Journal articles on the topic 'GATA-1'
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1
Sartori, Daniel J., Christopher J. Wilbur, Simon Y. Long, Matthew M. Rankin, Changhong Li, Jonathan P. Bradfield, Hakon Hakonarson, Struan F. A. Grant, William T. Pu, and Jake A. Kushner. "GATA Factors Promote ER Integrity and β-Cell Survival and Contribute to Type 1 Diabetes Risk." Molecular Endocrinology 28, no. 1 (January 1, 2014): 28–39. http://dx.doi.org/10.1210/me.2013-1265.
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Abstract Pancreatic β-cell survival remains poorly understood despite decades of research. GATA transcription factors broadly regulate embryogenesis and influence survival of several cell types, but their role in adult β-cells remains undefined. To investigate the role of GATA factors in adult β-cells, we derived β-cell-inducible Gata4- and Gata6-knockout mice, along with whole-body inducible Gata4 knockouts. β-Cell Gata4 deletion modestly increased the proportion of dying β-cells in situ with ultrastructural abnormalities suggesting endoplasmic reticulum (ER) stress. Notably, glucose homeostasis was not grossly altered in Gata4- and Gata6-knockout mice, suggesting that GATA factors do not have essential roles in β-cells. Several ER stress signals were up-regulated in Gata4 and Gata6 knockouts, most notably CHOP, a known regulator of ER stress-induced apoptosis. However, ER stress signals were not elevated to levels observed after acute thapsigargin administration, suggesting that GATA deficiency only caused mild ER stress. Simultaneous deletion of Gata4 and CHOP partially restored β-cell survival. In contrast, whole-body inducible Gata4 knockouts displayed no evidence of ER stress in other GATA4-enriched tissues, such as heart. Indeed, distinct GATA transcriptional targets were differentially expressed in islets compared with heart. Such β-cell-specific findings prompted study of a large meta-analysis dataset to investigate single nucleotide polymorphisms harbored within the human GATA4 locus, revealing several variants significantly associated with type 1 diabetes mellitus. We conclude that GATA factors have important but nonessential roles to promote ER integrity and β-cell survival in a tissue-specific manner and that GATA factors likely contribute to type 1 diabetes mellitus pathogenesis.
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Crispino, John D., and Marshall S. Horwitz. "GATA factor mutations in hematologic disease." Blood 129, no. 15 (April 13, 2017): 2103–10. http://dx.doi.org/10.1182/blood-2016-09-687889.
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Abstract GATA family proteins play essential roles in development of many cell types, including hematopoietic, cardiac, and endodermal lineages. The first three factors, GATAs 1, 2, and 3, are essential for normal hematopoiesis, and their mutations are responsible for a variety of blood disorders. Acquired and inherited GATA1 mutations contribute to Diamond-Blackfan anemia, acute megakaryoblastic leukemia, transient myeloproliferative disorder, and a group of related congenital dyserythropoietic anemias with thrombocytopenia. Conversely, germ line mutations in GATA2 are associated with GATA2 deficiency syndrome, whereas acquired mutations are seen in myelodysplastic syndrome, acute myeloid leukemia, and in blast crisis transformation of chronic myeloid leukemia. The fact that mutations in these genes are commonly seen in blood disorders underscores their critical roles and highlights the need to develop targeted therapies for transcription factors. This review focuses on hematopoietic disorders that are associated with mutations in two prominent GATA family members, GATA1 and GATA2.
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Capo-chichi, Callinice D., Jennifer L. Smedberg, Malgorzata Rula, Emmanuelle Nicolas, Anthony T. Yeung, Richard F. Adamo, Andrey Frolov, Andrew K. Godwin, and Xiang-Xi Xu. "Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells." Stem Cells International 2010 (2010): 1–15. http://dx.doi.org/10.4061/2010/602068.
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Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment.Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (−/−) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (−/−) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (−/−) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages.Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.
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Dore, Louis C., Timothy M. Chlon, Zan Huang, and John Crispino. "Identification of a GATA Switch In Megakaryocytic Development." Blood 116, no. 21 (November 19, 2010): 2605. http://dx.doi.org/10.1182/blood.v116.21.2605.2605.
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Abstract Abstract 2605 GATA family transcription factors play critical roles in various mammalian developmental processes, including hematopoiesis. In particular, GATA-1 expression is necessary for proper terminal differentiation of mast cells, red blood cells, eosinophils, and megakaryocytes. GATA-2 is required for proliferation and survival of hematopoietic stem and progenitor cells, and is also expressed in erythroid precursors, mast cells, and early megakaryocytes. In developing erythrocytes, GATA-2 and GATA-1 are responsible for temporal control of a multi-factor transcriptional regulatory network that involves (a) GATA-2 positively regulating its own gene transcription, (b) GATA-2 positively regulating the expression of the Gata1 gene, (c) GATA-1 positively regulating its own gene transcription, and (d) GATA-1 negatively regulating Gata2 gene transcription. During this sequence of events, a “GATA switch” occurs, wherein GATA-1 replaces GATA-2 at canonical GATA binding sites within the regulatory regions of the Gata2 and Gata1 genes, as well as at many other genomic loci that encode genes responsible for proliferation or differentiation of erythroid progenitors. Similarly, in early megakaryocytic progenitors, GATA-2 promotes proliferation and suppresses expression of alternative-lineage genes; subsequent activation of GATA-1 precipitates terminal differentiation with concomitant downregulation of proliferative genes and activation of megakaryocyte-specific genes. The presence or role of a GATA switch in megakaryocytes has not yet been formally investigated. To address the role of the GATA switch in megakaryocytic differentiation, we performed massively parallel sequencing of chromatin immunoprecipitation (ChIP-Seq) material for GATA-2 and GATA-1 before or after GATA-1 restoration in the GATA1-null megakaryocytic progenitor cell line, G1ME. We obtained 22 million unique GATA-2 tags and 10 million unique GATA-1 tags and identified 14985 and 5102 high-confidence GATA-2 and GATA-1 binding sites, respectively. Additionally, we used 13 million tags from ChIP for H3K4me3 to identify 24909 genomic sites enriched for the presence of trimethylated lysine-4 on histone H3. Trimethylated H3K4 marks nearly half of all GATA-1 bound sites and one-third of GATA-2 bound sites. Over 40% of the sites bound by GATA-1 in differentiating G1ME cells were also bound by GATA-2 in proliferating G1ME cells, indicating that a GATA switch does indeed occur during megakaryocyte development. Coordinated analyses of these occupancy data with previously published gene expression datasets show that the lists of bound genes are significantly enriched for differentially expressed genes and the data depict a generally antagonistic relationship between GATA-2 and GATA-1. Interestingly, we find that even among genes that don't contain GATA switch sites, greater than 40% of those bound by GATA-1 were also occupied by GATA-2 at distinct sites. To further characterize the occupied loci, we surveyed the genomic regions bound by GATA-1 and GATA-2 to detect motifs enriched in the sequences surrounding the peak calls. As expected, we found that over 80% contained the canonical WGATAR binding motif. In contrast to reports of motifs enriched in GATA-1 ChIP studies in erythroid cells, we failed to observe significant enrichment of LRF binding motifs. Rather, the GATA-1 and GATA-2 bound regions in megakaryocytes are strongly enriched for motifs that match the binding sites for Ets family transcription factors. Finally, we have found that these genomic regions are indeed occupied by one or more Ets factors in proliferating G1ME cells. Together, these data establish the presence of a GATA switch in megakaryocyte development and provide novel insights into coordinated gene regulation by GATA factors and the differences between the closely related erythroid and megakaryocyte lineages. Disclosures: No relevant conflicts of interest to declare.
