Normal GATA1 Research Articles

Difference in megakaryocyte expression of GATA-1, IL-6, and IL-8 associated with maintenance of platelet counts in patients with plasma cell neoplasm with dysmegakaryopoiesis

Dysmegakaryopoiesis is a diagnostic criterion for myelodysplastic syndrome (MDS), which accompanies thrombocytopenia. Recently, dysmegakaryopoiesis was reported in patients with plasma cell neoplasm (PCN). Although these patients maintained normal platelet counts, disease progressed, with most bone marrow cells being replaced by plasma cells. Several studies reported that dysmegakaryopoiesis was induced by inflammatory mechanisms; however, the exact mechanism underlying dysmegakaryopoiesis remains unknown. This study aimed to investigate whether changes in the megakaryocytic expression of GATA-1, pro-inflammatory cytokines (interleukin [IL]-6 and IL-8), and CD9 affect platelet counts and dysmegakaryopoiesis in patients with PCN and MDS. A total 114 patients were examined and categorized into four groups: MDS with dysmegakaryopoiesis (34 patients), PCN without dysmegakaryopoiesis (36 patients), PCN with dysmegakaryopoiesis (19 patients), and lymphoma without bone marrow infiltration (25 patients). Expression of GATA-1, IL-6, IL-8, and CD9 in megakaryocytes was assessed by immunohistochemical (IHC) staining of paraffin-embedded bone marrow sections. Localized expression of transcription factor and cytokines was observed in megakaryocytes. Furthermore, expression of GATA-1, IL-6, and IL-8 significantly differed (all p values < 0.05). Decreased GATA-1 expression was identified in MDS. Decreased IL-6 expression was observed in PCN and MDS. Moreover, decreased IL-8 expression was associated with dysmegakaryopoiesis, regardless of whether platelet counts were maintained. In conclusion, PCN patients with dysmegakaryopoiesis had normal platelet counts, and their megakaryocytes showed decreased IL-6 and IL-8 expression and normal GATA-1 expression. The differences in the megakaryocytic expression of cytokines in PCN and MDS with dysmegakaryopoiesis may be applicable to future therapeutic strategies.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

Elucidating the mechanisms underlying GATA-1 activity

The fact that GATA-1 binds to gene regulatory elements that contain GATA sites, and thereby ultimately regulates the expression of hundreds of genes involved in hematopoietic development is well established. However, at a molecular level, the steps between DNA binding and changes in the expression levels of target genes are only partly understood, and the goal of our work is understand at a mechanistic level the changes in chromatin structure and occupancy and the protein interactions that are made to drive these changes in gene expression. We have recently shown that, surprisingly, the localization of GATA1 to its target sites in chromatin is dependent on acetylation of specific lysines in the protein, and have elucidated the structural basis for this requirement [1,2]. Our data point towards a model for gene regulation in which not only are the post-translational modifications of histones essential for establishing gene expression patterns, but equivalent modifications in DNA-binding transcription factors are also an integral part of whatever ‘code’ underlies these patterns. Concurrently, we have also determined the molecular details underlying the recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex to GATA1-dependent gene promoters [3], an event that is essential for normal GATA1 activity. Combined with data that explore the molecular makeup of the NuRD complex, these results provide a glimpse into the mechanisms through which complex coregulator complexes are recruited to target genes and begin to map out the molecular events that drive gene regulation both in erythropoiesis and, by extension, in a range of other tissues.

