A Mutation in the Translation Initiation Codon of Gata-1 Disrupts Megakaryocyte Maturation and Causes Thrombocytopenia

A mutation in the translation initiation codon of Gata-1 disrupts megakaryocyte maturation and causes thrombocytopenia

Ian J. Majewski*†, Donald Metcalf*‡, Lisa A. Mielke*, Danielle L. Krebs*, Sarah Ellis§, Marina R. Carpinelli*, Sandra Mifsud*, Ladina Di Rago,* Jason Corbin*, Nicos A. Nicola*, Douglas J. Hilton*, and Warren S. Alexander*‡

*The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia; †Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia; and §Peter MacCallum Cancer Centre, Trescowthick Research Laboratories, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia

Contributed by Donald Metcalf, July 27, 2006 We have generated mice from a N-ethyl-N-nitrosourea mutagen- progenitors in fetal life, an abnormality that resolves spontane- esis screen that carry a mutation in the translation initiation codon ously at birth. This phenotype resembles the transient myelo- of Gata-1, termed Plt13, which is equivalent to mutations found in proliferative disorder associated with trisomy 21 that resolves patients with acute megakaryoblastic leukemia and Down syn- shortly after birth and is followed later in life by AML-M7 DS drome. The Gata-1 locus is present on the X chromosome in in a proportion of affected children (18). humans and in mice. Male mice hemizygous for the mutation We report here a N-ethyl-N-nitrosourea (ENU)-induced mu- (Gata-1Plt13͞Y) failed to produce red blood cells and died during tation in the translation initiation codon of Gata-1, designated embryogenesis at a similar stage to Gata-1-null animals. Female Plt13, which is analogous to mutations detected in patients with mice that carry the Plt13 mutation are mosaic because of random AML-M7 DS and transient myeloproliferative disorder (10–13). inactivation of the X chromosome. Adult Gata-1Plt13/؉ females Gata-1Plt13͞Y mice failed to produce red blood cells and died were not anemic, but they were thrombocytopenic and accumu- during embryogenesis at a similar stage to Gata-1-null animals. lated abnormal megakaryocytes without a concomitant increase in Female mice that carry the Plt13 mutation, mosaic because of megakaryocyte progenitor cells. Gata-1Plt13/؉ mice contained large lyonization of the X chromosome, were born at the expected numbers of blast-like colony-forming cells, particularly in the fetal frequency. Gata-1Plt13/ϩ mice displayed thrombocytopenia and liver, but also in adult spleen and bone marrow, from which accumulation of abnormal megakaryocytes. No significant in- continuous mast cells lines were readily derived. Although the crease in megakaryocyte progenitor cell numbers was observed equivalent mutation to Gata-1Plt13 in humans results in production in Gata-1Plt13/ϩ mice, but accumulation of abnormal blast-like of GATA-1s, a short protein isoform initiated from a start codon colony-forming cells was evident in the fetal liver and persisted downstream of the mutated initiation codon, Gata-1s was not throughout adult life in the bone marrow and spleen. Molecular detected in Gata-1Plt13/؉ mice. analyses showed that, unlike cells in humans bearing mutations in the initiation codon of GATA1, Gata-1Plt13/ϩ mice expressed N-ethyl-N-nitrosourea mutagenesis ͉ platelet no detectable Gata-1s protein.

