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Prepublished online as a Blood First Edition Paper on May 17, 2002; DOI 10.1182/blood-2002-02-0387.
HEMATOPOIESIS
From the Division of Hematology/Oncology, Department of
Medicine, Children's Hospital and Harvard Medical School, Boston;
Howard Hughes Medical Institute, Boston; Department of Pediatric
Oncology, Dana-Farber Cancer Institute, Boston, MA; and Division of
Medical Genetics, Department of Medicine, University of Washington,
Seattle.
Transcription factor GATA-1 is essential for the development of
erythroid cells and megakaryocytes. Each of its 2 zinc fingers is
critical for normal function. The C-terminal finger is necessary for
DNA binding. The N finger mediates interaction with FOG-1, a cofactor
for GATA-1, and also modulates DNA-binding affinity, notably at complex
or palindromic GATA sites. Residues of the N finger-mediating
interaction with FOG-1 lie on the surface of the N finger facing away
from DNA. Strong sequence conservation of residues facing DNA suggests
that this other surface may also have an important role. We report here
that a syndrome of X-linked thrombocytopenia with thalassemia in humans
is caused by a missense mutation (Arg216Gln) in the GATA-1 N finger. To
investigate the functional consequences of this substitution, we used
site-directed mutagenesis to alter the corresponding residue in GATA-1.
Compared with wild-type GATA-1, Arg216Gln GATA-1 shows comparable
affinity to single GATA sites but decreased affinity to palindromic
sites. Arg216Gln GATA-1 interacts with FOG-1 similarly with wild-type GATA-1. Arg216Gln GATA-1 supports erythroid maturation of GATA-1 erythroid cells, albeit at reduced efficiency compared with wild-type GATA-1. Together, these findings suggest that residues of the N finger
of GATA-1-facing DNA contribute to GATA-1 function apart from
interaction with the cofactor FOG-1. This is also the first example of
The zinc finger transcription factor GATA-1 is
expressed in erythroid, megakaryocytic, eosinophilic, and mast cells
and plays multiple roles in hematopoiesis (reviewed in
Orkin1). For instance, GATA-1 is essential for the
differentiation of erythroid and megakaryocytic precursors, as
demonstrated by gene targeting in the mouse.2-4
Members of the GATA family are notable for the presence of 2 closely spaced, homologous zinc fingers of the
C2-C2 class.5 Although highly
similar to each other, the 2 fingers have different functions, both
with respect to DNA binding and interaction with other
proteins.1 In vitro DNA-binding experiments demonstrate that the C-terminal zinc finger (C finger) is required for binding to
WGATAR GATA consensus sites.6 Whereas the N-terminal zinc finger (N finger) is not required for DNA binding per se, its presence
increases the stability of the interaction between GATA-1 and specific
DNA sequences, particularly complex or palindromic GATA
sites.6,7 Functional studies show that the zinc fingers of
GATA-1 are individually required for normal erythroid development. For
example, an erythroid cell line (G1E) that is developmentally arrested
because of the lack of GATA-1 differentiates on the introduction of
wild-type GATA-1, but not GATA-1 lacking either finger.8 Similar findings have been reported recently in transgenic
mice.9
Several protein interactions of GATA-1 are mediated through the zinc
finger domain. Such interactions are mediated by the C finger or by
both zinc fingers. However, the N finger specifically mediates
association with a zinc finger protein cofactor, FOG-1 (friend of
GATA-1).10,11 Structural predictions guided by nuclear magnetic resonance (NMR) studies suggest that the N finger
structure may participate simultaneously in interactions with DNA and
with FOG-1.12 Site-specific mutagenesis has defined
several amino acid residues of the N finger that are critical to the
interaction of GATA-1 with FOG-1. These residues lie on the same face
of the predicted structure of the N finger, suggesting that the N
finger may be viewed as having a FOG-1 surface and another potentially in apposition to DNA. Indeed, amino acid substitutions of residues on
the FOG-1 surface of the finger have been identified in patients with
X-linked thrombocytopenia with (or without) anemia.13,14 These mutations perturb the association of GATA-1 with its cofactor, FOG-1. Sequence comparisons across species reveal that the entire N
finger of GATA-1 is highly conserved with respect to GATA-1 and other
GATA factors. Despite the high degree of conservation of residues on
the presumptive DNA face of GATA-1, there are no findings to date that
address the biologic relevance of the DNA-binding contribution of its N finger.
