Page 379 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 26 Biology of Erythropoiesis, Erythroid Differentiation, and Maturation 315

GATA1 protein itself. In fact, the structure of all the GATA proteins Another gene of the GATA family important for erythroid
is so well conserved among different family members and in evolution differentiation is GATA2. Both GATA1 and GATA2 are expressed
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that GATA1 embryonic stem cells are rescued not only by reintro- early in multipotential progenitors; however, their expression ratios
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duction of the GATA1 gene itself but also by introducing any other change as the cells differentiate (see Table 26.1), suggesting that
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member of the GATA family, such as GATA3. The lineage-specific the ratio of these two factors may be important at specific stages
action of GATA1 in regulating gene expression is achieved through of erythroid differentiation. Knock-out experiments with both of
the presence of lineage-specific regulatory sequences in the promoter these genes have borne this out. Thus in contrast to GATA2, which
regions of the target genes. Therefore the relative concentration of is expressed at high levels in early cells and affects expansion of all
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GATA1, as opposed to the levels of a few key regulatory partners, may hematopoietic lineages, GATA1 expression increases as differen-
establish a lineage-permissive microenvironment. Furthermore, the tiation advances and seems to be the obligatory factor required for
existence of lineage-specific regulatory sequences in the GATA1 gene survival and terminal differentiation of erythroid cells. In mice with
itself ensures that such concentrations are achieved only in the right targeted disruption of GATA1, erythropoiesis proceeds only up to
cell. Although GATA1 is expressed in erythroid, megakaryocytic, mast, the stage of proerythroblasts; these mice die early and fail to mature
dendritic, and eosinophilic cells, its level of expression differs greatly further. 509,510 Furthermore, transgenic mice with partial loss of func-
among the various cell types, with erythroid cells expressing the most. tion (knockdown alleles, GATA1 LOW ) of GATA1 show that erythroid
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Three DNase hypersensitive sites (HS) have been recognized within differentiation is dose-dependent with respect to GATA1. High
the 8 Kb upstream and the first intron of the murine GATA1 gene, levels of GATA1 are necessary to form complexes with its cofactor
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defined as HSI, HSII, and HSIII. Targeted deletion mutants in the FOG-1 and with the other proteins described earlier (LM02, SCL,
mouse have shown that each of these sites functions as an enhancer or Hsp70) during terminal erythroid differentiation.
in different cell types. HSI is required for GATA1 expression in The realization that minute differences in transcription factor
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megakaryocytes, mast cells, and also for upregulation of GATA1 concentrations are required for lineage specification under physiologic
expression during the process of antigen presentation in dendritic conditions supports the idea that the differentiation system allows
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cells and during the progression of erythroid maturation. HSIII more flexibility in both the choice and the reversibility of pathway
is capable of sustaining low levels of GATA1 expression in erythroid commitment toward a specific lineage. For example, a CFU-E was
and dendritic cells. HSII, which is dispensable for erythroid and thought to have no other choice than to become an erythroid cell
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megakaryocyte expression, is absolutely required for gene expres- or to die. More recently, experiments with forced expression of
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sion in eosinophils. All of the 317 bp of HSI are required for transcription factors in fully committed or even mature cells have
GATA1 expression in megakaryocytes, but only the first 5′ 62 bp demonstrated that the system has some degree of plasticity and that
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are needed for erythroid-specific reporter activity. The HSI region forced expression of FOG-1 into mast cells may turn them into
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contains a canonical minimal erythroid activation sequence, and erythroblasts, whereas forced expression of GATA1 into common
point mutations in the GATA site, but not in the E-box, abolish myeloid progenitor cells induces their transdifferentiation into
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HSI function in both erythroid and megakaryocytic cells. Of note, MEP. (It is foreseen that future experiments will demonstrate that
GATA1 mRNA has an unusually long half-life (>9 hours). Two any cell type may be turned into an erythroblast by overexpression
GATA1 bands, corresponding to the native and processed (acetylated of an appropriate combination of transcription factors.) All of these
and phosphorylated) forms of the protein, have been detected by manipulations were performed in vitro. Of interest, experimentally
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Western blot analysis. The processed form binds DNA with higher decreased expression of GATA1 in progenitor cell compartments in
affinity than the native form. Furthermore, although the half-life of vivo does not alter the frequency of individual compartments (i.e.,
the native form is short (approximately 0.5 hour) and stabilized by does not decrease MEP by increasing the granulocyte/macrophage
EPO, the processed form is extremely stable (half-life >6 hours) and progenitor) but results in alternative differentiation pathways.