ata-1 is a transcription factor that is essential for appropri- Results Gate development of multiple hematopoietic lineages includ- Plt13, an ENU-Induced Mutation in Gata-1. The founder of the PLT13 ing erythroid, megakaryocytic, eosinophilic, and mast cells (1– pedigree was a thrombocytopenic female mouse (platelet count ϫ 6 4). In megakaryocyte development, Gata-1 and the of 495 10 per milliliter) identified among the G1 offspring of transcriptional cofactor Friend of Gata-1 (Fog-1) regulate ex- ENU-treated male BALB͞c mice. Several female offspring of pression of genes that are critical for megakaryocyte maturation this founder exhibited a similarly reduced platelet count, and and platelet function, including the receptor for thrombopoietin expansion of the pedigree by breeding affected G2 females with (c-mpl), platelet factor 4 (pf4), cell-surface integrin GPIb␣, and wild-type BALB͞c mice revealed that whereas Ϸ50% of female transcription factor NF-E2 (nuclear factor erythroid 2) (5–8). mice were thrombocytopenic, no affected males were produced Mice that lack Gata-1 expression only in the megakaryocyte (Fig. 1A). This result established the heritability of the throm- lineage (⌬neo⌬HS) are viable but display marked thrombocy- bocytopenia and suggested that the Plt13 mutation was X-linked, topenia and abnormal megakaryocytopoiesis (9). potentially with hemizygous lethality. In humans, GATA1 mutations have been associated with In human populations, mutations in GATA1 have been asso- X-linked anemia and thrombocytopenia. Recently, GATA1 mu- ciated with X-linked thrombocytopenia (19–22). Accordingly, ϩ tations have been identified in patients with acute megakaryo- genomic DNA was isolated from affected Gata-1Plt13/ mice for blastic leukemia accompanying Down syndrome (AML-M7 DS) analysis of Gata-1 by DNA sequencing. A single base pair change (10, 11). The mutations occur in the second exon of GATA1 and (T3A transversion) was identified in the second exon of Gata-1 are either small insertions or deletions that introduce premature in affected mice, which disrupts the translation initiation codon stop codons or mutations that disrupt splicing or the start codon of GATA1 (10–13). These mutations all prevent translation of full-length GATA1 and result in the synthesis of a shorter Author contributions: I.J.M., D.M., L.A.M., D.L.K., S.E., M.R.C., N.A.N., D.J.H., and W.S.A. designed research; I.J.M., D.M., L.A.M., D.L.K., S.E., M.R.C., S.M., L.D.R., and J.C. performed GATA1 isoform (GATA1s) that is initiated at Met-84. research; I.J.M., D.M., L.A.M., D.L.K., S.E., M.R.C., D.J.H., and W.S.A. analyzed data; and The N terminus of Gata-1 contains a highly acidic region, I.J.M. and W.S.A. wrote the paper. deletion of which reduces transactivation activity (14). Never- The authors declare no conﬂict of interest. theless, expression of Gata-1s at high levels is able to drive Abbreviations: ENU, N-ethyl-N-nitrosourea; AML-M7 DS, acute megakaryoblastic leukemia megakaryocytic maturation (7, 15, 16), and mice engineered to and Down syndrome. express Gata-1s using the endogenous Gata-1 promoter (Gata- ‡To whom correspondence may be addressed. E-mail: [email protected] or alexandw@ ⌬ 1 e2) show no signs of anemia or thrombocytopenia in adult life wehi.edu.au. (17). These mice accumulate hyperproliferative megakaryocytic © 2006 by The National Academy of Sciences of the USA

14146–14151 ͉ PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606439103 Downloaded by guest on September 30, 2021 Table 1. Peripheral blood proﬁle of Gata-1Plt13/؉ mice Genotype

Blood cell parameter Gata-1ϩ/ϩ Gata-1Plt13/ϩ

Platelet count (ϫ10Ϫ6 per ml) 1,009 Ϯ 139 473 Ϯ 143* Mean platelet volume, ﬂ 7.1 Ϯ 1.0 8.3 Ϯ 0.8 Red cell count (ϫ10Ϫ9 per ml) 11.1 Ϯ 0.3 10.4 Ϯ 0.8 Hematocrit, % 53.3 Ϯ 1.7 52.4 Ϯ 3.9 White cell count (ϫ10Ϫ6 per ml) 9.2 Ϯ 1.6 9.1 Ϯ 1.9 No. of neutrophils 1.3 Ϯ 0.3 1.3 Ϯ 0.4 No. of lymphocytes 7.6 Ϯ 1.4 7.5 Ϯ 1.6 No. of monocytes 0.06 Ϯ 0.02 0.06 Ϯ 0.02 No. of eosinophils 0.16 Ϯ 0.02 0.10 Ϯ 0.04

Means Ϯ SD are shown (n ϭ 54–58 mice per group). Statistical analysis was by ANOVA with correction for multiple testing (*, P Ͻ 0.001).