We show here that a previously reported family with X-linked
thrombocytopenia and Patients and genetic mapping
Transfections
Electrophoretic mobility shift analyses Double-stranded probes containing either a palindromic GATA site present in the chicken GATA-1 promoter (5'-GCGCTATCAGATAAGGCCTTG -3' and 5'-CAAGGCCTTATCTGATAGCGC-3') or a single GATA site patterned from this site (5'-GCGCTCAGAGATAAGGCCTTG-3' and 5'-CAAGGCCTTATCTCTGAGCGC-3') were used as previously described7,10 to study GATA-1 dissociation from DNA in electrophoretic mobility shift analyses. Briefly, nuclear extracts of COS cells transfected with pXM-GATA-1, pXM-Arg216Gln-GATA-1, or pXM vector alone were incubated with 32P-labeled double-stranded probe (palindromic GATA site or single GATA site) for 20 minutes. After 100-fold molar excess, unlabeled competitor probe was added (t = 0 minute), and reaction mixtures were loaded onto polyacrylamide gels and run under established conditions.6 Quantities of bound probe and free probe were measured by Storm PhosphorImager analysis using ImageQuant software (Molecular Dynamics, Piscataway, NJ).Coimmunoprecipitation assays COS cells were cotransfected with pXM-GATA-1, pXM-Arg216Gln-GATA, or pXM vector alone and either FLAG-FOG-1 (a gift from S. G. Katz) or FLAG vector alone (pEFrFLAGPGKpuropAV18, a gift from D. Huang). Immunoprecipitation with anti-FLAG antibody and detection of FLAG proteins and GATA-1 by Western blot were performed as described earlier,10 except that the ECL+ Kit (Amersham Pharmacia Biotech, Piscataway, NJ) was used for final visualization in Western blots.G1E-ER2 clones G1E cells were cultivated as described.8 Using the vector pGD-G1/ER-puro, we created pGD-Arg216Gln-G1/ER-puro using site-directed mutagenesis as described above. The wild-type or mutant vector was used to retrovirally infect G1E cells as described.10 GATA-1 was detected by the N6 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) on Western blots as described.8Northern blot analyses Northern blots with cDNA probes were performed with total RNA samples obtained from G1E-ER2 clones as detailed earlier.10 In that study, ABC-me was described as the GATA-1 target clone HD2 before it was renamed later.17
Syndrome of X-linked thrombocytopenia with thalassemia maps to the GATA-1 locus We previously determined16 that a syndrome of X-linked thrombocytopenia with thalassemia maps to band p11-12 on the human X chromosome, a region that includes a number of genes involved in hematopoiesis, including GATA-1. Noting defects in the megakaryocytic and erythroid lineages, 2 lineages that require GATA-1 for normal development, we investigated whether the GATA-1 locus was altered in affected males and obligate carrier females.Sequencing of the GATA-1 exons, splice junctions, and promoter showed
that affected males and obligate carrier females had a GATA-1 allele
with a transition mutation in codon 216 (CGG>CAG), recoding arginine
(Arg) as glutamine (Gln; Figure 1A). The
missense mutation segregated with disease in the XLTT family and was
not found in 100 control X chromosomes studied. Arg216 is within the N
finger of GATA-1 and is strictly conserved in GATA-1 orthologs in
various species (Figure 1B). The NMR structure of the GATA-1 N finger
(National Center for Biotechnology Information [NCBI] structure
1GNF12) indicates that this residue lies on the face of
the N finger that faces DNA
Arg216Gln GATA-1, like wild-type GATA-1, binds to FOG-1 To determine whether Arg216Gln mutation affects protein interaction with FOG-1, we performed coimmunoprecipitation experiments with different GATA-1::FOG-1 complexes. We have shown previously that a shortened and FLAG-tagged version of FOG-1, which spans zinc fingers 5 and 6, interacts with wild-type GATA-1 on the overexpression of both proteins in COS cells.10 Interaction is markedly reduced, however, with the Val205Gly mutation on the FOG face.10 Based on the structure model, we predicted that Arg216Gln GATA-1 would associate normally with FOG-1. To assess this hypothesis, we tested the interaction of full-length, rather than shortened, versions of these proteins.Wild-type murine GATA-1 or GATA-1 mutants were coexpressed with
FLAG-FOG-1 (full-length murine FOG-1) in COS cells.