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Grass, Jeffrey A., Huie Jing, Shin-Il Kim, Melissa L. Martowicz, Saumen Pal, Gerd A. Blobel, and Emery H. Bresnick. "Distinct Functions of Dispersed GATA Factor Complexes at an Endogenous Gene Locus." Molecular and Cellular Biology 26, no. 19 (October 1, 2006): 7056–67. http://dx.doi.org/10.1128/mcb.01033-06.
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ABSTRACT The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a “GATA switch” abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at −77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the −77 kb region. The −77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the −77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.
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Bouchard, Marie France, Hiroaki Taniguchi, and Robert S. Viger. "Protein Kinase A-Dependent Synergism between GATA Factors and the Nuclear Receptor, Liver Receptor Homolog-1, Regulates Human Aromatase (CYP19) PII Promoter Activity in Breast Cancer Cells." Endocrinology 146, no. 11 (November 1, 2005): 4905–16. http://dx.doi.org/10.1210/en.2005-0187.
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Cancers, including that of the breast, are the result of multiple contributing factors including aberrant gene expression. Indeed, the CYP19 gene encoding P450 aromatase, the key enzyme for estrogen biosynthesis, is up-regulated in breast tumors predominantly via the cAMP-responsive gonad-type PII promoter, ultimately leading to increased intratumoral estrogen production and tumor growth. Thus, identifying the molecular factors involved in aromatase PII promoter regulation is essential for our understanding and treatment of the disease. Because we have previously shown activity of the murine aromatase PII promoter to be markedly up-regulated by GATA factors with respect to the gonads, we hypothesized that GATA factors are also key determinants of human PII promoter-driven aromatase transcription in breast tumors. We now show that GATA3 and GATA4 are indeed expressed in several breast cancer cells lines. Consistent with the cAMP dependence of the PII promoter, activation elicited by GATA3 or GATA4 alone and the striking synergism between GATA3 or GATA4 and the nuclear receptor liver receptor homolog (LRH)-1 was intimately linked to forskolin treatment or overexpression of protein kinase A (PKA) catalytic subunit. PKA-mediated phosphorylation increases the interaction between GATA3 and LRH-1 and the requirement for PKA in aromatase PII promoter stimulation involves at least three specific amino acid residues: GATA3 Ser308, GATA4 Ser261, and LRH-1 Ser469. Finally, we show that the human LRH-1 promoter is itself a target for GATA factors. Thus, taken together, our results suggest that GATA factors likely contribute to aberrant aromatase expression in breast tumors through two distinct, yet complementary mechanisms.
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Doré, Louis C., Timothy M. Chlon, Christopher D. Brown, Kevin P. White, and John D. Crispino. "Chromatin occupancy analysis reveals genome-wide GATA factor switching during hematopoiesis." Blood 119, no. 16 (April 19, 2012): 3724–33. http://dx.doi.org/10.1182/blood-2011-09-380634.
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Abstract There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.
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Muntean, Andrew G., and John D. Crispino. "Differential requirements for the activation domain and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development." Blood 106, no. 4 (August 15, 2005): 1223–31. http://dx.doi.org/10.1182/blood-2005-02-0551.
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Abstract GATA1 is mutated in patients with 2 different disorders. First, individuals with a GATA1 mutation that blocks the interaction between GATA-1 and its cofactor Friend of GATA-1 (FOG-1) suffer from dyserythropoietic anemia and thrombocytopenia. Second, children with Down syndrome who develop acute megakaryoblastic leukemia harbor mutations in GATA1 that lead to the exclusive expression of a shorter isoform named GATA-1s. To determine the effect of these patient-specific mutations on GATA-1 function, we first compared the gene expression profile between wild-type and GATA-1–deficient megakaryocytes. Next, we introduced either GATA-1s or a FOG-binding mutant (V205G) into GATA-1–deficient megakaryocytes and assessed the effect on differentiation and gene expression. Whereas GATA-1–deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s–expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies show that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1.
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Onodera, Koichi, Tohru Fujiwara, Yasushi Onishi, Ari Itoh-Nakadai, Yoko Okitsu, Noriko Fukuhara, Kenichi Ishizawa, Ritsuko Shimizu, Masayuki Yamamoto, and Hideo Harigae. "GATA-2 Regulates Dendritic Cell Differentiation." Blood 126, no. 23 (December 3, 2015): 2382. http://dx.doi.org/10.1182/blood.v126.23.2382.2382.
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Abstract (Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms. (Method) Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology). (Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP. To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells. (Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. Disclosures Fujiwara: Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.
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Hosoya-Ohmura, Sakie, Naomi Mochizuki, Mikiko Suzuki, Osamu Ohneda, Kinuko Ohneda, and Masayuki Yamamoto. "GATA-4 Incompletely Substitutes for GATA-1 in Promoting Both Primitive and Definitive Erythropoiesis in Vivo." Journal of Biological Chemistry 281, no. 43 (August 30, 2006): 32820–30. http://dx.doi.org/10.1074/jbc.m605735200.
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Vertebrate GATA transcription factors have been classified into two subgroups; GATA-1, GATA-2, and GATA-3 are expressed in hematopoietic cells, whereas GATA-4, GATA-5, and GATA-6 are expressed in mesoendoderm-derived tissues. We previously discovered that expression of GATA-2 or GATA-3 under the transcriptional control for the Gata1 gene eliminates lethal anemia in Gata1 germ line mutant mice (Gata1.05/Y). Here, we show that the GATA-4 expression by the same regulatory cassette prolongs the life span of Gata1.05/Y embryos from embryonic day 12.5 to 15.5 but fails to abrogate its embryonic lethality. Gata1.05/Y mice bearing the GATA-4 transgene showed impaired maturation of both primitive and definitive erythroid cells and defective erythroid cell expansion in fetal liver. Moreover, the incidence of apoptosis was observed prominently in primitive erythroid cells. In contrast, a GATA-4-GATA-1 chimeric protein prepared by linking the N-terminal region of GATA-4 to the C-terminal region of GATA-1 significantly promoted the differentiation and survival of primitive erythroid cells, although this protein is still insufficient for rescuing Gata1.05/Y embryos from lethal anemia. These data thus show a functional incompatibility between hematopoietic and endodermal GATA factors in vivo and provide evidence indicating specific roles of the C-terminal region of GATA-1 in primitive erythropoiesis.