quency at embryonic day 10.5 [Gata-1ϩ͞Y (10), Gata-1Plt13͞Y (7), Gata-1ϩ/ϩ (8), and Gata-1Plt13/ϩ (8)]; however, by embryonic day 13.5 Gata-1Plt13͞Y embryos were present at a reduced frequency [Gata-1ϩ͞Y (15), Gata-1Plt13͞Y (6), Gata-1ϩ/ϩ (17), and Gata-1Plt13/ϩ (12)], were smaller than wild-type embryos, were pallid consistent with severe anemia, and upon histological examination exhibited regions of necrosis and little or no fetal liver (data not shown). Whereas no Gata-1Plt13͞Y mice survived to birth, female heterozygote (Gata-1Plt13/ϩ) embryos exhibited no evidence of anemia or other abnormalities and were born at the expected frequency. MEDICAL SCIENCES

Abnormal Megakaryocytopoiesis in Gata-1Plt13/؉ Mice. Analysis of peripheral blood from a cohort of female Gata-1Plt13/ϩ mice confirmed a 60% reduction in the number of circulating platelets when compared with wild-type littermates, but there was no significant increase in mean platelet volume (Table 1). Although Gata-1 is an essential regulator of red blood cell maturation, affected mice displayed normal numbers of red blood cells and Fig. 1. The Plt13 mutation affects the translation initiation codon of Gata-1. a normal hematocrit. White blood cell counts were within the (A) The PLT13 founder (G1, arrow) was identiﬁed because of a low platelet normal range (Table 1). count (495 ϫ 106 per milliliter) and bred to test the heritability of the The spleen and bone marrow of Gata-1Plt13/ϩ mice demon- phenotype. G2 mice that displayed a reduced platelet count (ﬁlled circles) strated a significant accumulation of megakaryocytes (Figs. 2A were selected for breeding, and data from their offspring (G3) were separated and 3A), and a uniform finding in all Gata-1Plt13/ϩ mice was the by sex to demonstrate that the phenotype was X-linked. Platelet counts from presence of small numbers of single megakaryocytes in the liver G3 mice were tabulated (mean Ϯ SD), and mice that had a platelet count Ͻ800 ϫ 106 per milliliter were considered affected. (B) DNA sequence from and lungs of adult mice. The majority of megakaryocytes ap- exon 2 of Gata-1 is shown for wild-type (Gata-1ϩ/ϩ) and affected (Gata-1Plt13/ϩ peared abnormal, commonly with hyperchromatic nuclei sur- and Gata-1Plt13͞Y) animals. The translation initiation codon of Gata-1 is un- rounded by scant areas of cytoplasm. Electron microscopic derlined. (C) 293T cells were transfected with expression constructs for wild- examination revealed abnormalities in the demarcation mem- type Gata-1 (WT), Gata-1Plt13 (PLT13), or vector alone (V). Lysates were sepa- brane system with mutant megakaryocytes containing large rated by SDS͞PAGE, and a Western blot was performed with an antibody that disorganized clumps of membrane or lacking a demarcation recognizes the C terminus of Gata-1. Filled circles mark full-length Gata-1, and membrane system entirely (Fig. 2B). Modal DNA ploidy in bone an open circle marks truncated Gata-1. marrow megakaryocytes was increased from 16 N in wild-type mice to 32 N in Gata-1Plt13/ϩ females, and the mutant mice (ATG3AAG) (Fig. 1B). This mutation was not detected in showed an increased abundance of megakaryocytes with high unaffected littermates and was absent in stock BALB͞c mice as DNA (64 N and 128 N) content (Fig. 3B). ͞ well as other inbred mouse strains including C57BL 6 and Megakaryocytes in spleens of wild-type mice stained promi- ͞ Plt13 129 Sv. In transfected 293T cells, the Gata-1 cDNA directed nently with antibodies directed to either the N or C terminus of synthesis of a 40-kDa protein that was recognized by an antibody Plt13/ϩ Ϸ Gata-1 protein (Fig. 4A). Random X inactivation in Gata-1 directed against the C terminus of Gata-1 but which was 10 females would be expected to result in half the megakaryocytes kDa smaller than Gata-1 expressed from a wild-type cDNA (Fig. Plt13 Plt13 expressing the wild-type allele and half expressing Gata-1 . 1C). Unlike wild-type Gata-1, the Gata-1 protein did not Plt13/ϩ react with an antibody that recognizes the N terminus of the However, only 10–20% of Gata-1 megakaryocytes stained protein (data not shown), and no protein of wild-type Gata-1 size with the N-terminal antibody, which detects protein from the was detected in transfected cells expressing the mutant allele. wild-type allele. This observation suggests that the increased Plt13 ϩ These data suggest that the Plt13 mutation prevents expression number of megakaryocytes in Gata-1 / tissues was due to the of full-length Gata-1, analogous to mutant GATA1 alleles in selective accumulation of mutant cells. Furthermore, the ma- ϩ patients with AML-M7 DS (10, 11). jority of megakaryocytes in Gata-1Plt13/ mice exhibited no staining with either N- or C-terminal Gata-1 antibodies (Fig. The Gata-1Plt13 Mutation Results in Hemizygous Lethality. Gata- 4B), suggesting that if the truncated Gata-1s protein is expressed 1Plt13͞Y embryos were present at the expected Mendelian fre- in mutant megakaryocytes in vivo then the amount is below

