myb induces myeloid factors (PU.1, C/EBP), while the v-Myb mutant induces SCL and GATA-1 in transformed blastoderm cells [32]. The c-Myb C-terminus can interact with its own N-terminus [33], likely affecting LZR accessibility for myeloid factors, activating myeloid-specific genes. Inaccessible Myb-LZR might favor formation of c-Myb complexes with erythroid factors, activating erythroid-specific genes. This molecular switch thus directs hematopoietic progenitors into lineage-specific development [32]. ERYTHROID TRANSCRIPTION FACTORS GATA-1 The GATA-1 gene, located on chromosome Xp11.23 [34], is expressed in erythroid cells, megakaryocytes, eosinophils, mast cells and Sertoli cells in the testis [35]. GATA-1 null mice show complete ablation of embryonic erythropoiesis due to arrested maturation and apoptosis of erythroid precursors at the proerythroblast stage [36], supporting its key role in erythroid commitment (Table 1). These mice also present blocked megakaryocyte development in mid- maturation and die by E11.5. However, GATA-1-negative ES cells can develop into other hematopoietic lineages. Forced expression of GATA-1 in an early myeloid cell line promotes megakaryocytic differentiation, suggesting that GATA-1 may affect both lineage selection and late erythroid maturation [11,37,38]. GATA-1 is expressed as two distinct transcripts in hematopoietic cells and in the testis, directed by different first exons/promoters. The coding exons are common to both transcripts [28]. In primitive erythroid cells, GATA-1 expression is regulatedbya5¢ enhancer, whereas its expression in definitive erythroid cells requires an additional element located in the first intron. Together, these two elements form the GATA-1 locus hematopoietic regulatory domain (HRD) [28]. Fig. 2. The erythroid transcription-activation complex. SCL and E47 bind an E box (CAGGTA), about 10 bp upstream from a GATA motif. LMO2 and Lbd1 bridge between SCL1 and GATA-1. However, GATA-1 binds DNA more commonly in a nonspecific orientation, with FOG as its cofactor. 3610 C. Perry and H. Soreq (Eur. J. Biochem. 269) Ó FEBS 2002 The C-terminal and the N-terminal zinc-fingers (CF and NF, respectively) in GATA-1 are required for recognition of the GATA motif and DNA binding as well as for physical interaction with other transcription factors. The highly conserved NF is essential for interaction with the GATA-1 coactivator FOG (Friend Of GATA) as well as with EKLF, LMO2 and C/REB binding protein (CBP), and enhances the specificity and stability of binding of the two-finger DNA binding domains to palindromic GATA recognition sequences [27,39]. CF is indispensable for GATA-1 func- tion, while NF is indispensable for definitive but not for primitive erythropoiesis. This suggests that different GATA-1 functional domains are required for target gene activation in primitive and definitive erythropoiesis [28]. Thus, both transcriptional regulatory elements and protein functional domains may ensure proper lineage specification in primitive and definitive erythropoiesis. GATA motifs may appear by themselves or occur in a specific orientation from an E box motif. Thus, the genomic orientation of GATA motifs and their neighboring sequences bears important functional implications. It has been speculated that GATA-1 binds isolated GATA motifs in a nonspecific orientation, in which FOG is the cofactor. In addition, GATA-1 binds GATA-E box elements, in which SCL and other components cooperate with GATA-1 (Fig. 2) [27]. For example, the pentameric erythroid tran- scription-activation complex includes SCL and E12/E47 that binds an E-box, about 10 bp upstream from a GATA motif, as well as LMO2 and Lbd1 bridging between SCL1 and GATA-1 [13]. GATA-3 is normally restricted to lymphoid precursors and committed T cells. Its overexpression in murine hematopoietic stem cells arrests proliferation, induces erythroid and megakaryocyte differentiation and inhibits development of myeloid and lymphoid precursors. This apparent functional redundancy among the GATA proteins suggests that lineage determination by individual GATA proteins is developmental-stage dependent [40]. Friend of GATA (FOG) FOG is a complex zinc-finger protein. It associates with GATA-1 NF through at least one of its nine fingers (usually finger 6). FOG is coexpressed with GATA-1 in fetal liver, embryonic erythroblasts, mast cells, megakaryocytes and adult spleen [41] and cooperates with GATA-1 to promote erythroid and megakaryocytic differentiation. Mutated GATA-1 that is unable to interact with FOG, fails to support terminal erythroid maturation due to deregulated expression of multiple GATA-1 target genes, such as the a-andb-globins and band 3, but not EKLF or FOG itself [27]. FOG does not modulate GATA-1 DNA binding specificity, or activation properties. Rather, it recruits additional nuclear factors, perhaps via its other fingers. Mice lacking FOG exhibit blocked erythripoiesis, similar to GATA-1-deficient mice. However, the NF domain, which mediates GATA)1 interactions with coactivators such as FOG, was found to be dispensable