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Lo, Ann, Weiming Zheng, Yimei Gong, John R. Crochet, and Lisa M. Halvorson. "GATA transcription factors regulate LHβ gene expression." Journal of Molecular Endocrinology 47, no. 1 (May 13, 2011): 45–58. http://dx.doi.org/10.1530/jme-10-0137.
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The GATA family of transcription factors are critical determinants of cell differentiation as well as regulation of adult gene expression throughout the reproductive axis. Within the anterior pituitary gland, GATA factors have been shown to increase glycoprotein α-subunit gene promoter activity; however, nothing has been known about the impact of these factors on expression of the gonadotropin β-subunits. In this study, we demonstrate expression of both GATA2 and GATA4 in primary mouse gonadotropes and the gonadotrope cell line, LβT2. Based on the transient transfection in fibroblast cells, GATA factors increase LH β-subunit gene (LHβ) promoter activity alone and in synergy with the orphan nuclear receptors steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1). The GATA response was localized to a DNA regulatory region at position −101 in the ratLHβgene promoter which overlaps with a previously described cis-element for pituitary homeobox-1 (Pitx1) and is flanked by two SF-1/LRH-1 regulatory sites. As determined by gel shift, GATA and Pitx1 can compete for binding to this element. Furthermore, mutation analysis revealed a requirement for both the GATA/Pitx1 and the SF-1/LRH-1 cis-elements in order to achieve synergy. These studies identify a novel role for GATA transcription factors in the pituitary and reveal additional molecular mechanisms by which precise modulation ofLHβgene expression can be achieved.
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Burda, Pavel, Nikola Curik, Juraj Kokavec, Dana Mikulenkova, Arthur Skoultchi, Jiri Zavadil, and Tomas Stopka. "PU.1 Relieves Its GATA-1-Mediated Repression near Cebpa and Cbfb During Transdifferentiation of Murine Erythroleukemia - Tool of Inducing Leukemic Blasts to Differentiate." Blood 114, no. 22 (November 20, 2009): 547. http://dx.doi.org/10.1182/blood.v114.22.547.547.
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Abstract Abstract 547 Transcription factors GATA-1 and PU.1 interact on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that co-express GATA-1 and PU.1 are blocked at the blast stage but respond to down-regulation of PU.1 or up-regulation of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode two key hematopoietic transcription factors. Inhibition of GATA-1 by siRNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb co-occupied by PU.1 and GATA-1 in the leukemic blasts. Substantial derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we demonstrate that either activation of PU.1 or inhibition of GATA-1 efficiently reverse the transcriptional block imposed by GATA-1 and lead to activation of a myeloid transcriptional program directed by PU.1. The mechanism of PU.1 and GATA-1 in leukemic state and upon leukemia differentiation involves the following putative steps: at myeloid genes such as Cebpa, PU.1 binds directly to DNA but is repressed by GATA1 that binds directly to PU.1 molecules on DNA. Activation of PU.1-ER and stable levels of GATA-1 create excess of availabel PU.1, which is not paired by availabel GATA-1 on DNA, allowing thus gene activation. Similarly, on erythroid genes such as Nfe2, GATA1 is bound to DNA, but is repressed by PU.1 that binds to this GATA-1 molecule. Activation of GATA1-ER creates an excess of availabel GATA-1 which is not paired on DNA by availabel PU.1, also allowing gene activation. Our mechanistic study implicates that transcription factor manipulation, such as inhibition of GATA-1 or activation of PU.1 in erythroleukemias, may represent an efficient tool of inducing leukemic blasts to differentiate. (Grants NR9021-4, 10310-3, 2B06077) Disclosures: No relevant conflicts of interest to declare.
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Stachura, David L., Stella T. Chou, and Mitchell J. Weiss. "An Early Block to Erythro-Megakaryocytic Development Conferred by Loss of Transcription Factor GATA-1." Blood 106, no. 11 (November 16, 2005): 1732. http://dx.doi.org/10.1182/blood.v106.11.1732.1732.
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Abstract Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 is required for the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythro-megakaryocytic development. To investigate these functions further, we studied hematopoietic differentiation of Gata1− murine embryonic stem cells on OP9 stroma with the cytokine thrombopoietin (TPO) present. Initially, the Gata1− cultures generated a wave of mutant megakaryocytes, but these were rapidly overgrown by a unique population of TPO-dependent blasts that continued to proliferate for more than 6 months in culture. These immature Gata1− cells arose reproducibly in culture without growth lag or crisis, indicating that they derive directly from loss of GATA-1 and not from random genetic events acquired during cell culture. The cells express transcription factors GATA-2, FOG-1 and PU.1 and exhibit the surface phenotype Lin−, Sca1−, IL7R−, CD41+, cKit+, CD9+, and GPIblow. Importantly, upon restoration of GATA-1 function, these cells undergo both erythroid and megakaryocytic differentiation, as assessed by morphology, ultrastructural analysis and the induction of lineage-specific markers. Clonal analysis shows that individual cells maintain the capacity for erythro-megakaryocytic differentiation. Hence, we term this unique population G1ME for Gata1− -Megakaryocyte-Erythroid. To determine if G1ME cells are present in vivo, we analyzed E13.5 fetal livers of Gata1−/Gata1wild-type chimeric embryos. Flow cytometry analysis demonstrates an expanded population of cells expressing the G1ME surface phenotype. Individual cells within this population also exhibit TPO-dependency, extensive proliferative capacity and GATA-1-dependent biphenotypic erythro-megakaryocytic maturation in vitro. Our findings indicate that the loss of GATA-1 impairs the maturation of a specific megakaryocyte-erythroid progenitor. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia (AMKL), a clonal malignancy with features of both erythroid and megakaryocyte maturation.
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Hong, Wei, Hongxin Wang, Yuhuan Wang, John K. Choi, Suresh G. Shelat, Mortimer Poncz, and Gerd A. Blobel. "The GATA-1/FOG-1/NuRD Axis Is Essential for Normal Hematopoiesis." Blood 110, no. 11 (November 16, 2007): 375. http://dx.doi.org/10.1182/blood.v110.11.375.375.