Majewski et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14147 Downloaded by guest on September 30, 2021 Fig. 2. Gata-1Plt13/ϩ mice exhibit megakaryocytosis and abnormal megakaryocyte maturation. (A) Accumulation of megakaryocytes in the bone marrow and spleen of Gata-1Plt13/ϩ mice. In Gata-1Plt13/ϩ tissues a signiﬁcant number of megakaryocytes demonstrated hyperchromatic nuclei and a reduced amount of cytoplasm (arrows). (B) Electron micrographs of megakaryocytes from wild-type mice demonstrate the expansive cytoplasm and complex demarcation membrane system that are characteristic of these cells. Gata-1Plt13/ϩ megakaryocytes have large, multilobed nuclei but generally have reduced cytoplasm with a poorly developed demarcation membrane system. (Scale bar: 5 ␮m.)

detection and likely to be considerably lower than the amount of 2). This extramedullary hematopoiesis was associated with a wild-type Gata-1 expressed in Gata-1ϩ megakaryocytes. slight splenomegaly [Gata-1Plt13/ϩ spleen weight, 172 Ϯ 29 mg (n ϭ 12); Gata-1ϩ/ϩ spleen weight, 106 Ϯ 12 mg (n ϭ 4)]. Hematopoietic Progenitor Cells in Gata-1Plt13/؉ Mice. Despite the Significant numbers of unusual colonies containing cells with increased numbers of megakaryocytes in Gata-1Plt13/ϩ mice, a blast-like appearance distinguished the Gata-1Plt13/ϩ bone there was a reduction in the number of megakaryocyte progen- marrow and spleen cultures from those of wild-type mice, and itor cells in the bone marrow, and, although colony numbers similar colonies were found at a much higher frequency in fetal were elevated in the spleen and feral liver (Table 2), colony size liver cultures (Table 2). These colonies differed from typical was unaffected. The numbers of colonies containing granulo- blast colonies in that none were multicentric. Individual blast- cytes, macrophages, or eosinophils were relatively normal in like colonies isolated from agar cultures could be maintained for cultures of Gata-1Plt13/ϩ bone marrow stimulated by stem cell at least several months as continuous cell lines in liquid cultures factor plus IL-3 plus erythropoietin (Table 2). Spleen cell stimulated by stem cell factor plus IL-3, but they failed to grow cultures from Gata-1Plt13/ϩ mice revealed significantly elevated in the absence of added cytokines. Cytocentrifuge preparations numbers of granulocyte and macrophage progenitor cells (Table of cells from the continuous lines revealed a distinctive mast cell morphology (Fig. 5A). An analysis of the cell surface phenotype demonstrated that the cells were expressing c-Kit, CD9, and CD41 but were negative for other lineage markers (Ter119, B220, Gr1, and Mac1). Examination of GATA-1 mRNA from 15 cell lines confirmed expression of Gata-1Plt13 mRNA and absence of wild-type Gata-1 mRNA (Fig. 5B). As observed in primary Gata-1Plt13/ϩ megakaryocytes (Fig. 4B), no Gata-1 protein was detected in these cells, consistent with little or no expression of Gata-1s from the mutant allele (Fig. 5C). Discussion The founder of the Plt13 pedigree was identified in an ENU mutagenesis screen because of its significantly reduced platelet count. Upon sequencing, the Plt13 mutation was found to disrupt the translation initiation codon of Gata-1. Mutations that disrupt the initiation codon of the human GATA1 mRNA result in the production of a truncated GATA1 protein, GATA1s, which lacks the N-terminal transactivation domain (23). Patients with such mutations exhibit clonal accumulation of megakaryoblasts that resolves shortly after birth. However, in Ϸ20% of such cases, AML-M7 DS develops within 4 years of life (18). Mice engi- Fig. 3. The number and DNA content of megakaryocytes are increased in neered to express Gata-1s in place of full-length Gata-1 (Gata- ⌬ Gata-1Plt13/ϩ mice. (A) The number of megakaryocytes enumerated from 1 e2 mice) are born at the expected frequency and exhibit normal microscopic examination of histological sections of bone marrow (BM) and red blood cell production. Consistent with the transient myelo- spleen from wild-type (open bars) and Gata-1Plt13/ϩ (filled bars) mice expressed proliferative disorder in humans, Gata-1⌬e2 mice transiently as the number of megakaryocytes per 10 high-power microscopic fields (ϫ600 accumulate large numbers of hyperproliferative megakaryocyte for bone marrow and ϫ200 for spleen). (B) The DNA content of wild-type Plt13/ϩ progenitor cells in the yolk sac and fetal liver. This abnormality (open bars) and Gata-1 (filled bars) bone marrow megakaryocytes was ⌬e2 analyzed by flow cytometry using CD41 staining in combination with pro- resolves, and adult Gata-1 mice display normal megakaryo- pidium iodide. Mean values are presented from two to four mice per point, cytopoiesis and platelet numbers (17). Plt13/ϩ and error bars represent standard deviations. *, P Ͻ 0.05; **, P Ͻ 0.001. In contrast, although Gata-1 mice display myeloprolif- Statistical analysis was by ANOVA and pairwise comparison with Bonferroni’s eration in the fetal liver, this phenotype is associated with correction for multiple testing. accumulation of blast-like colony-forming cells with mast cell

14148 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606439103 Majewski et al. Downloaded by guest on September 30, 2021 Fig. 4. Analysis of Gata-1 protein expression in megakaryocytes. Spleen sections were stained with antibodies that recognize the N terminus (in cyan) and the C terminus (in red) of Gata-1. CD41 staining was used to identify splenic megakaryocytes (in green). (A) In wild-type mice megakaryocytic nuclei stain with both N- and C-terminal antibodies. (B) There is a signiﬁcant accumulation of megakaryocytes in Gata-1Plt13/ϩ mice, and many of these appear negative for Gata-1 staining (arrows).