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EPO-independent. Because the cell cycle of hemopoietic cells in Although the numbers of cells phenotypically recognizable as MEP
vivo is as short as 6 hours, erythroid cells accumulate GATA1 mRNA in these animals are much higher than normal, MEP with reduced
and protein as they proliferate. Because maturation is dependent GATA1 expression, unlike normal cells, also have the potential to
on the levels of GATA1 expressed by cells, the cellular GATA1 differentiate into mast cells. 513
content might represent the biologic clock that, by controlling Another factor with special importance in the erythroid lineage
the number of precursors, determines the cellular output of the is the CACCC binding protein designated EKLF (also known as
differentiation process. This hypothesis suggests that EPO-induced KLF1), which is expressed at all stages of erythropoiesis but binds
GATA1 processing through the ubiquitin–proteasome pathway is preferentially to CACCC sites in the β-globin promoter. EKLF is a
an important element in the regulation of erythroid differentiation. zinc finger protein that binds not only DNA, but also, after appropri-
On the other hand, the TRAIL-Bruton kinase death pathway has ate posttranslational modifications, is a key regulatory protein that
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as an end point caspase 3, the protein specifically responsible for modulates chromatin structure of the β-globin locus. Mice lacking
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GATA1 cleavage. However, caspase 3 is unable to cleave GATA1 if EKLF (EKLF ) die of a thalassemic-like defect because of severe
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the protein is complexed in the nucleus with the chaperone protein deficiency of β-globin expression. Microarray analysis of EKLF
heat shock protein 70 (Hsp70). EPO-receptor signaling counteracts erythroid cells and promoter-specific expression of reported genes
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the apoptotic pathway by favoring Hsp70-GATA1 colocalization in in EKLF cells have identified that the first GATA1-dependent
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the nucleus. The equilibrium between TRAIL and EPO-dependent molecular control of erythroid differentiation is followed by a second
control on Hsp70 localization may be perturbed under pathologic EKLF-dependent phase. 516,517 Primarily GATA1-dependent genes
conditions. As an example, defective nuclear localization of Hsp70 include, in addition to EPOR and those involved in the control of
and increased GATA1 cleavage is associated with dyserythropoiesis apoptosis, α- and δ-globin. EKLF-dependent genes, in addition to
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in myelodysplastic disorders. Similarly, by sequestering Hsp70, β-globin 518,519 and AHSP, are represented by those required for appro-
free α-globin promotes GATA1 degradation and induces ineffective priate membrane assembly, such as β-spectrin, ankyrin, and band 3
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erythropoiesis in β-thalassemia. The biochemical studies detailing (but not α-spectrin). These results are consistent with the notion that,
the link between EPO and TRAIL from one side and GATA1 from in erythroid differentiation, activation of α-globin gene expression
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the other are consistent with additional data indicating that EPO precedes that of β-globin and that loss of GATA1 binding sites
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signaling also induces GATA1 phosphorylation at Ser310 and that in the promoter of the gene is found in α-thalassemia, in the
this phosphorylation plays an important role in regulating GATA1 Greek nondeletion HPFH (guanine to adenine at nucleotide position
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function in erythroid cell lines. 505,506 Although GATA1 mutants −117 of γ-globin), and in δ-thalassemia (point mutation leading to
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expressing only the native form of GATA1 do not have a detectable G→A substitution at position +69 of the δ-globin gene), whereas
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erythroid phenotype under steady-state conditions, more studies loss of EKLF binding site is present in other forms of HPFH. In
on the response of these mice to erythroid stress will clarify the role addition to regulating globin gene expression directly, EKLF inhibits
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of GATA1 processing in stress erythropoiesis. γ-globin expression indirectly by activating BCL11A expression.