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Abstract GATA-1 controls the development of erythroid cells and megakaryocytes through its ability to activate and repress gene transcription. GATA-1 binds many nuclear proteins, but only a few of these associations have been examined in vivo. One important example is FOG-1, a critical cofactor that contributes to both gene activation and repression by GATA-1. Loss of FOG-1 generally phenocopies GATA-1 deficiency, impairing both erythroid and megakaryocytic differentiation. We reported previously that FOG-1 directly binds the NuRD protein complex, which contains histone deacetylase and chromatin remodeling activities. This provides one mechanism for GATA-1/FOG-1-mediated gene repression. Accordingly, ChIP profiling of the NuRD proteins MTA-2, RbAp46 and Mi-2β revealed the presence of these molecules at the Kit and Gata2 genes both of which are directly repressed by GATA-1 in a FOG-1-dependent manner. NuRD proteins were spread broadly across the Kit and Gata2 genes but were further enriched at sites occupied by GATA-1 and FOG-1 in vivo. Unexpectedly, we also observed NuRD components at GATA-1-activated genes including β-globin and Ahsp. Moreover, the ability of FOG-1 to augment GATA-1-induced transcription in transient transfection assays required NuRD binding. Hence, NuRD may be bi-functional, contributing to either gene activation or repression, depending on the transcriptional and cellular context. To study the role of the FOG-1/NuRD interaction in vivo we generated mice bearing missense mutations in the Fog-1(Zfpm1) gene that disrupt NuRD binding in the FOG-1 protein. Homozygous mutant mice are born at reduced Mendelian ratios. Surviving animals display ineffective erythropoiesis marked by splenomegaly and impaired erythroid maturation. In addition, homozygous mutant animals display macrothrombocytopenia with impaired platelet function. Thus, recruitment of NuRD by GATA-1 and FOG-1 is essential for both erythropoiesis and megakaryocytopoiesis. Ongoing studies include further phenotypic analysis of the mutant mice, including comparative gene expression analysis in stage-matched wild-type and mutant erythroid cells to identify critical NuRD-dependent GATA target genes, and to resolve whether NuRD is essential for both activation and repression by GATA-1 and FOG-1 in vivo. An important open question under investigation is how recruitment of the NURD complex can lead to suppression of some genes and the enhanced expression of others. The FOG-1/NuRD mutant mice provide useful tools to dissect transcription pathways initiated by GATA-1. Moreover, given the role of GATA-1 mutations in congenital anemias and megakaryoblastic leukemias, enzymatic components of the NuRD complex may provide novel targets for pharmacologic manipulation to treat these disorders.
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Chou, Stella T., Eugene Khandros, L. Charles Bailey, Kim E. Nichols, Christopher R. Vakoc, Yu Yao, Zan Huang, et al. "Graded repression of PU.1/Sfpi1 gene transcription by GATA factors regulates hematopoietic cell fate." Blood 114, no. 5 (July 30, 2009): 983–94. http://dx.doi.org/10.1182/blood-2009-03-207944.
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GATA-1 and PU.1 are essential hematopoietic transcription factors that control erythromegakaryocytic and myelolymphoid differentiation, respectively. These proteins antagonize each other through direct physical interaction to repress alternate lineage programs. We used immortalized Gata1− erythromegakaryocytic progenitor cells to study how PU.1/Sfpi1 expression is regulated by GATA-1 and GATA-2, a related factor that is normally expressed at earlier stages of hematopoiesis. Both GATA factors bind the PU.1/Sfpi1 gene at 2 highly conserved regions. In the absence of GATA-1, GATA-2 binding is associated with an undifferentiated state, intermediate level PU.1/Sfpi1 expression, and low-level expression of its downstream myeloid target genes. Restoration of GATA-1 function induces erythromegakaryocytic differentiation. Concomitantly, GATA-1 replaces GATA-2 at the PU.1/Sfpi1 locus and PU.1/Sfpi1 expression is extinguished. In contrast, when GATA-1 is not present, shRNA knockdown of GATA-2 increases PU.1/Sfpi1 expression by 3-fold and reprograms the cells to become macrophages. Our findings indicate that GATA factors act sequentially to regulate lineage determination during hematopoiesis, in part by exerting variable repressive effects at the PU.1/Sfpi1 locus.
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Takahashi, Satoru, Ritsuko Shimizu, Naruyoshi Suwabe, Takashi Kuroha, Keigyou Yoh, Jun Ohta, Shigeko Nishimura, Kim-Chew Lim, James Douglas Engel, and Masayuki Yamamoto. "GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies." Blood 96, no. 3 (August 1, 2000): 910–16. http://dx.doi.org/10.1182/blood.v96.3.910.
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Abstract GATA-1 germline mutation in mice results in embryonic lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether the obligate presence of GATA-1 in erythroid cells is due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor complementary DNAs (cDNAs) placed under the transcriptional control of the GATA-1gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1–deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryonic lethal phenotype of the GATA-1 mutation. However, adult mice rescued with the latter transgenes developed anemia, while GATA-1 transgenic mice did not. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis. The results also show that there are biochemical distinctions among the hematopoietic GATA proteins and that during adult hematopoiesis the hematopoietic GATA factors are not functionally equivalent.
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Takahashi, Satoru, Ritsuko Shimizu, Naruyoshi Suwabe, Takashi Kuroha, Keigyou Yoh, Jun Ohta, Shigeko Nishimura, Kim-Chew Lim, James Douglas Engel, and Masayuki Yamamoto. "GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies." Blood 96, no. 3 (August 1, 2000): 910–16. http://dx.doi.org/10.1182/blood.v96.3.910.015k29_910_916.
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GATA-1 germline mutation in mice results in embryonic lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether the obligate presence of GATA-1 in erythroid cells is due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor complementary DNAs (cDNAs) placed under the transcriptional control of the GATA-1gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1–deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryonic lethal phenotype of the GATA-1 mutation. However, adult mice rescued with the latter transgenes developed anemia, while GATA-1 transgenic mice did not. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis. The results also show that there are biochemical distinctions among the hematopoietic GATA proteins and that during adult hematopoiesis the hematopoietic GATA factors are not functionally equivalent.
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Stachura, David L., Stella T. Chou, and Mitchell J. Weiss. "Early block to erythromegakaryocytic development conferred by loss of transcription factor GATA-1." Blood 107, no. 1 (January 1, 2006): 87–97. http://dx.doi.org/10.1182/blood-2005-07-2740.
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Abstract Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 drives the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythromegakaryocytic differentiation. To investigate these functions further, we stimulated Gata1- murine embryonic stem-cell-derived hematopoietic cultures with thrombopoietin, a multistage cytokine. Initially, the cultures generated a wave of mutant megakaryocytes. However, these were rapidly overgrown by a unique population of thrombopoietin-dependent blasts that express immature markers and proliferate indefinitely. Importantly, on restoration of GATA-1 function, these cells differentiated into both erythroid and megakaryocytic lineages, suggesting that they represent bipotential progenitors. Identical cells are also present in vivo, as indicated by flow cytometry and culture analysis of fetal livers from Gata1- chimeric mice. Our findings indicate that loss of GATA-1 impairs the maturation of megakaryocyte-erythroid progenitors. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia, a clonal malignancy with features of both erythroid and megakaryocyte maturation.
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Katsumura, Koichi Ricardo, Irene M. Ong, Andrew W. DeVilbiss, Rajendran Sanalkumar, and Emery H. Bresnick. "GATA Factor-Dependent Positive-Feedback Circuit Controls Acute Myeloid Leukemia Cell Proliferation: Mechanisms and Targets for Therapeutic Intervention." Blood 128, no. 22 (December 2, 2016): 1706. http://dx.doi.org/10.1182/blood.v128.22.1706.1706.