potential rather than excessive numbers of megakaryocyte pro- unlike the Gata-1Plt13/ϩ progenitors, the colonies derived from genitors. In adult hematopoietic tissues, the abnormal blast-like Gata-1low mice also contained megakaryocytes and erythroid colony-forming cells persist in Gata-1Plt13/ϩ mice and are accom- cells (2). panied by an accumulation of megakaryocytes without signifi- High-level expression of the Gata-1Plt13 mRNA in 293T cells cant expansion of megakaryocyte progenitors. The Gata-1Plt13 confirmed that full-length Gata-1 protein could not be produced MEDICAL SCIENCES megakaryocytes exhibit morphological abnormalities and higher from this allele but resulted instead in the production of a than normal modal DNA ploidy, and the mice were thrombo- truncated protein predicted to initiate from Met-84 that was cytopenic, suggesting that the dysmorphic megakaryocytes are equivalent in size to Gata-1s. Analysis of Gata-1 protein from the unable to efficiently release platelets. Inefficient platelet pro- endogenous Gata-1Plt13 mRNA in cells derived from Gata- duction may result from reduced expression of Gata-1 target 1Plt13/ϩ mice demonstrated no detectable expression of Gata-1s genes including NF-E2 and GpIb␣, both of which are known to in megakaryocytes or in cell lines derived from blast-like colo- have a critical role in megakaryocyte maturation and platelet nies, suggesting that translation is initiated at Met-84 with low shedding (24, 25). The thrombocytopenia and megakaryocytosis efficiency in vivo in Gata-1Plt13/ϩ mice, or that the truncated in Gata-1Plt13/ϩ mice resemble more the phenotype of mice protein is unstable and rapidly degraded. Consistent with little lacking Gata-1 expression in megakaryocytes, such as Gata- or no expression of Gata-1 from the Gata-1Plt13 allele, Gata- 1⌬neo⌬HS and Gata-1low, than that of mice that express Gata-1s. 1Plt13͞Y males died at a similar stage to Gata-1-null embryos (1, However, the excessive number of megakaryocytes in Gata- 3) and exhibited no signs of fetal red blood cell production. 1⌬neo⌬HS and Gata-1low mice is due to the presence of hyperpro- Thus, unlike cells in humans bearing mutations in the trans- liferative megakaryocyte progenitors, and the megakaryocytes lation initiation codon of GATA1 or Gata-1⌬e2 mice, which that accumulate are immature with low DNA content (6, 9), express Gata-1s, there is no detectable expression of full-length whereas in Gata-1Plt13/ϩ mice megakaryocyte progenitor cell Gata-1 or Gata-1s from the Gata-1Plt13 allele, and this is highly numbers are not elevated, and the megakaryocytes that accu- likely to account for the differences in the phenotypes observed mulate have large and complex nuclei with a high DNA content. between these distinct mouse and human models. In the Gata- The blast-like colonies observed in Gata-1Plt13/ϩ mice failed to 1⌬e2 mice, deletion of exon 2 removes two additional AUG grow in the presence of thrombopoietin, were uniformly negative codons that are upstream of the Met-84 translation initiation site for acetylcholinesterase activity, and had mast cell morphology for Gata-1s (17). These start sites are out of frame with the when grown in liquid culture. Highly proliferative mast cell Gata-1 ORF and may reduce the efficiency of translation of progenitors have also been identified in Gata-1low mice; however, Gata-1s in Gata-1Plt13/ϩ mice. Interestingly, only one of these

Table 2. Hematopoietic progenitor cells in Gata-1Plt13/؉ mice No. of colonies Gata-1 Tissue genotype Blast͞mast G GM M Eo Meg

Bone marrow ϩ͞ϩ 16 Ϯ 317Ϯ 10 5 Ϯ 211Ϯ 42Ϯ 118Ϯ 3 Plt13͞ϩ 29 Ϯ 516Ϯ 39Ϯ 314Ϯ 51Ϯ 17Ϯ 2 Spleen ϩ͞ϩ 1 Ϯ 1 0.2 Ϯ 0.3 0 0.5 Ϯ 0.5 0 0.5 Ϯ 1 Plt13͞ϩ 13 Ϯ 32Ϯ 21Ϯ 25Ϯ 2 0.1 Ϯ 0.3 4 Ϯ 2 Fetal liver ϩ͞ϩ 8 Ϯ 812Ϯ 87Ϯ 92Ϯ 407Ϯ 8 Plt13͞ϩ 313 Ϯ 97 18 Ϯ 15 23 Ϯ 17 93 Ϯ 55 0 33 Ϯ 13

Data represent the means and SD of colony numbers in cultures from 3–11 mice of each genotype stimulated with stem cell factor plus IL-3 plus erythropoietin. Data have been standardized to colonies per 25,000 cultured cells. GM, granulocyte–macrophage colonies; G, granulocyte colonies; M, macrophage colonies; Eo, eosinophil colonies; Meg, megakaryocyte colonies.