Linking Transcription Factor Pathways to Disease-Causing GATA1 Mutations

Abstract 2371Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. Seven such mutations are reported. All of these give rise to amino acid substitutions within the amino terminal zinc finger (NF) of GATA1, producing a range of clinical phenotypes. Thus, V205M, G208R, and D218Y cause severe anemia and thrombocytopenia; G208S, R216Q, and D218G cause thrombocytopenia with minimal anemia; R216W gives rise to thrombocytopenia and congenital erythropoietic porphyria. One of these mutations, R216Q, occurs at the DNA binding interface and alters the ability of GATA1 to recognize a subset of cis motifs in vitro. Other mutations, including V205M, G208S, D218G, and D218Y, occur outside the DNA binding domain of the NF and inhibit interactions with the GATA1 cofactor FOG1 as determined by in vitro binding assays. However, these two mechanisms do not easily explain the broad spectrum of phenotypes associated with the mutations. For example, how do two substitutions of the same residue bring about disparate phenotypes?We examined the effects of each mutation on erythroid maturation, lineage-specific gene expression, in vivo target gene occupancy, and cofactor recruitment by introducing altered forms of GATA1 into murine GATA1-null proerythroblasts. The V205M, G208R, and D218Y mutations severely impaired erythroid maturation, recapitulating patient phenotypes. The G208S mutation also severely impaired erythroid maturation, causing a more pronounced defect than that expected from the clinical presentation. In contrast, R216Q and D218G produced mild effects in erythroid cells consistent with patient phenotypes. The porphyria-associated mutation R216W also produced relatively subtle effects in erythroid cells. We note that among the mutants, failure to activate gene expression strongly correlated with failure to repress gene expression. ChIP assays revealed that the V205M, G208R, and D218Y mutations impaired GATA1 target site occupancy. This indicates that despite normal DNA binding in vitro, the association with cofactor complexes is required for stable binding to chromatinized target sites in vivo. In contrast, the G208S mutant exhibited relatively normal chromatin occupancy, but reduced recruitment of FOG1 and SCL/Tal1 to GATA1-bound sites at erythroid genes. D218G also perturbed cofactor recruitment without greatly affecting GATA1 binding to its target genes. Notably, this mutation diminished SCL/Tal1 recruitment without significantly altering FOG1 occupancy. This implicates the SCL/Tal1 transcription complex in the pathogenesis of disorders caused by certain GATA1 mutations. Moreover, by uncoupling GATA1 chromatin occupancy and cofactor recruitment, G208S and D218G offer potentially useful tools for unraveling site-specific mechanisms of GATA1-regulated gene expression. Finally, both the R216Q and R216W mutants displayed relatively normal GATA1 chromatin occupancy and FOG1 and SCL/Tal1 recruitment at most sites. R216W presents as porphyria, and selective defects in the regulation of heme biosynthetic genes have yet be uncovered. Given that R216Q presents as thrombocytopenia, defects caused by this mutation may be revealed only in the context of megakaryocytes. Studies using similar rescue assays of a GATA1-null megakaryocyte-erythroid progenitor line are underway and will be discussed.In concert, our results reveal that in vivo analysis of GATA1 in its native environment provides mechanistic insights not obtainable from in vitro studies. Moreover, they demonstrate the usefulness of gene complementation assays for the dissection of transcription pathways surrounding normal and altered GATA1 to improve our understanding of disease. Disclosures:No relevant conflicts of interest to declare.

Development of Acute Megakaryoblastic Leukemia in Down Syndrome Is Associated with Sequential Epigenetic Changes That Target the Down Syndrome Critical Region on Chromosome 21,

Abstract 3451Down syndrome (DS) is associated with an increased frequency of Acute Megakaryoblastic Leukemias (AMKL). In the context of DS, AMKL is usually preceded by the development of a transient myeloproliferative disorder (TMD). Up to 10% of DS children may develop this disorder, and approximately 20–30% of those with TMD go on to develop AMKL, which may or may not be preceded by a return to normal hematopoiesis. While trisomy 21 (tri21) alone is not sufficient to develop AMKL, it is clear that the acquisition of somatic mutations in GATA1 is a requirement for the development of DS-AMKL. Much has been studied in relation with this genetic mutation, however not much is known about the epigenetic changes accompanying the development of AMKL in DS patients. Given the step-wise nature of the development of DS-AMKL, we hypothesized that distinct and cumulative epigenetic changes would be associated with each of the phases of the disease. In order to address this hypothesis we performed DNA methylation profiling of a cohort of 7 primary DS-AMKL samples using the latest design of the HELP microarray assay, which covers ∼230,000 CpG sites at ∼22,000 RefSeq promoter regions. In order to better understand how aberrant epigenetic patterns become established during the development of this disease we compared these DNA methylation profiles to those of mononuclear cells from normal fetal liver (FL-MNC) (n=4), tri21 FL-MNC (n=4) and TMD blasts (n=6). We identified a set of 711 unique genes that displayed significant loss of methylation in Tri21 FL-MNC with respect to control FL-MNC (FDR < 5% and methylation delta ≥ 25%). However, when tri21 FL-MNC were compared to the GATA1s positive TMD blasts, we observed that TMD samples acquired significant hypermethylation of 346 unique genes at the same significance cutoff. A direct comparison of the genes affected by these two opposing waves of methylation changes demonstrated that different sets of genes were being targeted at each stage. Next we compared the DNA methylation profiles of TMD and DS-AMKL blasts, and found them to be virtually identical, indicating that an epigenetic imprint becomes established at the TMD phase, which is still present at the leukemic phase. Finally we compared DS-AMKL to a cohort of 8 non-DS AMKL, and observed that DS-AMKL samples display significant hypomethylation of 267 unique genes, indicating that these two diseases are not only genetically but also epigenetically distinct. In determining the location of the epigenetic changes acquired at the different stages of the disease we found that, similar to what had been previously reported for gene expression, DNA methylation changes did not localize solely to chr 21, but that these changes were distributed along all chromosomes. However, detailed analysis of chr 21 revealed that changes on this chromosome preferentially targeted a specific region on the long arm, the Down Syndrome Critical Region (DSCR). This region becomes significantly hypomethylated in Tri21 FL-MNC and remains hypomethylated through all the stages of the disease. Several groups have sought to identify candidate genes on the DSCR that may contribute to the pathogenesis of the disease. Two genes, BACH1 and CXADR, have been identified in common by two different studies as being overexpressed in DS-AMKL vs. Non-DS AMKL. Both of these genes displayed aberrant hypomethylation at their promoter regions in Tri21 FL MNC, TMD and DS-AMKL samples: such hypomethylation was absent from all the control samples. Finally, given that GATA1 mutations are a requirement for the development of DS-AMKL, we compared the different aberrant DNA methylation signatures to the normal GATA1 targets identified by ChIP sequencing experiments, and found 20–25% overlap. In summary, we have completed the first comprehensive DNA methylation study of the developmental phases of DS-AMKL and have demonstrated that a) specific epigenetic changes are associated with the different stages of the disease, b) these changes occur in ‘waves’ of sequential hypo and hypermethylation, c) aberrant DNA methylation on chr 21 preferentially targets the DSCR and results in aberrant overexpression of the associated genes, and d) epigenetic profiles of TMD and DS-AMKL are virtually identical. These observations lead us to believe that an epigenetic memory established at the TMD stage may be at least in part contributing to the development of full-blown AMKL at a later time point. Disclosures:No relevant conflicts of interest to declare.