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Abstract Recent studies have established a relationship between GATA-2, a master regulator of hematopoiesis, and acute myeloid leukemia (AML). Loss-of-function mutations and elevated GATA2 expression are implicated in AML. While inhibitory GATA2 mutations occur in patients with primary immunodeficiency, which progresses to AML, GATA2 can be overexpressed in AML patients with poor prognosis. Many questions remain unanswered regarding mechanisms by which GATA2 deregulation results in AML. We reported that the Ras-p38α pathway induces multi-site GATA-2 phosphorylation in proerythroblasts, and Ser192 is required for the phosphorylation. Because RAS is mutated in 20% of AML patients, and elevated GATA2 expression correlates with poor prognosis, we tested whether this pathway functions in AML. GATA-2 was phosphorylated in the steady-state in AML cell lines, and oncogenic Ras(G12V) expression induced GATA-2 hyperphosphorylation in a p38/ERK-dependent manner. GATA-2 contains a sequence that conforms to the MAPK docking site termed "DEF motif". Mutation of S192 or the DEF motif abrogated Ras(G12V)-mediated GATA-2 hyperphosphorylation. We analyzed function using a Mouse Aortic Endothelial (MAE) cell line, in which GATA-2 and Ras(G12V) synergistically induce expression of specific GATA-2 target genes, e.g. Hdc. The DEF mutation abrogated GATA-2 hyperphosphorylation by Ras(G12V) and attenuated GATA-2 activity to induce Hdc expression in the presence of Ras(G12V) in MAE cells (50% of control, p < 0.05). GATA-2 hyperphosphorylation was also induced by expression of constitutively-active (ca) p38α and caMEK-1. caMEK-1-induced hyperphosphorylation was suppressed by the p38 inhibitor, SB203580 (p < 0.05) while the MEK inhibitor U0126 did not influence cap38α-induced hyperphosphorylation. The phosphatase inhibitor okadaic acid induced GATA-2 hyperphosphorylation, and p38 was more important than ERK in this context. These data support a model in which a hierarchical network of kinases controls GATA-2 phosphorylation and function in AML. Phospho-proteomics is being used to establish interconnectivity between network components and temporal aspects of the multi-step signaling mechanism. ChIP-Seq analysis in Kasumi-3 AML cells revealed GATA-2 occupancy at IL1B and CXCL2. IL1B and CXCR2, encoding the CXCL2 receptor, are implicated in AML. GATA-2 downregulation decreased expression of these genes (IL1B: 81.5% decrease, p < 0.001, CXCL2: 73% decrease, p < 0.001). Inhibition of ERK or p38 attenuated GATA-2 phosphorylation, decreased GATA-2 occupancy at GATA2, IL1B, and CXCL2, and lowered mRNA expression of these genes. To determine the consequences of the reduced occupancy, FAIRE was used to quantitate accessibility at the occupancy sites. Under conditions of reduced GATA-2 occupancy, accessibility decreased 50-65% at the GATA2 -77 enhancer and GATA-2 occupancy sites of IL1B and CXCL2 (p < 0.05). IL-1β and CXCL2 stimulated GATA-2 phosphorylation via p38 and ERK activation in Kasumi-1 and primary AML cells and increased GATA-2 chromatin occupancy and target gene expression. These results reveal a Ras-p38/ERK-GATA-2-IL-1β/CXCL2 axis that constitutes a positive-feedback circuit. We tested whether this circuit impacts Kasumi-1 cell proliferation. GATA2 downregulation reduced the proliferative rate (p < 0.001), which was partially rescued with recombinant CXCL2 (p < 0.01). To determine whether the signal-dependent mechanism can be extrapolated to human AML patients, we analyzed AML datasets from TCGA and GEO. GATA2 and CXCL2 expression was elevated in AML (p < 0.01). GATA2, IL1B, and CXCL2 mRNA levels correlated in M5 AML (GATA2 vs. IL1B: p=0.0015, GATA2 vs. CXCL2: p=0.0046, IL1B vs. CXCL2: p=0.000001). Among the patients with normal karyotype AML, high GATA2 and CXCL2 expression correlated with significantly reduced survival (p=0.003). In aggregate, our results establish a Ras-p38/ERK-GATA-2-IL-1β/CXCL2 axis in AML. It is attractive to propose that the GATA-2-dependent positive-feedback circuit is deployed upon ectopic elevation of GATA-2 activity in certain AML contexts and may harbor potential therapeutic targets. Given the profound capacity of this mechanism to alter GATA-2 activity, it is crucial to consider the GATA-2 activation state, rather than mRNA or total GATA-2 protein levels in clinical scenarios. Disclosures No relevant conflicts of interest to declare.
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Chlon, Timothy M., Louis C. Dore, and John Crispino. "The Leukemic Isoform GATA-1s Is Deficient In Chromatin Occupancy: Implications for AMKL." Blood 116, no. 21 (November 19, 2010): 3643. http://dx.doi.org/10.1182/blood.v116.21.3643.3643.
