Through development and adult life tissue-restricted transcription factors, like the GATA family, coordinate differentiation and proliferation in a cell context-specific manner to ensure that appropriate numbers of terminally mature cells arise from stem/progenitor cells. In the human hemopoietic system, these processes generate ~1010 new mature blood cells daily. Moreover, the number and mix of cells produced can be altered rapidly as required. When this process goes awry, diseases like leukemia can occur. To date, there is an incomplete understanding of how tissue-restricted transcription factors coordinate differentiation with cell cycle exit during maturation of lineage-restricted progenitor and precursor cells.
The DNA-binding zinc finger transcription factor GATA1 plays key roles in myelopoiesis. Enforced GATA1 expression specifies erythroid and megakaryocytic lineages from primary multipotential myeloid progenitors and committed myelomonocytic and lymphoid progenitors (22
). Once the lineages are specified, continued GATA1 expression is necessary for expression of many red cell-and megakaryocyte-specific genes in terminally maturing precursor cells (48
). In mice, germ line ablation of GATA1 function results in embryonic lethality at embryonic day 11.5 (E11.5) from fatal anemia due to a block in erythroid differentiation at the proerythroblast stage accompanied by apoptosis (16
). Similarly, deletion of an upstream enhancer in the GATA1 locus (ΔneoΔHS mice), resulting in near complete loss of megakaryocyte GATA1 expression, specifically blocks terminal megakaryocyte differentiation and proplatelet formation, and mice suffer from thrombocytopenia (48
). However, in contrast to cell death in GATA1-null erythroid cells, immature GATA1-deficient megakaryoblasts accumulate in bone marrow (10-fold) and spleen (100-fold) and show abnormal growth when cultured in vitro. This suggests that although GATA1 is required for gene expression in both terminally maturing red cells and megakaryocytes, it interfaces with cell cycle and apoptotic machinery differently in the two cell types.
How does GATA1 execute these functions in hematopoietic cells? A number of experimental approaches including transactivation assays (31
), induction of partial megakaryocyte differentiation of a myeloid multipotential cell line 416B (59
), and rescue of erythroid differentiation in GATA1-null erythroid cells (4
) have identified three functional domains in GATA1: two highly conserved N-terminal and C-terminal cysteine-rich zinc-binding fingers (Cys-X2
) and a less well conserved N-terminal activation domain.
The C-terminal zinc finger (Cf) and adjacent C-terminal basic residues are required for high-affinity binding to all WGATAR DNA sequences (17
). Deletion of Cf completely abolishes GATA1 function in all assays, suggesting that DNA binding is absolutely required (47
). Although physical interactions between the Cf and other hematopoietic (GATA2-3, EKLF, and PU.1) and widely expressed (Sp1 and CBP) (3
) transcriptional regulators have been reported, their importance in Cf function remains unclear.
Initial transactivation and 416B differentiation assays suggested that the N-terminal zinc finger (Nf) was dispensable for GATA1 function (31
). However, rescue of erythroid differentiation of GATA1-null erythroid cells and definitive erythropoiesis in transgenic mice required the Nf (47
). The Nf cooperates with the Cf to bind tandem WGATAR sequences (51
) and binds independently to GATC DNA motifs (38
). It also interacts with the proteins FOG-1, c-MYB, and STAT-3 (13
). Structural studies show that the Nf has two surfaces: one interacts with DNA and the other binds FOG-1 (26
). Point mutations affecting DNA binding (R216Q) (70
) or interaction with FOG-1 (e.g., V205G) (7
) impede terminal megakaryocyte and red cell differentiation. This results in severe thrombocytopenia accompanied by dysplastic megakaryopoiesis and variable dyserythropoietic anemia.
The functional role of the third proposed functional domain, the N terminus, is still unresolved. Reporter gene transactivation assays in fibroblasts originally defined an activation domain in the N-terminal 63 amino acids (31
). However, this domain is dispensable for partial megakaryocyte differentiation of 416B cells (60
) and erythroid differentiation of GATA1-null erythroid cells (67
). In contrast, studies in transgenic mice suggest that the N-terminal 84 amino acids are only dispensable for erythropoiesis when the mutant protein is severalfold overexpressed (47
). Discrepancies between the erythroid rescue studies may either reflect differences in the GATA1 N-terminal deletions tested or the assays employed or a combination of both.
The most convincing evidence of involvement of the N terminus in megakaryopoiesis has come from studies in children with megakaryocytic preleukaemia (transient myeloproliferative disease [TMD]) and acute megakaryoblastic leukemia (AMKL) in Down syndrome (DS) (1
). Although the DNA mutations are varied, the functional consequences are predicted to always result in exclusive production of a shorter GATA1 protein (GATA1s) with an N-terminal 84-amino-acid truncation. DS TMD and AMKL (pre)leukemic blasts show partial megakaryocyte differentiation and exhibit altered growth. This suggests that deletion of the N-terminal domain in combination with trisomy 21 alters megakaryocyte cell fate. However, the mechanisms by which GATA1 generates appropriate numbers of megakaryocytes and coordinates terminal megakaryocyte differentiation remain unclear.
To further probe GATA1 function, we established a rescue assay of primary GATA1-deficient fetal megakaryocyte progenitors to identify domains of GATA1 required for coordinated platelet production and megakaryocyte growth. In particular, we have contrasted the role of the N terminus with the interaction of GATA1 with FOG-1.