Majewski et al. PNAS ͉ September 19, 2006 ͉ vol. 103 ͉ no. 38 ͉ 14149 Downloaded by guest on September 30, 2021 reactions containing 20 ng of DNA, 2 mM MgCl2,50␮M dNTPs, 2 units of Taq polymerase, and 10 pmol of each primer (for exon two of Gata-1: forward, 5Ј-TCTCAGTGACAGATTCG- GAGAA-3Ј; reverse, 5Ј-GCTAACCATCTCTATGGCAACC- 3Ј). PCR products were sequenced by using Big Dye Terminator V3.1 (Applied Biosystems, Carlsbad, CA) before processing on an ABI 3700 sequence analyzer.

RT-PCR. The Gata-1 ORF was amplified by PCR from bone marrow cDNA and cloned into the pEFBOS mammalian expression vector incorporating a C-terminal Flag epitope, and a Plt13 mutant version was generated via PCR mutagenesis. To distinguish between wild-type and mutant mRNA, RT-PCR primers were designed to flank the second exon of Gata-1 (forward, 5Ј-CTTGGGATCACCCTGAACTC-3Ј; reverse, 5Ј- GCTCTTCCCTTCCTGGTCTT-3Ј), and the resulting PCR products were digested with NcoI.

Immunoprecipitation and Western Blotting. 293T cells grown in DMEM with 10% FCS were transfected with expression constructs by using FuGENE 6 reagent (Roche Diagnostics, Indi- anapolis, IN). Cells were lysed after 48 h in KALB buffer (1 mM EDTA͞150 mM NaCl͞50 mM Tris͞1% Triton X-100, pH 7.4), and proteins were separated by SDS͞PAGE. Protein was trans- ferred to a PVDF membrane and blotted with an antibody directed against the C terminus of Gata-1 (M-20, Santa Cruz Fig. 5. Cell lines derived from Gata-1Plt13/ϩ mice have a mast cell phenotype. Biotechnology, Santa Cruz, CA). Lysates prepared in KALB (A) Cells from a continuous mast cell line derived from a blast-like colony from buffer were immunoprecipitated overnight with biotinylated ϩ Gata-1Plt13/ bone marrow were cytocentrifuged and stained with May– goat anti-human GATA-1 (R & D Systems, Minneapolis, MN). Grunwald–Giemsa. (B) RT-PCR was performed on RNA extracted from continuous cell lines and from bone marrow (BM) with primers that ﬂank the site of Hematology. Manual or automated counts were performed on the Plt13 mutation (468-bp product). Digestion with NcoI cleaves the wild- type PCR product, resulting in two fragments (381 bp and 87 bp), but not the blood collected into EDTA. In vitro colony assays for enumer- product from the Gata-1Plt13 allele. (C) Lysates from cell lines were immuno- ation of hematopoietic progenitors were performed as described precipitated with an antibody that binds to the C terminus of Gata-1 (ϩ)ora (27). Bone marrow (2.5 ϫ 104), spleen (5 ϫ 104), or fetal liver control IgG from goat (Ϫ). Western blotting with the C-terminal M20 antibody (2.5 ϫ 103) cells were cultured for 7 days in 1 ml of 0.3% agar demonstrated that Gata-1s protein (open circle) was absent in mast cell lines in DMEM supplemented with 20% FCS and recombinant ϩ from Gata-1Plt13/ mice, whereas the protein was detectable in lysates from cytokines. Individual colonies were isolated then maintained in transfected 293T cells. IgG heavy chain is marked by an arrow. liquid culture in DMEM with 10% FCS and recombinant