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Abstract Abstract 3643 GATA-1 is a zinc finger transcription factor that regulates the differentiation of megakaryocytes and erythrocytes from the megakaryocyte-erythrocyte progenitor (MEP). Mutations in GATA1 are associated with hematologic malignancies of these two related lineages. Acquired mutations that lead to expression of the short isoform of GATA-1, termed GATA-1s, are associated with Acute Megakaryocytic Leukemia in children with Down syndrome (DS-AMKL). Moreover, inherited mutations in the N-finger of GATA1, such as V205M, cause a set of related diseases characterized by dyserythropoietic anemia and thrombocytopenia. These latter mutations disrupt recruitment of the essential cofactor FOG-1 and thus promote disease by interfering with the ability of the GATA-1:FOG complex to properly regulate gene expression. Despite the fact that V205 lies along the surface of the zinc finger opposite to DNA, previous studies suggest that FOG-1 may regulate the chromatin binding activity of GATA-1 at a subset of sites. In contrast to V205 mutation, the precise mechanisms by which GATA-1s contributes to disease is poorly understood. Previous studies have shown that GATA-1s uncouples megakaryocyte proliferation from differentiation, likely by an inability of GATA-1s to properly repress expression of a subset of GATA-1 target genes. We hypothesized that both inherited and acquired GATA1 mutations contribute to disease by interfering with not only target gene activation or repression, but also with GATA-1 chromatin binding. In order to define the chromatin binding activity of GATA-1 and its disease associated mutants, we performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-Seq) for wild-type GATA-1, GATA-1s and GATA-1V205G in the G1ME cell line. G1ME cells, which were derived from GATA-1 null ES cells, approximate an MEP in that they can differentiate into either erythroid cells (in the presence of EPO) or megakaryocytes (in the presence of TPO) upon reconstitution with GATA-1. We expressed GATA-1, GATA-1s, or GATA-1V205G in G1ME cells by retroviral transduction and subjected the cells to ChIP for GATA-1. The resulting DNA was sequenced on the Illumina GAII, yielding 11.8M, 10.4M, and 8.5M uniquely mapped reads. Analysis of the datasets using QuEST yielded 2367, 963, and 4130 peaks, respectively, with an FDR of <0.4%. A search for genes within 50kb of each peak in each data set revealed GATA occupancy of 1699 (GATA-1), 704 (GATA-1s), and 2757 (GATA-1V205G) genes. These results show that GATA-1s indeed binds significantly fewer genes in vivo. Surprisingly, these results also show that GATA-1V205G binds more genes, suggesting that loss of the GATA-1:FOG-1 interaction leads to increased promiscuity of GATA-1 binding to chromatin. Next, we used the non-biased motif finder MEME to identify specific transcription factor binding motifs in each data set. This analysis revealed the presence of canonical GATA binding sites in 82% (GATA-1), 65% (GATA-1s), and 85% (GATA-1V205G) of the peaks. Moreover, we identified Ets-family transcription factor binding motifs in 49%, 43%, and 41% of the peaks, respectively. Other motifs that were discovered at lower frequency include CACCC-box motifs and the Gfi1b binding site. DAVID pathway analysis of the three different GATA-1 datasets demonstrated that although most pathways are conserved among the three proteins, the “Acute Myeloid Leukemia” pathway was altered by the GATA-1s mutation. Among the genes in this pathway that are bound by GATA-1 but not GATA-1s are Kit, Grb2, and Sos1. Other genes that are bound by GATA-1, but not by GATA-1s, include Lmo2, Ikaros, Klf1, Ldb1, and Trp53. Taken together, our data report the novel discovery that the N-terminus of GATA-1, which has not previously been implicated in DNA binding, is essential for proper binding of GATA-1 to chromatin. In addition, we reveal that one key function of FOG-1 is to restrict binding of GATA-1 to a subset of loci in vivo. Future studies will focus on defining the role of the N-terminus in chromatin binding and on determining how FOG- modulates occupancy in vivo. Given that loss of the N-terminus is an essential step in leukemogenesis, the identification of GATA sites that fail to be bound by GATA-1s will provide important new insights into the mechanisms of this malignancy. Disclosures: No relevant conflicts of interest to declare.
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Huang, Junbin, Lifeng Huang, Yingsi Lu, Chengming Zhu, and Chun Chen. "Identification of a Novel Indel Mutation in GATA-1 Gene In Vivo and Verified Its Defective Function in Vitro." Blood 134, Supplement_1 (November 13, 2019): 4801. http://dx.doi.org/10.1182/blood-2019-126762.
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GATA-1 is a zinc finger TF encoded by the GATA-1 gene located on the short-arm of the X-chromosome. Most of GATA-1 mutations were located in exon2 or 3. We describe a male infant presented with dyserythropoietic anemia, who is harbored a novel GG deletion of exon6 in GATA-1 gene and varified its disrupted function in vitro. We confirmed that: (1) by using an unique GATA-1 antibody, which corresponded to amino acids 394-413 of human GATA1, the mutation of the proband led to GATA-1 protein and mRNA defective expressions in both peripheral blood and bone marrow; (2) demonstrated the expression of the GATA-1 mutated form is restricted to erythroblasts and red blood cells, consisted with the proband's abnormal erythropoiesis; (3) temporary transfection of GATA-1-wt(wild type) and GATA-1-indel in Hela cell line, which has no expression of GATA-1 originally, resulted in normal and defective expression both in protein and mRNA leve, respectively; (4) in a manipulated K562 cell line, that its GATA-1 had been knocked down by shRNA, stable transfection GATA-1-indel can not rescue K562 from GATA-1 deficiency.. Our reseach provide new insights into the clinically relevant in vivo function of the C-terminal domain of GATA-1 in human hematopoiesis and the results were validated in two seprerated cell line. Disclosures No relevant conflicts of interest to declare.
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Burda, Pavel, Nikola Curik, Juraj Kokavec, Vit Pospisil, Arthur I. Skoultchi, Jiri Zavadil, and Tomas Stopka. "Fog1 and Cebpa Are DNA Targets of GATA-1/PU.1 Antagonism during Leukemia Differentiation." Blood 110, no. 11 (November 16, 2007): 4121. http://dx.doi.org/10.1182/blood.v110.11.4121.4121.
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Abstract PU.1 and GATA1 are hematopoietic lineage-specific transcription factors that play key roles in normal myeloid and erythroid differentiation respectively. Inappropriate expression of PU.1 in proerythroblasts causes its binding to GATA-1 on DNA resulting in a block of erythroid differentiation and development of murine erythroleukemia (MEL) (Rekhtman 1999). Activation of a conditional transgene of GATA-1 (fused with the ligand binding domain of the estrogen receptor, ER) in MEL cells disrupts PU.1-mediated repression in chromatin leading to re-intiation of erythroid differentiation and cell cycle arrest (Choe 2003, Stopka 2005). In this study we show that MEL cells can also be induced to express myeloid differentiation programs upon PU.1-ER activation. Gene expression microarray analysis of GATA-1-ER MEL cells and PU.1-ER MEL cells treated with ER activators allowed us to identify mRNAs that are regulated by both GATA-1 and PU.1 including the set of GATA-1 targets repressed by PU.1, as well as the set of PU.1 targets repressed by GATA-1 in MEL cells. The targets of mutual antagonism of PU.1 and GATA-1 consisted of lineage specific transcription factors, differentiation markers and genes that cause cell cycle arrest and antiapoptotic regulators previously associated with myeloid and erythroid cell differentiation. To determine if PU.1 and GATA-1 directly regulate the lineage specific transcription factor genes, we performed chromatin immunoprecipitation (ChIP) and analyzed the ChIP samples on microarrays and by qPCR. We found that PU.1 and GATA-1 are localized near PU.1 binding sites in the genes for myeloid transcription factors Cebpa and Cbfb and near GATA-1 binding sites in the genes for erythroid transcription factors Fog1 and Nfe2. In addition, further ChIP experiments delineated chromatin architecture near the binding sites for PU.1 and GATA-1 including histone H3 content and acetylation of histone H3K9. We propose that the mutual antagonism of PU.1 and GATA-1 in inhibiting the respective differentiation programs is rendered through specific changes in chromatin structure around lineage specific transcription factors genes. These changes may contribute to the block to differentiation evident in leukemias.
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Letting, D. L., Y. Y. Chen, C. Rakowski, S. Reedy, and G. A. Blobel. "Context-dependent regulation of GATA-1 by friend of GATA-1." Proceedings of the National Academy of Sciences 101, no. 2 (December 26, 2003): 476–81. http://dx.doi.org/10.1073/pnas.0306315101.