in primitive erythropoiesis. Therefore, FOGs contribution to primitive erythropoiesis appears to be independent of GATA-1. FOG null mice also display ablation of the megakaryocytic lineage, unlike loss of GATA-1 which blocks megakaryo- cyte development in midmaturation. This points at addi- tional, GATA-1 independent role of FOG during the earliest stages of megakaryocyte development. Thus, the early, independent functions of FOG differ from its later, GATA-1 dependent role during erythroid and megakaryo- cyte maturation [42]. FOG may also function as a repressor. A FOG homo- logue in Drosophila, u-shaped, was found to repress the action of a GATA-like factor, pannier. A second mamma- lianFOG,mFOG2,isexpressedinheart,neuronsand gonads in the adult with somewhat broader expression during embryogenesis [43]. Ectopic expression of mFOG2 inhibits red cell formation and maturation in intact Xenopus embryos and reduces xGATA-1 and xSCL levels in ventral marginal zone explants, while xGATA-2 levels remain unchanged [43,44] (Table 1). In murine erythroleukemia cells, FOG1 represses the GATA-1-induced activity of the transferrin receptor-2 (TfR-2)-promoter [45]. A Xenopus FOG homologue, xFOG, contains a short peptide motif (PIDLSK), which is highly conserved among FOG proteins and mediates interactions with the transcrip- tion corepressor CtBP [46]. In Xenopus embryos, FOG2 with a mutated CtBP binding site stimulated red cell formation dramatically [44], although, knock-in mice expressing a FOG1 variant, which is unable to bind CtBP have normal erythropoiesis [47]. It was suggested that FOG:GATA-1 complexes may repress transcription of GATA-2, which promotes progenitor proliferation over differentiation in committed erythroblasts, limiting the number of cells with erythropoietic fate and preventing depletion of pluripotent stem cells. In the absence of FOG, GATA-1 might fail to shut off GATA-2 transcription and erythropoiesis might be stalled at a blast-phase. Once cells are committed, FOG may cooperate with GATA-1 in erythroid maturation. Familial X-linked dyserythropoietic anemia due to a substitution of methionine for valine at residue 205, in a highly conserved region of GATA-1 NF, interrupts the GATA)1:FOG1 interaction and inhibits the ability of GATA-1 to rescue erythroid differentiation in a GATA-1 deficient erythroid cell line [48]. This results in severe fetal anemia and anemia with severe thrombocytopenia at birth and thereafter, as well as cryptorchidism, in the male offspring. The substitution Ser208 fi Gly or Gly218 fi Asp in GATA-1 NF domain, was reported in families with recessive X-linked thrombocytopenia and X-linked macro- thrombocytopenia, respectively. The replaced residues are involved in GATA-1:FOG1 direct interactions and the mutation partially disrupts this interaction [49,50]. Table 2 lists these mutations and their clinical consequences, which together confirm the vital role played by specific domains in the corresponding transcription factors during in vivo erythroid and megakaryocyte development. Erythroid Kruppel-like factor (EKLF) This zinc finger protein plays an essential role in the regulation of b-globin gene expression [51]. The b-globin locus regulation has recently been exten- sively reviewed [52–54]. The b-globin gene is part of the globin cluster, the genes of which are arranged in the order of their expression during development. Regulation of the b-globin tissue- and developmental stage-specific expression ismediatedbyitspromoteraswellasbydistalregulatory Ó FEBS 2002 Transcriptional regulation of erythropoiesis (Eur. J. Biochem. 269) 3611 sequences, the most prominent of which is the locus control region (LCR). The LCR consists of several DNase 1 hypersensitive domains (HS sites). In erythroid cells, where the gobin genes are transcriptionally active, the locus shows higher DNase 1 sensitivity, indicating an open and access- ible chromatin structure (euchromatin). Tissue- and stage- specific expression of the various globin genes is determined by the interactions between the LCR and the specific globin gene promoters, interactions mediated by recruiting chro- matin modifying, coactivators and transcription complexes [52]. EKLF expression is remarkably restricted to erythroid, megakaryocytic and mast cells [55]. The human EKLF gene was located to chromosome 19p13, a region deleted in some cases of human erythroleukemia [56]. Human EKLF encodes a 362 residues protein that includes three C 2 H 2 type zinc fingers at its C-terminus. It shares 69% overall identity and 93% identity with the three C-terminal zinc finger domains of mouse EKLF. Each finger includes three key amino acids that form sequence-specific contacts with three DNA residues. The N-terminal of the protein is rich in proline and acidic residues [57]. EKLF, like other members of the Kruppel family, binds a CACC consensus-sequence in regulatory elements of many erythroid-specific genes, including adult b-globin, often closely spaced from a GATA site (Fig. 2). GATA proteins interact physically and functionally with Kruppel-like