Immunoprecipitation of nuclear extracts with anti-FLAG antibody
demonstrates that Arg216Gln GATA-1, like wild-type GATA-1, forms a
complex with FLAG-FOG-1 (Figure 2, lanes
5 and 4, respectively). As expected, interaction is not observed with
DNA binding is altered in Arg216Gln GATA-1 Because Arg216 lies on the predicted DNA-facing side of the N finger, we characterized the DNA-binding properties of Arg216Gln GATA-1. Previous studies6,7,18 show that the C finger is required for GATA-1 to bind DNA, whereas the N finger stabilizes the interaction between GATA-1 and palindromic GATA sites. Accordingly, a mutation in the DNA face of the N finger could be expected to dissociate more easily from a palindromic site than would wild-type GATA-1. In contrast, as suggested from binding studies in which the entire N finger is deleted,7 this mutation would not be expected to show a difference in dissociation from a single GATA site.Using electrophoretic mobility shift assays, we assessed the
dissociation profiles of wild-type and Arg216Gln GATA-1 proteins with
single and palindromic GATA-site targets. We used site-directed mutagenesis to introduce the Arg216Gln mutation into pXM-GATA-1, an
expression vector containing the complete cDNA of murine GATA-1. Nuclear extracts were prepared from COS cells transfected with expression vectors encoding wild-type or Arg216Gln GATA-1. Using radioactively labeled, double-stranded DNA probes encoding either the
palindromic GATA site from the chicken GATA-1 promoter or a single GATA
site modeled after this site (Figure 3,
top), we assembled GATA-1::DNA complexes in vitro.
Following the addition of the respective unlabeled (cold) competitor probe, we observed the dissociation of GATA-1::DNA complexes over time by following the electrophoretic mobility shift of bound probe to free probe (Figure 3, middle panels). (Note: the "slant" in the migration of either the bound or free probe across time points is due merely to the different times at which the samples are loaded onto the gel, which is running continuously.) PhosphorImager quantitation (Figure 3, bottom panels) demonstrates that wild-type and Arg216Gln GATA-1 dissociate at similar rates from a single GATA site (left graph), whereas only wild-type GATA-1 and not Arg216Gln shows a slower rate of dissociation from a palindromic GATA site (right). Indeed, it seems as though Arg216Gln GATA-1 shows similar binding characteristics on the palindromic site and the single site, as predicted. Experiments with other point mutants in this region (Pro213Gly, Leu214Phe, Arg216Ala, Arg216Glu, Leu230Glu, Leu230Phe) also demonstrate this altered DNA-binding profile without disruption of FOG-1 binding (not shown). Of note, the dissociation rates seen in these experiments are similar to those previously reported.7,10 Extracts from COS cells transfected with pXM vector alone did not bind palindromic or single GATA site probes (not shown). Arg216Gln GATA-1 allows erythroid maturation to proceed in a GATA-1 null cell line We next examined the biologic implications of the Arg216Gln mutation in G1E cells, which lack GATA-1 and undergo late erythroid maturation on restoration of GATA-1 function. We have shown previously in these cells that an intact N finger is required for erythroid maturation,8 probably because of recruitment by GATA-1 of FOG-1,10,11 which, like GATA-1, is required for normal erythropoiesis.11Wild-type GATA-1 or Arg216Gln GATA-1 was stably introduced into G1E
cells as a GATA-1/ER fusion (murine GATA-1 cDNA fused in frame to the
human estrogen receptor ligand-binding domain).11,19 Synchronous GATA-1-dependent maturation can then be induced by the
addition of hormone (
To examine the qualitative differences between wt G1E-ER2 and Arg216Gln G1E-ER2, we selected for further analysis 2 clones of each group that showed higher levels of differentiation. Arg216Gln clones exhibited morphologic changes similar to those observed in wild-type clones and showed a comparable degree of benzidine staining in individual benzidine-positive cells (Figure 4C). No benzidine-positive cells were observed in parental G1E cells. The 4 clones studied here expressed GATA-1/ER protein at similar magnitudes (Figure 4D). Northern blot analysis of a panel of transcription factors and
erythroid markers (as performed similarly in Crispino et
al10) shows that Arg216Gln G1E-ER2 clones display changes
in gene expression similar to wt G1E-ER2 clones following activation of
GATA-1 (Figure 5). Transcripts of adult
globins, the erythroid transporter band 3, the heme-regulated
eukaryotic initiation factor-2
Here we demonstrate that a human syndrome of X-linked
thrombocytopenia with thalassemia maps to a missense mutation
(Arg216Gln) in the transcription factor GATA-1, a factor required for
the normal development of erythroid cells and platelets. Affected males, who have a single mutant allele, have a reduction in platelet levels with disproportionately increased bleeding times and an increase
in the Our work is informative with respect to the function of GATA-1 in 2 respects. First, it reveals a physiologic role for the DNA face of the
N finger of the protein. Second, it reveals a novel mechanism for