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Divine, Joyce K., Lora J. Staloch, Hanna Haveri, Christina M. Jacobsen, David B. Wilson, Markku Heikinheimo, and Theodore C. Simon. "GATA-4, GATA-5, and GATA-6 activate the rat liver fatty acid binding protein gene in concert with HNF-1α." American Journal of Physiology-Gastrointestinal and Liver Physiology 287, no. 5 (November 2004): G1086—G1099. http://dx.doi.org/10.1152/ajpgi.00421.2003.
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Transcriptional regulation by GATA-4, GATA-5, and GATA-6 in intestine and liver was explored using a transgene constructed from the proximal promoter of the rat liver fatty acid binding protein gene ( Fabpl). An immunohistochemical survey detected GATA-4 and GATA-6 in enterocytes, GATA-6 in hepatocytes, and GATA-5 in neither cell type in adult animals. In cell transfection assays, GATA-4 or GATA-5 but not GATA-6 activated the Fabpl transgene solely through the most proximal of three GATA binding sites in the Fabpl promoter. However, all three factors activated transgenes constructed from each Fabpl site upstream of a minimal viral promoter. GATA factors interact with hepatic nuclear factor (HNF)-1α, and the proximal Fabpl GATA site adjoins an HNF-1 site. GATA-4, GATA-5, or GATA-6 bounded to HNF-1α in solution, and all cooperated with HNF-1α to activate the Fabpl transgene. Mutagenizing all Fabpl GATA sites abrogated transgene activation by GATA factors, but GATA-4 activated the mutagenized transgene in the presence of HNF-1α. These in vitro results suggested GATA/HNF-1α interactions function in Fabpl regulation, and in vivo relevance was determined with subsequent experiments. In mice, the Fabpl transgene was active in enterocytes and hepatocytes, a transgene with mutagenized HNF-1 site was silent, and a transgene with mutagenized GATA sites had identical expression as the native transgene. Mice mosaic for biallelic Gata4 inactivation lost intestinal but not hepatic Fabpl expression in Gata4-deficient cells but not wild-type cells. These results demonstrate GATA-4 is critical for intestinal gene expression in vivo and suggest a specific GATA-4/HNF-1α physical and functional interaction in Fabpl activation.
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Weiss, Mitchell J. "Transcriptional Regulation of Erythropoiesis." Blood 114, no. 22 (November 20, 2009): SCI—7—SCI—7. http://dx.doi.org/10.1182/blood.v114.22.sci-7.sci-7.
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Abstract Abstract SCI-7 Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas. Disclosures No relevant conflicts of interest to declare.
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Chou, Stella T., Charles Bailey, Yu Yao, Zan Huang, Kim Nichols, John Crispino, Ross Hardison, Gerd Blobel, Chris Vakoc, and Mitchell J. Weiss. "Graded Repression of PU.1 Expression by GATA Factors Influence Hematopoietic Progenitor Cell Fate." Blood 112, no. 11 (November 16, 2008): 2469. http://dx.doi.org/10.1182/blood.v112.11.2469.2469.
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Abstract Hematopoietic cell fate is determined by combinatorial interactions between nuclear proteins that activate and repress transcription. This principle is illustrated by the transcription factors GATA-1 and PU.1, which promote erythro-megakaryocytic and granulocyte-macrophage development respectively. These two proteins interact physically to cross-antagonize each other’s activities, creating regulatory loops for hematopoietic differentiation. Previously, we showed that loss of GATA-1 causes expansion of bipotental megakaryocyte erythroid progenitors (MEPs) from embryonic stem cells or fetal liver derived hematopoietic progenitors. These cells, termed G1ME, for GATA-1-null megakaryocyte-erythroid, proliferate continuously in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. G1ME cells express GATA-2, a GATA-1-related protein normally found in multipotential hematopoietic progenitors and stem cells. These cells also express moderate levels of PU.1 mRNA, approximately 1/3 of that expressed in the myeloid cell line 416B. Upon retroviral restoration of GATA-1, GATA-2 is downregulated and PU.1 mRNA decreases rapidly. Microarray analysis of GATA-1-rescued G1ME cells revealed repression of PU.1 and many of its downstream target genes, raising the possibility of direct PU.1/Sfpi1 gene repression by GATA-1. Chromatin immunoprecipitation (ChIP) studies identified two GATA factor-binding sites at the PU.1/Sfpi1 locus. In the absence of GATA-1, when the PU.1/Sfpi1 gene is active, these sites are occupied by GATA-2. Retrovirally expressed GATA-1 replaces GATA-2 at these sites, repressing PU.1 transcription during concomitant erythro-megakaryocytic maturation. These findings resemble the “GATA-factor switch” described at other loci such as Gata2 and Kit where GATA-2 and GATA-1 compete for the same cis elements to activate and repress transcription respectively. To test this, we used siRNA to repress GATA-2 expression in G1ME cells by about 60%. Strikingly, this caused PU.1 to be upregulated 4-fold, indicating that GATA-2 also represses PU.1/Sfpi1, but to a lesser extent than GATA-1. Moreover, G1ME cells expressing GATA-2 siRNA differentiated into macrophages, as evidenced by morphology, expression of numerous cell-type specific markers and massive induction of macrophage specific genes including myeloperoxidase, Mac-1, and C/EBPα. Our findings illustrate two new insights into the transcriptional control of hematopoietic cell differentiation: First, cross-antagonism between GATA-1 and PU.1 not only occurs at the level of protein-protein interaction, but also through direct transcriptional repression. Second, in addition to having opposite effects on transcription of the same target gene as described previously, GATA-2 and GATA-1 can act cooperatively and successively to exert repressive effects of different magnitudes that gradually restrict gene expression during hematopoietic development. In this model, hematopoietic progenitors express GATA-2 and low levels of PU.1 that maintain the multipotential state but are not sufficient for myelopoiesis. Repression of GATA-2 in the absence of GATA-1 raises PU.1 levels to stimulate granulocyte-macrophage development. In contrast, activation of GATA-1 causes PU.1 to be fully repressed, promoting erythrocyte and megakaryocyte differentiation. Our data illustrate how lineage fate and hematopoietic differentiation are influenced by the stoichiometry between GATA-1, GATA-2, and PU.1 in multipotential progenitors.
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Zhang, Pu, Xiaobo Zhang, Atsushi Iwama, Channing Yu, Kent A. Smith, Beatrice U. Mueller, Salaija Narravula, Bruce E. Torbett, Stuart H. Orkin, and Daniel G. Tenen. "PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding." Blood 96, no. 8 (October 15, 2000): 2641–48. http://dx.doi.org/10.1182/blood.v96.8.2641.
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Abstract The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1–estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1–mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein–protein interactions. These findings contribute to understanding how protein–protein interactions participate in hematopoietic differentiation and leukemogenesis.