proteins to regulate gene expression [58]. Competition assays show that EKLF favors binding to the human and murine adult type of b-globin CACC element over the CACC elements in the murine fetal bh1- globin, human c-globin or the erythropoietin receptor (EpoR) gene promoters. Naturally occurring adult type b-globin CACC box mutations result in reduced b-globin expression and b-thalassemia, due to poor EKLF binding (Table 2) [57,59]. EKLF null mice die before E16 of severe anemia and b-globin deficiency. Embryonic erythropoiesis and embry- onic e and f globin genes expression is normal [60], demonstrating the pivotal role of EKLF in the activation of the adult b-globin gene in the late stages of erythropoiesis. Overexpressing EKLF induces an earlier switch from fetal to adult type globin [61]. EKLF deficient mice that carry a complete copy of the human b-globin locus display elevated levels of the human fetal c-globin mRNA, in addition to b-globin deficiency (Table 1). Elevated fetal type c-globin levels, in adult life, were reported in carriers of point mutations within the b-globin promoter CACC box [57,59]. This may indicate a role for EKLF in silencing c-globin expression, or in the c-tob-globin switching process. EKLF activation of the b-globin gene is dramatically enhanced in the presence of the DNase 1 HS2 of the gene LCR [62]. Within the LCR, EKLF was found to activate HS3 directly. One model for the globin chromatin opening proposes that factor binding at HS3 initiates the process, allowing the spreading of open chromatin, binding of other trans-acting factors throughout the LCR, and looping out intervening DNA to establish the LCR holocomplex [53]. A protein complex that can activate transcription of a chromatin-assembled b-globin, in an EKLF-dependent fashion, was purified and named EKLF coactivator remodeling complex-1 (E-RC1) [63]. This suggests that the function of EKLF as an activator of transcription is to attract the complex to the b-globin promoter. Reintroducing EKLF into an EKLF-null erythroid cell line, which harbors a copy of the human b-globin locus, resulted in enhanced differentiation and hemoglobinization, as well as reduced proliferation. This may point to a role for EKLF in cell cycle regulation and hemoglobinization, in addition to regulation of b-globin gene expression [64]. J2E cells transfected with antisense EKLF cDNA show normal proliferation but reduced expression of b-globin and two rate-limiting heme synthesis enzymes as well as defective hemoglobinization in response to erythropoietin stimulation [65]. This may suggest EKLF regulation of other genes involved in hemoglobin synthesis. Basic Kruppel-like factor (BKLF) The BKLF protein is found in erythroid cells, fibroblasts and brain. It binds CACC motifs through three highly conserved C-terminal Kruppel-like zinc fingers and interacts with the corepressor CtBP to repress EKLF promoter activation in vitro [46,66]. BKLF erythroid expression depends on EKLF, so that EKLF deficient mice express significantly reduced levels of BKLF in erythroid cells and normal BKLF levels in the brain [66]. Table 2. Translocated or mutated transcription factor genes in human pathologies. Gene Motif Pathology Molecular background SCL bHLH T cell acute lymphoblastic leukemia (ALL) t1; 14, t1; 3, t1; 5, t1; 7 LMO2 LIM domain Childhood T cell ALL t11; 14 GATA-1 Zinc finger Familial dyserythropoietic anemia (with cryptorchidism) V205M at GATA)1 (interrupting interaction with FOG) Recessive X-linked thrombocytopenia G208S at GATA)1 (interrupting interaction with FOG) EKLF and target genes Zinc finger b-Thalassemia b-globin promoter CACC box mutations Erythroleukemia del 19p13 SHP-1 (BKLF-activated?) Polycythemia vera SHP-1 is down-regulated in CFU-E; hematopoietic progenitor hyper-susceptible to growth factors? 3612 C. Perry and H. Soreq (Eur. J. Biochem. 269) Ó FEBS 2002 EKLF null mice express elevated levels of fetal globins, perhaps due to missing EKLF upregulation of BKLF in erythroid cells. This suggests that BKLF represses the expression of embryonic and fetal globin genes, both of which contain a CACC box in their promoters [55]. BKLF deficient mice display a myeloproliferative disorder and an overall phenotype that resembles that of mice mutated for the protein tyrosine phosphatase SHP-1, suggesting a role for BKLF in regulation of SHP-1 expression [55]. SHP-1 is expressed in erythroid progenitors, and is downregulated during terminal differentiation. It inactivates complexes of growth factors and their receptors, including factors known to guide proliferation and differen- tiation in erythroid progenitors. Polycytemia vera is a clonal myeloproliferative disorder, leading to hyperproliferation of erythroid, myeloid and megakaryocytic cells. Sixty percent of polycytemia vera patients have diminished expression of SHP-1 in CFU-E populations (Table 2), suggesting that repression of this inactivator of growth factor complexes renders the hematopoietic progenitors in polycytemia vera patients more susceptible to growth effects [67]. Neptune and other KLF