GATA-1 N finger has a role in binding DNA We have shown that the Arg216Gln mutation perturbs the DNA face of the N-terminal zinc finger, thereby reducing the affinity of GATA-1 for palindromic GATA sites. The opposite FOG-binding face of the N finger is unaffected as Arg216Gln GATA-1 binds normally to FOG-1. Several human mutations identified in X-linked thrombocytopenic syndromes, some with concomitant anemia, map to the FOG face and show no alteration in DNA-binding properties. Therefore, the mutation seen in this family provides the first evidence in humans of an anticipated, but undocumented, role for the N finger in DNA-binding and function in vivo.Determining the function of the DNA face of the N finger has broader
implications for understanding the mechanism of GATA-1 action. The
strong conservation of the residues on this face across organisms
suggests that this region is likely to be important. To date, a number
of "complex" sites that depend on N-finger binding have been
identified in vertebrate globin genes, including the chicken
The bleeding disorders that brought patients in this family to clinical attention underscore the importance of the N finger in normal platelet development. We have found previously that GATA-1 is critical for the creation of functional platelets.3,4 Unfortunately, evaluation of the consequences of the Arg216Gln mutation on megakaryocyte development and platelet biogenesis is precluded by the lack of a suitable experimental system for in vitro study. Furthermore, loss of these patients to follow-up prevents more detailed analysis of platelet function with the Arg216Gln mutation. Of note, the mild thalassemia seen in this family is not reproduced in
the G1E differentiation model. Ribonuclease protection assays examining
the ratio between With the identification of naturally occurring mutations on both sides
of the N finger that result in distinct biochemical alterations and
distinguishable phenotypes, it is now possible to distinguish the
FOG-binding and DNA-binding functions of the N finger. This is
important in assigning targets of GATA-1 that depend on interaction
with FOG-1 and those independent of FOG-1. As we have previously shown,
slightly different phenotypes are observed in GATA-12
versus FOG-111 knockout mice. Like GATA-1 Phenotype resulting from Arg216Gln GATA-1 reveals a new
mechanism of  -thalassemia in
humans. While this work was in preparation, an example of
-thalassemia caused by the mutation of a general transcription
factor, TFIIH, was reported.28
We thank John Crispino for the Val205Gly/ER expression construct.
Submitted February 11, 2002; accepted April 15, 2002.
Prepublished online as Blood First Edition Paper, May 17, 2002; DOI 10.1182/blood-2002-02-0387.
Supported in part by grants from the National Institutes of Health and by a grant from NASA to the Washington Space Grant program. S.H.O. is an Investigator of the Howard Hughes Medical Institute.
C.Y. and K.K.N. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stuart H. Orkin, Children's Hospital, Division of Hematology/Oncology, 300 Longwood Ave, Boston, MA 02115; e-mail: stuart_orkin{at}dfci.harvard.edu.
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© 2002 by The American Society of Hematology.
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  C. L. Balduini, E. De Candia, and A. Savoia Why the disorder induced by GATA1 Arg216Gln mutation should be called "X-linked thrombocytopenia with thalassemia" rather than "X-linked gray platelet syndrome" Blood, October 1, 2007; 110(7): 2770 - 2771. [Full Text] [PDF]
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 A. T. Nurden and P. Nurden Inherited thrombocytopenias Haematologica, September 1, 2007; 92(9): 1158 - 1164. [Full Text] [PDF]
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V. N. Tubman, J. E. Levine, D. R. Campagna, R. Monahan-Earley, A. M. Dvorak, E. J. Neufeld, and M. D. Fleming X-linked gray platelet syndrome due to a GATA1 Arg216Gln mutation Blood, April 15, 2007; 109(8): 3297 - 3299. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
32]P or [
that is, it lies on the surface that does
not contact the GATA-1 cofactor FOG-1. Consistent with this location,
Arg216 was not altered in an unbiased mutagenesis screen
-globin/
(within normal limits). (B) Partial sequence
of the N finger of GATA-1. Arg216 is conserved across GATA-1 orthologs
of various species. Here, amino acid numbering relates to the human,
rat, and mouse polypeptides. Val205, mutated to methionine in familial
dyserythropoietic anemia with thrombocytopenia,
200 to 248 GATA-1, in which the entire N finger is deleted (lane 6).
No GATA-1 was immunoprecipitated in various control experiments (lanes
1, 2, 3, 7).
-globin gene silencer.
erythroid cell line
(G1E) shows that Arg216Gln GATA-1 directs erythroid differentiation but
at a slightly weaker extent relative to that of wild-type GATA-1. Thus,
we observe how a specific mutation on the DNA face of the N finger
demonstrates an effect on erythropoiesis. Unlike mutation of a residue
on the FOG face (Val205Met), which leads to dyserythropoietic anemia
with thrombocytopenia in humans and poor induction of G1E cell
differentiation,