cytokines. upstream AUG codons is evident in the human GATA-1 mRNA, potentially explaining the capacity of human GATA1 alleles with DNA Ploidy. Bone marrow was collected from femurs and tibias mutations in the usual translation initiation codon to express into 1 ml of CATCH buffer (Hank’s balanced salt solution with GATA1s. A germ-line mutation in the second exon of GATA1 3% BSA, 1.3 mM sodium citrate, 1 mM adenosine, 2 mM has recently been described in a family with heritable macrocytic theophylline, and 3% FCS) and stained with FITC-conjugated anemia in the absence of trisomy 21 (26). In contrast to the anti-CD41 antibody (Becton Dickinson, Franklin Lakes, NJ) on ͞ murine model (Gata-1⌬e2), this finding would suggest that the ice. Samples were incubated with propidium iodide (0.05 mg ml production of GATA-1s, in the context of GATA1 mutation, is in 3.4 mM sodium citrate) for1honice. Cells were washed twice ␮ unable to support normal erythropoiesis in humans. Thus, in CATCH buffer, aggregates were removed with a 100- m ␮ ͞ species differences and precise levels of expression of Gata-1 and sieve, and samples were treated with 50 g ml RNase H for 30 Gata-1s appear critical to the specific effects of Gata-1 mutations min at room temperature before analysis on a FACScan flow on regulation of megakaryocytopoiesis and erythropoiesis and cytometer (Becton Dickinson). the potential for development of megakaryoblastic leukemia. The development of mouse models with diverse mutations in Immunofluorescence Microscopy. Frozen spleen sections (5 ␮m) Gata-1, in which these variables are differentially affected, have were fixed sequentially in 4% paraformaldehyde and methanol, established powerful tools for dissecting the mechanism of washed with PBS͞1% BSA, and incubated for1hwith2␮g͞ml Gata-1 action in blood cell regulation in health and disease. rabbit anti-C-terminal GATA1 (Sigma) or 1 ␮g͞ml rat anti-N- terminal GATA1 (Santa Cruz Biotechnology). Slides were Materials and Methods washed and incubated with 2 ␮g͞ml Alexa Fluor 546-conjugated ENU Mutagenesis. Male BALB͞c mice were injected with 200–400 anti-rabbit antibody (Invitrogen, Carlsbad, CA) and 1 ␮g͞ml mg͞kg ENU (Sigma, St. Louis, MO) and then mated to un- Cy5-conjugated anti-rat antibodies (Jackson ImmunoResearch, ␮ ͞ treated BALB͞c females to produce G1 offspring. At 7 weeks of West Grove, PA) for 1 h then washed, blocked with 1 g ml age G1 mice were bled and platelet numbers were determined by purified rat IgG2a (Becton Dickinson), and incubated with 1 using an Advia 120 automated hematological analyzer (Bayer, ␮g͞ml FITC-conjugated anti-CD41 antibody (Becton Dickin- Tarrytown, NY). son) and 0.5 ␮g͞ml DAPI (Sigma) for 30 min. Purified rabbit IgG (2 ␮g͞ml; Zymed, San Francisco, CA), rat IgG2a (1 ␮g͞ml; DNA Sequencing and Genotyping. Genomic DNA was isolated from Becton Dickinson), and FITC-conjugated rat IgG1 (1 ␮g͞ml; Gata-1Plt13/ϩ mice, and exonic regions were amplified by PCR in Becton Dickinson) were used as isotype control primary anti-