family members Neptune,aXenopus member of the Kruppel-like factor (KLF) family of zinc-finger transcription factors, can bind CACC as well as GC-rich DNA elements. Neptune shares 91% of its sequence, at the nuclear localization signal and zinc finger region, with another family member, the gut KLF-GKLF, and 76% with EKLF [68]. Neptune appears at sites of primitive erythropoiesis prior to xGATA-1. It is expressed in the ventral blood islands, in cells committed to primitive erythropoiesis, cranial ganglia and hatching and cement glands, as well as in peripheral red blood cells and spleen. Neptune specifically binds to CACC elements in the promoters of both embryonic and adult mouse b-globin genes, with minimal binding to CACC elements in the fetal c-globin gene promoter. Similarly to EKLF, neptune activates the human b-globin promoter and cooperates with xGATA-1 to enhance globin induction in animal cap explants, though by itself it fails to induce globin produc- tion. Globin gene regulation by xGATA-1 depends on neptune function in ventral marginal zones and animal caps, both sites of primitive erythropoiesis [68]. biklf, the zebrafish ortholog of neptune, is required for erythroid cell differentiation. biklf is expressed in the hatching gland and in the zabrafish homologue of the Xenopus ventral blood islands. Repressing biklf expression in zebrafish embryos results in embryonic anemia, sup- pressed expression of the embryonic globin and inhibition of GATA-1 expression, demonstrating conservation of func- tion during vertebrate evolution [69]. FKLF (human Fetal KLF) [70] activates embryonic (e) globin expression, and to a lesser extent the fetal (c) globin genes, through its interaction with these genes’ CACC boxes, but fails to activate other CACC box-containing erythroid genes. Murine FKLF-2 increases c-globin expression 100-fold. It activates the promoters of e-andb-globins, GATA-1 and heme synthesis enzyme genes to a much lower degree [71]. Thus, all globin genes contain CACC boxes in their regulatory domains, yet FKLF, FKLF-2 and EKLF activate the embryonic, fetal and adult globin genes, respectively. A four-step model for human b globin gene regulation has been suggested [52]; the first step involves partial unfolding of globin chromatin structure and generation of highly accessible LCR. It is mediated by erythroid-specific proteins, which bind to sequences throughout the globin locus. GATA-1, which is known to associate with histone acetyl-transferases, may be involved in this step. The disruption of the LCR chromatin structure allows binding of transcription factors such as EKLF and other KLF family members, GATA family members and the HLH proteins to the LCR HS sites, and the recruitment of chromatin-remodeling complexes and coactivators. In the third step, chromatin domains permissive for transcription are being established. Intergenic transcription was suggested to modify chromatin structure of an active gene domain, distinguishing it from an accessible but inactive one, that way separating the globin gene into developmental stage- specific chomatin domains. Finally, transcription complexes are being transferred from the LCR to individual glo- bin gene promoters within transcriptionally permissive domains, allowing the developmental stage-specific pattern of globin gene expression. The Fli-1 oncogene A member of the Ets family of transcription factors, Fli-1, was identified in Friend virus-induced erythroleukemia and affects the self-renewal of erythroid progenitor cells [72]. In pluripotent human hematopoietic cells, differentiation is followed by reduced Fli-1 expression and over expressing Fli-1 inhibits erythroid differentiation, impairs the cells’ ability to respond to specific erythroid inducers, such as hemin, and reduces the levels of GATA-1 [73]. In the erythroblastic cell line, HB60, Fli-1 expression is downregulated by erythropoietin (Epo), which induces terminal erythroid differentiation. Constitutive expression of Fli-1 blocks Epo-induced differentiation and enhances cell proliferation in HB60 cells, suggesting that Fli-1 targets erythroid cells to either proliferation or differentiation, in response to Epo [74]. Fli-1 binds a cryptic Ets consensus site within the retinoblastoma (Rb) gene promoter, repressing Rb expres- sion, which results in impaired terminal erythroid matur- ation and continuous presence of nucleated erythrocytes in peripheral blood [75]. Negative regulation of Rb by Fli-1 could destine erythroid progenitors to self-renewal, while Epo-induced repression of Fli-1 expression will enable differentiation [74]. PU.1 The putative oncogene Spi-1 (PU.1) protein product is a hematopoietic-specific Ets factor, promoting differentiation of lymphoid and myeloid lineages [76]. PU.1 expression in erythroid progenitors can induce erythroleukemia in mice. Like Fli-1, PU.1 blocks erythroid differentiation and restoration of terminal erythroid differentiation in murine erythroleukemia (MEL) cells requires PU.1 suppression [77,78]. PU.1 can interact directly with GATA-1 and repress GATA-1 mediated transcriptional activation. Both the Ó FEBS 2002 Transcriptional regulation of erythropoiesis (Eur. J. Biochem. 