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Chapter 28 Thrombocytopoiesis 343

that regulates chromosome segregation and anaphase progression factors have been identified in cis-acting regulatory elements of
by targeting key factors for degradation. Since some chromosomal essentially every megakaryocytic and erythroid gene that has been
missegregation occurs during megakaryocyte endomitosis, several studied. GATA1, the founding member of this family, is highly
groups have examined the expression levels and/or activity of certain expressed in erythroid and megakaryocytic cells and, to a lesser extent,
APC components and associated factors. These studies have shown in eosinophils and mast cells. GATA1 plays an essential role in ery-
no significant difference in protein levels of the core APC protein throid development, with loss of function resulting in blocked ery-
CDC27 or the kinetochore-associated signaling protein hsMAD2 throid maturation and apoptosis of erythroid progenitor cells. GATA1
in primary murine megakaryocytes undergoing polyploidization is also required for megakaryocyte maturation and growth control.
compared with nonendomitotic precursors. Haploinsufficiency Lineage-selective loss of GATA1 in megakaryocytes results in marked
of BUBR1, a key component of the spindle checkpoint, perturbs thrombocytopenia in mice with platelet counts of only ≈15% of
megakaryocyte development and polyploidization in mice, but does wild-type littermates. Megakaryocytes are present in the mutant
not cause alterations in circulating platelet counts. animals but have a disorganized DMS, paucity of platelet-specific
granules, reduced expression of multiple megakaryocyte-specific
genes (including GPIbα, GPIbβ, PF4, c-Mpl, and p45 NF-E2), and
Microtubule Regulation marked hyperproliferation as compared with wild-type mice. Gene
expression studies of GATA1-deficient versus wild-type murine
Microtubules play key roles in mitosis. Therefore factors that regulate megakaryocytes have revealed a large number of potential GATA1
their assembly have also been investigated as candidates involved in target genes, and many of these have been found to be bound by
megakaryocyte endomitosis. Protein regulator of cytokinesis 1 GATA1 in genome-wide chromatin occupancy studies. Mice contain-
low
(PRC-1) is involved in mitotic spindle elongation and cytokinesis. ing reduced megakaryocyte-specific expression of GATA1 (GATA1 )
However, no differences in PRC-1 levels were detected in primary develop myelofibrosis as they age, a frequent finding with disorders
murine megakaryocytes undergoing polyploidization compared with of MkP hyperproliferation. A GATA binding site mutation in the
nonendomitotic precursors. Stathmin is a microtubule-depolymerizing GPIbβ promoter has been described in a patient with Bernard-Soulier
factor that plays an important role in regulation of the mitotic syndrome, which is characterized by deficiency of the GPIb/IX/V
spindle. Levels of stathmin are inversely related to the level of ploidy complex and a bleeding diathesis. Taken together, these findings
of megakaryocytic cell lines and primary megakaryocytes. Inhibition suggest that GATA1 acts as master regulator of megakaryocyte matu-
of stathmin in K562 cells increases their propensity to undergo ration and proliferative control.
endomitosis when induced to differentiate into megakaryocytes, and
overexpression of stathmin prevents the transition from mitotic to
endomitotic cell cycles. Together, these findings support a possible Friend of GATA
role of stathmin in modulating endomitosis.
All vertebrate GATA factors contain two zinc fingers. The carboxyl
zinc finger mediates high-affinity DNA binding, whereas the amino
Contractile Ring Activity zinc finger stabilizes the DNA interaction at certain double GATA
sites. The amino zinc finger also interacts with friend of GATA
Cytokinesis requires the assembly and activity of a contractile ring. (FOG) proteins, a family of large multitype zinc finger transcriptional
The failure to complete cytokinesis during endomitotic cell cycles in cofactors. This interaction occurs on the surface of the zinc finger
megakaryocytes may involve functional defects in the Rho/Rock opposite to its DNA binding surface. FOG-1 (also called zfpm1), the
21
pathway. Silencing of nonmuscle myosin heavy chain IIB (Myh10) founding member, is expressed predominantly within erythroid and
by the transcription factor RUNX1 is also required for efficient megakaryocytic cells. Knockout of FOG-1 in mice results in embry-
22
megakaryocyte polyploidization. Further studies will be required to onic lethality caused by severe anemia from a block in erythroid
−
fully dissect the molecular pathways involved in megakaryocyte maturation similar to that observed in GATA1 mice. In addition,
−/−
endomitosis. FOG-1 mice have complete failure of megakaryocytopoiesis,
establishing FOG-1 as the first identified transcription-associated
TRANSCRIPTIONAL CONTROL OF factor selectively required to generate the entire megakaryocyte
lineage. FOG-1’s role in megakaryocyte and erythroid development
MEGAKARYOCYTOPOIESIS requires direct physical interaction with GATA factors. The discrep-
ancy between the relatively late block in megakaryocyte development
Since platelets do not contain nuclei, all transcriptional regulation of seen in GATA1-deficient animals and the complete loss of mega-
−/−
platelet-specific genes must occur at the level of the megakaryocyte. karyocytopoiesis in FOG-1 mice is explained by overlapping
Significant strides have been made in identifying key transcription FOG-dependent roles of GATA1 and GATA2 during early stages of
factors involved in megakaryocyte development and platelet-specific megakaryocytopoiesis.
gene expression. Importantly, mutations in a large number of these
factors are linked to various human thrombopoiesis disorders provid-
ing significant new insights into the pathogenesis of these diseases X-Linked Dyserythropoietic Anemia and
(see box on Inherited Causes of Thrombocytopenia). Thrombocytopenia Caused by GATA1 Mutations
GATA Family Transcription Factors Germline GATA1 mutations that impair binding to FOG-1 and/or
DNA have been identified in several families with X-linked macro-
thrombocytopenia and/or anemia (GATA1 is located on the X
GATA1 chromosome in both humans and mice). The first case, reported by
22a
Nichols et al., involved a woman with mild chronic thrombocyto-
GATA transcription factors comprise a family of zinc finger proteins penia who had two pregnancies with male offspring that were both
that bind the consensus DNA sequence (T/A)GATA(A/G). There are complicated by severe fetal anemia and thrombocytopenia requiring
six known members of the GATA family in vertebrates. GATA1, in utero transfusions. BM examination after birth revealed marked
GATA2, and GATA3 play roles predominantly, although not exclu- dyserythropoiesis and an overabundance of immature-appearing,
sively, within the hematopoietic system. GATA4, GATA5, and dysplastic megakaryocytes that share many of the features of GATA-
low
GATA6 are expressed in nonhematopoietic tissues and play diverse 1 murine megakaryocytes. Remarkably, sequencing of the GATA1
developmental roles within the cardiac, gastrointestinal, endocrine, gene from affected family members identified substitution of valine
and gonadal systems. Functionally important binding sites for GATA by methionine at codon 205 within the amino zinc finger. This