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314 Part IV Disorders of Hematopoietic Cell Development

factors implicated in the control of erythropoiesis are listed in Table zinc finger domains, only the first of which is required for GATA1
26.2. binding. The function of its other nine zinc finger domains is not
Studies of mice with targeted gene disruption have provided key clear because they appear to be dispensable in structure-function
insights into the complex molecular pathways that regulate hemato- studies, but they are well conserved in evolution. The GATA1-FOG-1
1,2
poiesis in general and erythropoiesis in particular. These studies, heterodimeric complex binds to the two flanking sites of the minimal
complemented by in vitro differentiation of mutated embryonic stem erythroid transcription activation domain. Experimentally induced
cells into different lineages, have provided clear evidence about dis- genetic mutations, such as GATA1 V205M , impairing GATA1-FOG-1
tinct regulatory requirements of primitive (yolk sac) versus definitive interaction in mice lead to impaired megakaryocytopoiesis and
(fetal liver and bone marrow) erythropoiesis, or of early versus late absence of definitive erythropoiesis, whereas primitive erythropoiesis
486
stages of erythroid differentiation. Because erythropoiesis is the first is normal. Rescue experiments indicate that GATA1 V205M newborns
differentiated lineage in embryonic yolk sac hematopoiesis and the are severely anemic with anisocytosis and spherocytosis with striking
predominant lineage in fetal liver hematopoiesis, factors that affect reduction mainly in the expression of genes encoding membrane
hematopoiesis in general will disturb erythropoiesis during early proteins, whereas expression of other erythroid-specific genes, such as
487
stages of development and lead to lethality at different gestational Alas2, was not affected. These results indicate that DNA binding of
days, depending on the defect. The time in development at which the GATA1-FOG-1 complex is necessary for activation of a subset of
disruption of each specific gene manifests its phenotype is used to GATA1 target genes in definitive erythroid cells but is dispensable for
establish a hierarchical control among the different transcription their activation in primitive erythroblasts. It should be emphasized
factors. The earliest disruption of erythroid differentiation is observed that GATA1-FOG-1 interaction, while activating the expression of
in mice lacking the bHLH factor TAL1/SCL, which is encoded by erythroid genes, inhibits target gene activation in testicular Sertoli
488
a gene initially identified on the basis of its localization in a chro- cells. This result provides insight into how one factor regulates
mosomal breakpoint region frequently associated with T-cell acute more than one differentiation program by suggesting that its func-
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478
leukemia. SCL embryos are bloodless and die very early, with tion, but not its expression, is different depending on the cellular
479
abrogation of both yolk sac and fetal liver erythropoiesis. Because context. (Whether GATA1-FOG-1 interaction inhibits erythroid
of the requirement for SCL in the formation of the transcription gene expression in myelomonocytic cells has not been investigated.)
complex with the nuclear protein Rbtn2/LMO2 rhombotin 2/LIM The CF domain, on the other hand, recognizes and binds to the
domain only 2 (Rbtn2/LMO2) and GATA1 (detailed later), it is GATA site localized in the core of the minimal erythroid transcrip-
not surprising that targeted disruption of Rbtn2 and LMO2 also tion sequence 10 bp downstream to the SCL binding site. SCL and
produces a bloodless phenotype. 480 GATA1 bind simultaneously to their respective sites of the core as
Mice lacking expression of GATA2, a member of the GATA multimeric complexes formed by SCL/E47/LMO2 on the one hand
family of transcription factors, exhibit an early and severe quantitative and by GATA1/LMO2 on the other. Binding of the two complexes
481
defect in hematopoiesis that influences all lineages. Other regula- to the core is stabilized by Lbd1, which forms a physical bridge
tory factors seem to totally spare embryonic (yolk sac) hematopoiesis between them. The paramount importance of the CF finger for
and have a specific effect only on fetal liver hematopoiesis, with GATA1 function is proved by the fact that GATA1 genes lacking
death occurring at later days (12.5 days postconception). In this the region encoding this domain are unable to rescue erythroid dif-
489
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category are the proto-oncogene c-Myb and the core-binding factors ferentiation in GATA1 embryonic stem cells, whereas minigenes
CBF-α 2 /AML1 and CBF-β. 482–484 Embryonic erythropoiesis is spared containing only the CF of either GATA1 or GATA2 are sufficient
490
in mice with targeted ablation of these genes. Both c-Myb, the to induce megakaryocytic differentiation of myeloid cell lines.