269) 3613 PU.1 DNA binding domain and transactivation domain are required for GATA-1 suppression and for blocking terminal differentiation in MEL cells. PU.1 does not seem to affect binding of other factors, such as FOG, to GATA-1, nor does it prevent GATA-1 DNA binding [78]. It is likely that PU.1 binds to assembled, DNA-bound GATA-1 complexes and represses their activity. Ectopic expression of PU.1 in Xenopus embryos blocks erythropoiesis. Exogenous GATA-1 is able to relieve this blockage of erythroid differentiation in MEL cells as well as in Xenopus embryos and explants, suggesting that lineage commitment decisions are regulated by their relative levels [78]. PU.1 can also bind to GATA-2 and EKLF, in vitro.As both PU.1 and GATA-2 are capable of blocking terminal erythroid differentiation, it is possible that these factors cooperate to stimulate self-renewal in early erythroid progenitors. Fli-1, known to block erythroid differentiation and suppress GATA-1 expression, was identified as a PU.1 target gene [73,79]. Signal transducer and activator of transcription (Stat) 5 Epo binding to its receptor (EpoR) leads to rapid activation of the transcription factor Stat5. Tyrosine phosphorylation of EpoR-bound Stat5 dimerizes the complex and translo- cates it to the nucleus, where it can induce the immediate early expression of the antiapoptotic gene bcl-x. Stat5 confers an antiapoptotic effect over erythroid cell lines; repressing stat5 expression increases apoptosis and inhibits growth of fetal liver erythroid precursors [80,81]. Decreased bcl-x expression and increased apoptosis in early erythroblasts suggests that Stat5 and bcl-x mediate the Epo antiapoptotic effect on erythroid pro- genitors [81]. Stat5a- and Stat5b-deficient mice are severely anemic due to decreased survival of fetal liver erythroid progenitors and show a marked increase in apoptosis at E13.5, when fetal liver cells are cultured with Epo. This is consistent with Stat5 mediating an Epo-dependent anti- apoptotic effect in fetal erythroid progenitors [81]. The anemia resolves during adult life in about half of Stat5- mutated mice, which then have near-normal hematocrit. However, they are deficient in generating high erythro- poietic response to hemolysis-induced stress and have persistent anemia despite compensatory expansion of their erythropoietic tissue, with erythroblasts failing to differentiate. Hematopoietic RING finger 1 (HERF1) During the initial development of definitive hematopoietic progenitors, the expression of HERF1 coincides with the appearance of definitive erythropoiesis. In adult mice, it is restricted to erythroid cells. Inhibition of HERF1 expression blocks terminal erythroid differentiation, whereas its over- expression induces erythroid maturation in MEL cells [82]. This suggests that HERF1 may have a role in the development of mature erythroid cells. Figure 3 lists some Fig. 3. Differentiation of committed erythroid progenitors. Shown are the transcription factors that affect erythrocyte precursors through their differentiation into erythroblasts. The exerted effects are marked in brackets. 3614 C. Perry and H. Soreq (Eur. J. Biochem. 269) Ó FEBS 2002 of these key transcriptional regulators of the erythropoietic process and notes at least part of their multi-element interactions during erythroid differentiation. DOWNSTREAM TARGET GENES Transcriptional regulation of erythropoietin Epo, a glycoprotein hormone, is not a transcription factor but activates intracellular signaling through binding to its receptor, EpoR. This stimulation upregulates the expression of globins, transferrin receptor and some membrane pro- teins that are characteristic of erythrocytes. It enhances the viability and maturation of erythroid progenitor cells, while Epo deprivation results in increased apoptosis [83]. Epo null mice die at E13.5 of severe anemia, when primitive erythroblasts die and are not being replaced by definitive erythropoiesis, accompanied by a dramatic increase in cell death. All this points at Epo’s major contribution to the survival, proliferation and differentiation of definitive erythroid progenitors [84]. The primary regulator of Epo expression in late fetal and postnatal life is oxygen tension. A hypoxia sensing mech- anism results in activation of the transcription factor Hypoxia inducible factor (HIF)1, which binds a 3¢ enhancer of the Epo gene, initiating its expression [85]. The mouse Epo 3¢ enhancer contains a DR2 element, a direct repeat of the hexameric sequence TGACC(C/T), adjacent to the HIF1 binding site. Coupled HIF1–DR2 sequences augment hypoxic induction of Epo gene reporter constructs, prob- ably through hepatocyte nuclear factor (HNF)4 [84,86]. During early erythropoiesis, the Epo gene is a direct transcriptional target of the retinoic acid receptor RXRa. Mouse embryos lacking RXRa are deficient in erythroid differentiation. Their Epo mRNA levels are reduced at E10.25 and E11.25 but can be induced by retinoic