14150 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606439103 Majewski et al. Downloaded by guest on September 30, 2021 bodies. Images were captured with a TCS-SP2 confocal micro- sections were stained with both methanolic uranyl acetate and scope (Leica, Bannockburn, IL). lead citrate before viewing in a transmission electron microscope at 60 kV (Hitachi, Tokyo, Japan). Images were collected using Electron Microscopy. After perfusion of mice with 2% glutaral- a Megaview-III CCD camera (Soft Imaging Systems, Mu¨nster, dehyde, 2.5% paraformaldehyde, 0.1 M sodium cacodylate, and Germany). 2 mM calcium chloride (pH 7.4), femurs were immersion-fixed for a further 4 h before rinsing in PBS and decalcifying in 270 We thank Erin Salt, Marc Sacco, Kelly Trueman, and Katya Gray for mM EDTA (pH 7.4) for 7 days at 4°C. Bones were washed three excellent animal husbandry and the staff of the microscopy core at the times in PBS͞5% sucrose for 15 min then postfixed in 2% Peter MacCallum Cancer Centre for technical assistance. This work was osmium tetroxide in PBS for 1 h and rinsed in distilled water supported by The Cancer Council Victoria and the Australian National before dehydration through graded concentrations of ethanol Health and Medical Research Council (Program Grant 257500). I.J.M. before infiltration and embedding in Spurr’s resin. Ultra thin is supported by an Australian Postgraduate Award.

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