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R E V I E W A R T I C L ETranscriptional regulation of erythropoiesis Fine tuning of combinatorial multi-domain elements Chava Perry1,2and Hermona Soreq1 1 Department of Biological Chemi

R E V I E W A R T I C L E

Transcriptional regulation of erythropoiesis

Fine tuning of combinatorial multi-domain elements

Chava Perry1,2and Hermona Soreq1

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

E M B R Y O N I C E R Y T H R O P O I E S I S

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] Hemahema-topoietic 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

B R O A D S P E C T R U M F A C T O R S

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]

SCLis 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, where x represents the

number of days post-conception.

(Received 14 March 2002, revised 2 May 2002, accepted 16 May 2002)

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]

SCLnull 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 myelopoihematopoiesis/erythropoi-esis,

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

LMO2expression 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.

CACC GC

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]

E R Y T H R O I D T R A N S C R I P T I O N F A C T O R S

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 regu lated by a 5¢ 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.

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- and b-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-lian FOG, mFOG2, is expressed in heart, neu rons and 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 Ser208fi 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

is mediated by its promoter as well as by distal regu latory

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 C2H2type 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- to b-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.

leukemia (ALL)

t1; 14, t1; 3, t1; 5, t1; 7

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?

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, a Xenopus 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]

Neptuneappears 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

neptunefunction 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

Xenopusventral 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 evolufunc-tion [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- and b-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

TheFli-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

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.

of these key transcriptional regulators of the erythropoietic

process and notes at least part of their multi-element

interactions during erythroid differentiation

D O W N S T R E A M T A R G E T G E N E S

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 CD34+ 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 localizatranscrip-tion, 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).

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

A C K N O W L E D G E M E N T S

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.

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