acid. The Epo gene enhancer was found to contain a DR2 element. DR2 represents a retinoic acid receptor binding site and a retinoic acid receptor transcriptional response element [84]. Surprisingly, the erythropoietic deficiency in RXRa null mice is transient. Epo is expressed at normal levels by E12.5 and erythropoiesis reaches normal levels by E14.5. HNF4, abundant in fetal liver hepatocytes, was shown to compete with RXRa for binding to the Epo gene enhancer DR2 element. Thus, Epo expression may be regulated by RXRa during early fetal erythropoiesis and then by HNF4 activity, a transition that may be responsible for switching the regulation of Epo expression from paracrine, retinoic acid control to hypoxic, HNF4-related control [84]. Acetylcholinesterase, a potential hematopoiesis/ erythropoiesis regulator A case study for a downstream regulator may be that of acetylcholinesterase (AChE). Primarily known to hydrolyze acetylcholine at brain synapses and neuromuscular junc- tions, its extended biological roles involve contribution to cell proliferation and differentiation in multiple tissues (reviewed in [87]). These include sites of hematopoiesis and osteogenesis, both known to share a common progenitor, as well as different tumor types [88–91]. One of the alternat- ively spliced transcripts of AChE, the ÔreadthroughÕ isoform (AChE-R), which is known to be upregulated in response to psychological and chemical stress, is induced by cortisol in CD 34+ hematopoietic progenitor cells. This cortisol- induced expression of AchE-R correlates with hematopoi- etic expansion, perhaps implying a role for AChE in bone marrow adaptive responses to stress [92]. AChE is also expressed in immature human megakaryo- cytes, where it is surprisingly localized to the nucleus [93]. Induction of differentiation in human megakaryoblasts suppresses AChE expression, as was reported for GATA-1 [93,94]. Transient suppression of ACHE gene expression in mouse hematopoietic multipotential progenitors, using an antisense oligonucleotide, induced AChEmRNA overex- pression, followed by cell expansion and suppressed apop- tosis [95]. Consensus DNA binding sites for hematopoietic transcription factors are extremely abundant along the three known regulatory domains in the ACHE locus. Among others, they include E2 and CACC boxes, glucocorticoid response elements and consensus binding sites for GATA-1, C/EBP and Stat5 [89,96–98]. Binding sites for the LMO2 complex with adjacent GATA-1 (though not 10 bp apart) and KLF motifs are found in the upstream enhancer, proximal promoter and intronic enhancer of the ACHE locus, suggesting multileveled control over its hematopoietic expression (Fig. 4). All this suggests that AChE may be either a downstream target for hematopoietic and/or erythroid-specific transcrip- tion factors or, in view of its surprising nuclear localization, that it is a transcription modifier by itself, affecting fate- determining crossroads. The apparent regulatory role of AChE in hematopoietic proliferation and differentiation at early developmental stages may be accompanied by a capacity for inducing proliferation at later, erythroid- commited stages, as was shown in megakaryoblasts [93]. Finally, this is a promising candidate for involvement with stress responses that induce erythropoietic development In conclusion, erythropoiesis is a highly complex process that is regulated by a finely tuned combination of transcription factors in a stage-specific and context-depend- ent manner. Several key characteristics of transcriptional regulation of erythropoiesis may be pointed out: Fig. 4. Erythroid transcription factor binding sites across the ACHE locus. Depicted is the reverse sequence of the cosmid inset (accession no. AF002993) including the ACHE gene and 22 kb of its upstream sequence. Exons (numbered above) and introns (numbered below) are marked. Arrows designate positions of the ACHE regulatory domains: distal enhancer domain (D.D), the proximal promoter (P.P) and the intronic enhancer (I.E), along the cosmid reverse sequence (nt 22 465 being the ACHE transcription start site). Consensus binding sites for the noted transcription factors are represented by wedges. LMO2 Complex ¼ LMO2 associated with DNA-bound SCL1-E47 and able to bridge binding to DNA-bound GATA-1 in the erythroid tran- scription-activation complex (see Fig. 2). Ó FEBS 2002 Transcriptional regulation of erythropoiesis (Eur. J. Biochem. 