cellular homologue of v-Myb proto-oncogene, and the heterodimeric In addition to forming heterodimers with LMO2, CF can form
transcription factor CBF are abundantly expressed early in normal complexes with Sp1 and PU.1, two factors essential for myelomono-
myelolymphoid cells, with decreasing expression as differentiation cytic differentiation. The GATA1-PU.1 complex is unable to bind
proceeds. Their expression pattern and their functional influence DNA, so its function might be to establish either an erythroid- or a
on growth factor receptor genes (i.e., IL-3, GM-CSF, CSF1, T-cell myeloid-permissive cellular environment depending on which factor
2
antigen receptor [TCR] α, β) may underlie their importance in the is expressed at the highest concentration. The presence of relatively
development of all definitive hematopoietic lineages. 1 higher concentrations of GATA1 would favor the formation of
Of paramount importance for adult erythropoiesis is the tran- GATA1-LMO2 complexes leading to activation of erythroid-specific
1
scription factor GATA1, the founder of the GATA family of factors. genes, whereas the presence of relatively higher concentrations of
The GATA1 protein controls erythroid differentiation at several levels PU.1 would lead mainly to the formation of the transcriptionally
by controlling (in cooperation with GATA2) the proliferative capacity inactive GATA1-PU.1 complexes.
of erythroid progenitor/precursor cells, the apoptotic rate of erythro- Although early experiments on cell lines failed to identify any
blasts, and the expression of lineage-specific genes. These effects are function for the N-terminal domain of GATA1, 490,491 knock-in
mediated through activation of expression of target genes by binding experiments in mice indicated that this domain, although dispensable
to specific sequences (WGATAR) present in the regulatory domains for primitive erythropoiesis, is required for appropriate production
489
of virtually any erythroid gene, including EPOR and GATA1 itself. of definitive red cells. A truncated GATA1 gene lacking the
However, WGATAR binding sites are also present in genes specific N-terminal domain is 10 times less efficient than the full-length
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489
for megakaryocytic, eosinophilic, mast cell, and dendritic lineages, as gene in rescuing erythroid differentiation in GATA1 mice. This
well as in genes expressed in testicular Sertoli cells. Insights into the experiment suggests that interaction of the N-terminal domain of
specificity of GATA1 in erythroid differentiation have been provided GATA1 with a suitable partner(s) is required for optimal definitive
by studies on the organization of WGATAR sites in erythroid-specific erythropoiesis. Structure function studies have identified that interac-
regulatory sequences. A minimal erythroid transcription-activation tion between the N-terminal domain of GATA1 and the product
sequence that consists of a core-binding motif flanked by two canoni- of the retinoblastoma (RB) gene is essential for proper terminal
cal GATA1 binding sites has been identified. The core-binding motif erythroid maturation, providing a unifying mechanism for the similar
is composed of one SCL binding site and one GATA binding site phenotype of several GATA1 and RB mouse mutants and of human
2
separated by 10 bp. Different domains of the GATA1 protein are diseases associated with mutations in these two genes. 492
responsible for binding to the core and the flanking sequences. At In addition to all the evidence pointing to GATA1 as exerting a
least three functional domains in the GATA1 protein have been predominant but ontogenetic-specific role in the control of erythroid
identified: two zinc finger domains (amino-terminal [N-terminal] differentiation, other evidence indicates that this gene exerts exquisite
finger [NF] and C-terminal finger [CF)]) and an active N-terminal control in the differentiation of other hemopoietic lineages, such as
494
495
1
493
domain. The NF domain is required for association with Friend megakaryocytes, mast cells, eosinophils, and dendritic cells.
of GATA1 (FOG-1), a protein encoded by a gene identified using The mechanism used by one single factor in guiding differentiation
485
GATA1 as bait in the two-hybrid yeast assay. FOG-1 contains 10 along different lineages does not rely on specific domains in the