The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish

AbstractThe nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

Trisomy 21 enhances human fetal erythro-megakaryocytic development

AbstractChildren with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes.

Clonogenic mast cell progenitors and their excess numbers in chimeric BALB/c mice with inactivated GATA-1.

In agar cultures of marrow cells from adult female BALB/c chimeric GATA-1(Plt13/+) mice, a high frequency of unusual dispersed colonies was noted. Analysis showed that these were colonies of mast cells and that mast cell colony-forming cells (progenitors) could be detected in clonal cultures of adult marrow, neonatal marrow, or fetal liver if the combined stimulus of stem cell factor and interleukin-3 was used. Mast cell progenitors were in active cell cycle and showed an extensive capacity for self-generation. Mast cell colonies both from control GATA-1(+/+) mice and GATA-1(Plt13/+) mice could generate growth factor-dependent cloned cell lines that grew for >18 months. Surprisingly, the majority of the excessive numbers of mast cell progenitors in chimeric GATA-1(Plt13/+) mice were transcribing the inactive Plt13 allele of GATA-1, suggesting that GATA-1 normally acts to restrict the emergence of committed mast cell progenitors. In sharp contrast, all eosinophil progenitors in these mice were transcribing the normal GATA-1 allele. No excess tissue mast cells were observed in GATA-1(Plt13/+) mice, suggesting that the excess mast cell progenitors in these mice might be generating mast cells with a defective in vivo proliferative or tissue homing capacity.

Mutations in GATA1 in both transient myeloproliferative disorder and acute megakaryoblastic leukemia of Down syndrome

Mutations in transcription factors often contribute to human leukemias by providing a block to normal differentiation. To determine whether mutations in the hematopoietic transcription factor GATA1 are associated with leukemia, we assayed for alterations in the GATA1 gene in bone marrow samples from patients with various subtypes of acute leukemia. Here we summarize our findings that GATA1 is mutated in the leukemic blasts of patients with Down syndrome acute megakaryoblastic leukemia (DS-AMKL). We did not find mutations in GATA1 in leukemic cells of DS patients with other types of acute leukemia, or in other patients with AMKL who did not have DS. Furthermore, we did not detect GATA1 mutations in DNAs from over 75 other patients with acute leukemia or from 21 healthy individuals. Since the GATA1 mutations were restricted to DS-AMKL, we also investigated whether GATA1 was altered in the “preleukemia” of DS, transient myeloproliferative disorder (TMD). TMD is a common myeloid disorder that affects 10% of DS newborns and evolves to AMKL in nearly 30% patients. We detected GATA1 mutations in TMD blasts from every infant examined. Together, these results demonstrate that GATA1 is likely to play a critical role in the etiology of TMD and DS-AMKL, and that mutagenesis of GATA1 represents a very early event in DS myeloid leukemogenesis. We hypothesize that disruption of normal GATA-1 function is an essential step in the initiation of megakaryoblastic leukemia in DS.

Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.