269) 3615 A single transcription factor may exert different effects on cell fate when expressed at different developmental stages. For example, SCL exerts a self-renewal, proliferative effect when expressed in early progenitors, but induces differen- tiation when expressed in more mature cells. Expression of a specific transcription factor at different developmental stages may also modulate lineage-commitment, and facili- tate interactions with different partner proteins. An intriguing compromise or antagonism between pro- liferation and differentiation emerges at several stages of the erythropoietic process. Determination of cell fate depends not only on the ability to express certain pro-differentiation factors, but also on the ability to repress other survival/ proliferation-inducing transcription factors (GATA-2, c-Myb, PU.1, Fli-1) at developmental crossroads. Both of these abilities are essential for erythroid differentiation. Genomic orientation and neighboring sequences may have functional implications for the interactions among the transcription factors. Many of the erythropoietic transcription factors are complex, multidomain proteins. Different domains of the same protein may be required to activate various target genes at different developmental stages. Therefore, the interplay among the various transcription factors involving their stage of expression, type of cells expressing them, the combination of factors present at a certain time point and the multidomain structure of many of these factors variegate the complex regulation of erythropoiesis. ACKNOWLEDGEMENTS Chava Perry MD, is the incumbent of a basic research fellowship from the Israel Ministry of Health. The study was supported by the US Army Medical Research and Materiel Command (DAMD 17-99-1-9547) and by Ester Neuroscience Ltd. REFERENCES 1. Palis, J. & Segel, G.B. (1998) Developmental biology of ery- thropoiesis. Blood Rev. 12, 106–114. 2. Migliaccio, A.R. & Migliaccio, G. (1998) The making of an ery- throid cell. Molecular control of hematopoiesis. Biotherapy 10, 251–268. 3. Dame, C. & Juul, S.E. (2000) The switch from fetal to adult ery- thropoiesis. Clin. Perinatol. 27, 507–526. 4. Palis, J., Robertson, S., Kennedy, M., Wall, C. & Keller, G. (1999) Development of erythroid and myeloid progenitors in the yolk sac andembryoproperofthemouse.Development 126, 5073–5084. 5. Orkin, S.H. (1996) Development of the hematopoietic system. Curr. Opin. Genet Dev. 6, 597–602. 6. Dzierzak, E. & Medvinsky, A. (1995) Mouse embryonic hema- topoiesis. Trends Genet. 11, 359–366. 7. Dzierzak, E., Medvinsky, A. & de Bruijn, M. (1998) Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo. Immunol. Today. 19, 228–236. 8. Medvinsky, A. & Dzierzak, E. (1996) Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86, 897–906. 9. Hershfield, M.S., Kurtzberg, J., Harden, E., Moore, J.O., Whang- Peng,J.&Haynes,B.F.(1984)Conversionofastemcellleukemia from a T-lymphoid to a myeloid phenotype induced by the ade- nosine deaminase inhibitor 2¢-deoxycoformycin. Proc. Natl Acad. Sci. USA 81, 253–257. 10. Begley, C.G., Visvader, J., Green, A.R., Aplan, P.D., Metcalf, D., Kirsch, I.R. & Gough, N.M. (1991) Molecular cloning and chromosomal localization of the murine homolog of the human helix-loop-helix gene SCL. Proc. Natl Acad. Sci. USA 88, 869–873. 11. Shivdasani, R.A. & Orkin, S.H. (1996) The transcriptional control of hematopoiesis. Blood 87, 4025–4039. 12. Hsu, H.L., Wadman, I. & Baer, R. (1994) Formation of in vivo complexes between the TAL1 and E2A polypeptides of leukemic Tcells.Proc.NatlAcad.Sci.USA91, 3181–3185. 13. Wadman, I.A., Osada, H., Grutz, G.G., Agulnick, A.D., Westphal, H., Forster, A. & Rabbitts, T.H. (1997) The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J. 16, 3145–3157. 14. Green,A.R.,Lints,T.,Visvader,J.,Harvey,R.&Begley,C.G. (1992) SCL is coexpressed with GATA-1 in hemopoietic cells but isalsoexpressedindevelopingbrain.Oncogene 7, 653–660. 15. Drake, C.J., Brandt, S.J., Trusk, T.C. & Little, C.D. (1997) TAL1/ SCL is expressed in endothelial progenitor cells/angioblasts and defines a dorsal-to-ventral gradient of vasculogenesis. Dev. Biol. 192, 17–30. 16. Shivdasani, R.A. (1997) Stem cell transcription factors. Hematol. Oncol. Clin. North Am. 11, 1199–1206. 17. Begley, C.G. & Green, A.R. (1999) The SCL gene: from case report to critical hematopoietic regulator. Blood 93, 2760–2770. 18. Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F.W. & Orkin, S.H. (1996) The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86, 47–57. 19. Shivdasani, R.A., Mayer, E.L. & Orkin, S.H. (1995) Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 373, 432–434. 20. Rabbitts, T.H. (1998) LMO T-cell translocation oncogenes typify genes activated by chromosomal translocations that alter transcription and developmental processes. Genes Dev. 12, 2651–2657. 21. Foroni, L., Boehm, T., White, L., Forster, A., Sherrington, P., Liao, X.B., Brannan, C.I., Jenkins, N.A., Copeland, N.G. & Rabbitts, T.H. (1992) The rhombotin gene family encode related LIM-domain proteins whose differing expression suggests multi- ple roles in mouse development. J. Mol. Biol. 226, 747–761. 22. Warren, A.J., Colledge, W.H., Carlton, M.B., Evans, M.J., Smith, A.J. & Rabbitts, T.H. (1994) The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 78, 45–57. 23. Yamada, Y., Warren, A.J., Dobson, C., Forster, A., Pannell, R. & Rabbitts, T.H. (1998) The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis. Proc. Natl Acad. Sci. USA 95, 3890–3895. 24. Vyas, P., McDevitt, M.A., Cantor, A.B., Katz, S.G., Fujiwara, Y. & Orkin, S.H. (1999) Different sequence requirements for expression in erythroid and megakaryocytic cells within a reg- ulatory element upstream of the GATA-1 gene. Development 126, 2799–2811. 