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Chapter 28 Thrombocytopoiesis 345

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Of note, Calligaris et al. previously reported that GATA1s is prolonged residence in the BM. The minimal chromosome regions
produced naturally at low levels in erythroid cells. They proposed deleted in Paris-Trousseau and Jacobsen syndromes associated with
that this might serve a regulatory role during normal hematopoiesis thrombocytopenia includes the genes for the ETS factors Fli-1 and
+
by acting as a dominant negative molecule at specific times/ ETS-1. Lentiviral expression of Fli-1 in CD34 cells from patients
environmental stimuli. Endogenous GATA1s has also been detected with Paris-Trousseau thrombocytopenia rescues megakaryocyte dif-
in normal mouse fetal liver megakaryocytes and adult human BM ferentiation in vitro, providing evidence that it is deficiency of Fli-1
megakaryocytes. Thus it has been proposed that the ratio of GATA1 that is the cause of impaired thrombopoiesis in these patients. Of
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to GATA1s plays a role in developmental aspects of megakaryocyto- interest, Raslova et al. have shown that in normal individuals,
poiesis and that acquired GATA1 mutations observed in DS-TMD expression of Fli-1 is mostly monoallelic in early megakaryocytic
+
−
and DS-AMKL, or germline mutations in the family described progenitors (CD41 /CD42 cells) but predominantly biallelic in later
earlier, perturb hematopoiesis by altering this ratio. stages. They propose that the different populations of megakaryocytes
seen in patients with Paris-Trousseau disorder arise from expression
of the normal allele in the normally differentiating megakaryocytes,
E26 Transformation Specific (ETS) Family and the deleted allele (leading to complete loss of Fli-1 expression)
Transcription Factors in the dying population of megakaryocytes.
Germline heterozygous missense Fli-1 gene mutations have
recently been reported in patients with a familial platelet defect. Most
A common feature of megakaryocyte-specific genes is the presence of of these mutations involve the DNA binding domain and abolish
tandem binding sites for GATA and ETS family transcription factors transcriptional activity. The small number of patients reported have
in their promoters and enhancers. The ETS transcription factor presented with mild macrothrombocytopenia and a storage pool-type
family is composed of a diverse group of proteins that share a common platelet defect.
ETS DNA-binding domain, which recognizes a GGAA core sequence.
Over 30 different ETS factors have been identified, at least 10 of
which (ELF1, ELF2, Fli-1, PU.1, TEL, GABPα, ETS1, ETS2, ETS-Related Gene (ERG)
ELK4, ERG) are expressed in megakaryocytes. Functional studies
have implicated several of these, including Fli-1, ETS1, ETV6 (TEL), ERG is closely related to Fli-1. Mouse studies show that ERG and
ERG, and GABPα, in megakaryocytopoiesis. Fli-1 play compensatory roles in murine thrombopoiesis. The ERG
gene is located on chromosome 21 in humans and has been suggested
to play a role in DS-TMD and AMKL.
Fli-1

The role of Fli-1 megakaryocytopoiesis is the best characterized of ETV6 (TEL)
the ETS factors in terms of its functional role in megakaryocyte
−/−
development. Fli-1 mice die during embryogenesis from hemor- Generation of a fusion protein between ETV6 and RUNX1 is the
rhage, likely caused by both vascular defects and dysmegakaryocyto- most frequent chromosome translocation in childhood pre-B cell
poiesis. Colony assays show an increased number of MkPs in acute lymphoblastic leukemia. Although ETV6 is required for the
−/−
Fli-1 embryos as compared with wild-type mice. However, the ontogeny of all definitive hematopoiesis, a conditional knock-out
megakaryocytes from these colonies are small, contain a high nuclear/ study of the ETV6 gene in mice demonstrates its specific requirement
cytoplasmic ratio, and have hypolobulated nuclei, disorganized for adult-stage megakaryocytopoiesis. Heterozygous germline ETV6
platelet demarcation membranes, and reduced number of α-granules. mutations have recently been described as a cause of autosomal domi-
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Expression of the late megakaryocyte marker gene GPIX is markedly nant thrombocytopenia. The mutations described to date involve
reduced, whereas expression of the early genes, TPO receptor, and either the DNA binding domain (and disrupt DNA interaction)
αIIb are normal or mildly reduced, consistent with a role of Fli-1 in or a common site in a linker region located between the DNA
late megakaryocyte maturation. Fli-1 is involved in the synergistic binding domain and the Pointed protein–protein interaction domain.
transcriptional activation of several megakaryocyte-specific genes by Megakaryocytes from these patients are generally small and contain
GATA1, FOG-1, and RUNX1. Different ETS factors act in a hypolobulated nuclei and underdeveloped cytoplasm. There is also
stage-specific manner during megakaryocytopoiesis, with GABPα variable red blood cell macrocytosis. Importantly, affected individuals
predominantly regulating genes active during early stages of mega- have increased risk for the development of hematologic malignancies.
karyocytopoiesis and Fli-1 during later stages.
Fli-1 has been implicated in the lineage commitment of bipotent
erythroid-MkP cells to the megakaryocyte pathway. Fli-1 expression RUNX1
is downregulated as bipotent cells commit to the erythroid lineage,
and its overexpression in the bipotent human erythroleukemia cell In 1999, Song et al. used positional cloning to identify the genetic
line K562 enhances the expression of several megakaryocyte-specific cause of a rare dominant disorder characterized by thrombocytopenia,
genes and induces a megakaryocyte phenotype. In addition, func- an aspirin-like functional platelet defect, and increased risk for
tional cross-antagonism occurs between Fli-1 and the erythroid- developing acute myelogenous leukemia (FPD/AML; OMIM
25
specific transcription factor EKLF. 601399). They identified nonsense mutations, intragenic deletions,
Paris-Trousseau syndrome (OMIM 188925) and Jacobsen syn- or missense mutations on one allele of the gene for RUNX1 (formerly
drome (OMIM 147791) are overlapping contiguous gene-deletion called AML-1 and CBFA2) that cosegregated with the disease in six
disorders in humans involving the long arm of chromosome 11 separate pedigrees. These mutations all resulted in loss of function,
(11q23). The constellation of findings in these syndromes includes indicating that haploinsufficiency of RUNX1 plays a causal role in
severe congenital cardiac abnormalities, trigonocephaly, mental this disorder. BM or peripheral blood from these patients were
retardation, dysmorphogenesis of the hands and face, and macro- characterized by reduced megakaryocyte colony formation, indicating
thrombocytopenia. The etiology of the thrombocytopenia in these that RUNX1 dosage affects megakaryocytopoiesis.
patients appears to be related to impaired platelet production, since RUNX1 is a member of an evolutionarily conserved family of
platelet survival time is normal. Examination of BM reveals significant transcription factors that share a conserved 128 amino acid domain in
dysmegakaryocytopoiesis with an abundance of micromegakaryocytes their amino half with homology to Drosophila runt gene. This region
and death of large numbers of megakaryocytes during terminal stages mediates binding to DNA (consensus [C/T]G[C/T]GGT), as well
of maturation. Peripheral blood platelets contain giant α-granules, as to its heterodimeric binding partner CBF-β via protein-protein
which are thought to arise from aberrant α-granule fusion during interactions. RUNX1 is the most frequently mutated transcription