25. Mead, P.E., Deconinck, A.E., Huber, T.L., Orkin, S.H. & Zon, L.I. (2001) Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins. Development 128, 2301–2308. 26. Vitelli, L., Condorelli, G., Lulli, V., Hoang, T., Luchetti, L., Croce, C.M. & Peschle, C. (2000) A pentamer transcriptional complex including tal-1 and retinoblastoma protein down- modulates c-kit expression in normal erythroblasts. Mol. Cell. Biol. 20, 5330–5342. 27. Crispino, J.D., Lodish, M.B., MacKay, J.P. & Orkin, S.H. (1999) Use of altered specificity mutants to probe a specific protein– protein interaction in differentiation: the GATA-1: FOG complex. Mol. Cell. 3, 219–228. 28. Shimizu, R., Takahashi, S., Ohneda, K., Engel, J.D. & Yama- moto, M. (2001) In vivo requirements for GATA-1 functional 3616 C. Perry and H. Soreq (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... Fujiwara, Y. , Hom, D.B & Orkin, S.H (1998) Failure of megakaryopoiesis and arrested erythropoiesis in mice lacking the GATA-1 transcriptional cofactor FOG Genes Dev 12, 1176–1188 Tevosian, S.G., Deconinck, A.E., Cantor, A.B., Rieff, H.I., Fujiwara, Y. , Corfas, G & Orkin, S.H (1999) FOG-2: a novel GATA-family cofactor related to multitype zinc-finger proteins Friend of GATA-1 and U-shaped Proc Natl Acad Sci USA... Devriendt, K., Matthijs, G., Van Hoof, A., De Vos, R., Thys, C., Minner, K., Hoylaerts, M.F., Vermylen, J & Van Geet, C (2001) Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1 mutation Blood 98, 85–92 Mehaffey, M.G., Newton, A.L., Gandhi, M.J., Crossley, M & Drachman, J.G (2001) X-Linked thrombocytopenia caused by a novel mutation of GATA-1 Blood 98, 2681–2688... 93–105 93 Lev-Lehman, E., Deutsch, V., Eldor, A & Soreq, H (1997) Immature human megakaryocytes produce nuclear-associated acetylcholinesterase Blood 89, 3644–3653 94 Dai, W & Murphy Jr, M.J (1993) Downregulation of GATA-1 expression during phorbol myristate acetate-induced megakaryocytic differentiation of human erythroleukemia cells Blood 81, 1214–1221 95 Soreq, H., Patinkin, D., Lev-Lehman, E., Grifman,... intramolecular regulation of c-Myb Genes Dev 10, 1858–1869 Zon, L.I., Tsai, S.F., Burgess, S., Matsudaira, P., Bruns, G.A & Orkin, S.H (1990) The major human erythroid DNAbinding protein (GF-1): primary sequence and localization of the gene to the X chromosome Proc Natl Acad Sci USA 87, 668– 672 Yamamoto, M., Takahashi, S., Onodera, K., Muraosa, Y & Engel, J.D (1997) Upstream and downstream of erythroid transcription... Y. , Browne, C.P., Cunniff, K., Goff, S.C & Orkin, S.H (1996) Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1 Proc Natl Acad Sci USA 93, 12355–12358 Visvader, J.E., Elefanty, A.G., Strasser, A & Adams, J.M (1992) GATA-1 but not SCL induces megakaryocytic differentiation in an early myeloid line EMBO J 11, 4557–4564 Shivdasani, R.A., Fujiwara, Y. ,... morphologic, and cytogenetic characteristics of 26 patients with acute erythroblastic leukemia Blood 80, 2873–2882 Perkins, A (1999) Erythroid Kruppel like factor: from fishing expedition to gourmet meal Int J Biochem Cell Biol 31, 1175– 1192 Merika, M & Orkin, S.H (1995) Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Kruppel family proteins Sp1 and... expression of the GATA-3 transcription factor affects cell fate decisions in hematopoiesis Exp Hematol 29, 971–980 Tsang, A.P., Visvader, J.E., Turner, C.A & Fujiwara, Y. , YuC., Weiss, M.J., Crossley, M & Orkin, S.H (1997) FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation Cell 90, 109–119 Tsang, A.P., Fujiwara, Y. , Hom,... differentiation-dependent increases in the morphogenically active 3¢ alternative splicing variant of acetylcholinesterase Mol Cell Biol 19, 788–795 90 Majumdar, M.K., Thiede, M.A., Mosca, J.D., Moorman, M & Gerson, S.L (1998) Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells J Cell Physiol 176, 57–66 91 Karpel, R., Ben Aziz-Aloya, R., Sternfeld, M., Ehrlich,... 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Transcriptional regulation of erythropoiesis (Eur J Biochem 269) 3617 domains during primitive and definitive erythropoiesis EMBO J 20, 5250–5260 Mucenski, M.L., McLain, K., Kier, A.B., Swerdlow, S.H., Schreiner, C.M., Miller, T.A., Pietryga, D.W., Scott, W.J Jr & Potter, S.S (1991) A functional c-myb gene is required for normal murine fetal hepatic... positive regulator of the Fli-1 gene involved in inhibition of erythroid differentiation in friend erythroleukemic cell lines Mol Cell Biol 19, 121–135 Bromberg, J & Darnell, J.E Jr (2000) The role of STATs in transcriptional control and their impact on cellular function Oncogene 19, 2468–2473 Socolovsky, M., Fallon, A.E., Wang, S., Brugnara, C & Lodish, H.F (1999) Fetal anemia and apoptosis of red cell progenitors . ARTICLE Transcriptional regulation of erythropoiesis Fine tuning of combinatorial multi-domain elements Chava Perry 1,2 and Hermona Soreq 1 1 Department of. is a clonal myeloproliferative disorder, leading to hyperproliferation of erythroid, myeloid and megakaryocytic cells. Sixty percent of polycytemia vera