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Zhang, Pu, Xiaobo Zhang, Atsushi Iwama, Channing Yu, Kent A. Smith, Beatrice U. Mueller, Salaija Narravula, Bruce E. Torbett, Stuart H. Orkin, and Daniel G. Tenen. "PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding." Blood 96, no. 8 (October 15, 2000): 2641–48. http://dx.doi.org/10.1182/blood.v96.8.2641.h8002641_2641_2648.
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The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1–estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1–mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein–protein interactions. These findings contribute to understanding how protein–protein interactions participate in hematopoietic differentiation and leukemogenesis.
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Kadauke, Stephan, Janine M. Lamonica, Alan Lau, Margaret Chou, and Gerd Blobel. "A Potential Epigenetic Bookmarking Function for the Hematopoietic Transcription Factor GATA-1." Blood 116, no. 21 (November 19, 2010): 2601. http://dx.doi.org/10.1182/blood.v116.21.2601.2601.
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Abstract Abstract 2601 Hematopoietic lineage choice decisions are stably maintained through many cell divisions. For example, erythroid precursor cells undergo several rounds of cell division during their maturation. During each mitosis, most transcription factors separate from chromatin causing transcription to cease globally. Mitosis therefore poses a challenge for transcription factors to re-associate with the appropriate target sites in chromatin of newborn cells. The epigenetic mechanisms that cement lineage stability and resist cell reprogramming during mitosis are poorly understood, although recent evidence supports the idea that “bookmarking” factors that remain associated with mitotic chromatin may play a role in this process. We therefore investigated whether the hematopoietic transcription factor GATA-1 might be retained at specific sites during mitosis. GATA-1 controls the expression of essentially all erythroid-specific genes and might therefore play a role in maintaining erythroid gene expression programs throughout the cell cycle. Surprisingly, we found that while a substantial fraction of GATA-1 dissociates from chromatin in mitosis, foci of high GATA-1 density are present within mitotic chromatin. To determine the exact locations of GATA-1 binding during mitosis, we developed a method to highly purify mitotic erythroid cells in sufficient quantities for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq). These experiments revealed that a subset of sites bound by GATA-1 during interphase is occupied continuously throughout mitosis. Importantly, continuously GATA-1-occupied sites are enriched at promoters and cis-regulatory elements of genes coding for key developmental regulators of hematopoiesis (e.g., Fog1/Zfpm1, Gata2, Lyl1) but are notably absent at erythroid physiological and structural genes (e.g., Hba, Hbb, Epb4.9). To examine the importance of mitotic chromatin binding by GATA-1, we engineered a version of GATA-1 bearing a mitosis-specific degron that targets GATA-1 for degradation during mitosis but not interphase. Preliminary results show that mitotically degraded GATA-1 fails to induce differentiation when expressed in GATA-1-null erythroblasts. This suggests an important mitotic function for GATA-1. Current work focuses on delineating the mechanism by which continuous chromatin occupancy of GATA-1 throughout mitosis ensures proper erythroid differentiation. The results will be presented and discussed at the meeting. Disclosures: No relevant conflicts of interest to declare.
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Johnson, Kirby D., Meghan E. Boyer, Jeong-Ah Kang, Amittha Wickrema, Alan B. Cantor, and Emery H. Bresnick. "Friend of GATA-1–independent transcriptional repression: a novel mode of GATA-1 function." Blood 109, no. 12 (June 15, 2007): 5230–33. http://dx.doi.org/10.1182/blood-2007-02-072983.
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Abstract The GATA-1–interacting protein Friend Of GATA-1 (FOG-1) is essential for the proper transcriptional activation and repression of numerous GATA-1 target genes. Although FOG-1–independent activation by GATA-1 has been described, all known examples of GATA-1–mediated repression are FOG-1 dependent. In the GATA-1–null G1E cell line, estrogen receptor ligand binding domain (ER) chimeras of either wild-type GATA-1 or a FOG-1–binding defective mutant of GATA-1 repressed several genes similarly upon activation with β-estradiol. Repression also occurred in a FOG-1–null cell line expressing ER–GATA-1 and during ex vivo erythropoiesis. At the Lyl1 and Rgs18 loci, we found highly restricted occupancy by GATA-1 and GATA-2, indicating that these genes are direct targets of GATA factor regulation. The identification of genes repressed by GATA-1 independent of FOG-1 defines a novel mode of GATA-1–mediated transcriptional regulation.
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Stopka, Tomas, Derek F. Amanatullah, and Arthur I. Skoultchi. "PU.1 and pRb Bind GATA-1 on DNA and Recruit a Histone H3K9 Methyl Transferase-Containing Complex to Repress the Erythroid Transcription Program." Blood 104, no. 11 (November 16, 2004): 1614. http://dx.doi.org/10.1182/blood.v104.11.1614.1614.
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Abstract Current work indicates that transcriptional repression is at least as important as transcriptional activation in normal development. Inappropriate or untimely transcriptional repression in immature hematopoietic cells is often the basis for a block to differentiation in hematologic malignancies. Activation of PU.1, a myeloid and B-cell specific transcription factor, in erythroid cells plays a key role in Friend virus-induced mouse erythroleukemia (MEL). Previous results from our laboratory showed that PU.1 blocks the erythroid differentiation-promoting activity of GATA-1 by binding directly to GATA-1 on DNA and inhibiting its transcriptional function. PU.1-mediated repression of GATA-1 on transiently transfected GATA-1 target genes is dependent on the corepressor pRb that also binds to PU.1 (Rekhtman et al., Genes & Dev 1999 and Mol Cell Biol 2003). To further investigate the mechanism of PU.1-mediated repression of GATA-1 in chromatin, we examined the occupancy of several GATA-1 target genes by PU.1 and pRb, as well as the state of core histone modifications at these loci in MEL cells by quantitative chromatin immunoprecipitation. These studies included both endogenous GATA-1 target genes and an exogenous GATA-1 target gene (alpha globin) integrated at a specific locus in MEL cells by Recombinase-Mediated Cassette Exchange. We found that GATA-1 sites at both the exogenous, integrated gene as well as at endogenous genes (including the regulatory regions of the alpha globin, beta globin, alas-e, eklf, p45 nf-e2) are occupied by a GATA1 - PU.1 - pRb complex in undifferentiated MEL cells. The presence of all three components of the complex is dependent on intact GATA-1 binding sites in the exogenous, integrated gene. The histone methyltransferase Suv39H1 and the histone H3MeK9 binding protein, HP1alpha, are also present at the repressed loci. During induced differentiation of MEL cells, PU.1, pRb, Suv39H1 and HP1alpha occupancy at these sites declines but GATA-1 continues to be present at its binding sites. The disruption of the repression complex at these loci during differentiation as well as during siRNA-mediated PU.1 knock down is associated with conversion of methylated H3K9 to acetylated H3K9 and significant transcriptional derepression of these GATA-1 target genes. These findings support a model for repression of GATA-1 by PU.1 at endogenous loci through recruitment of the corepressor pRb and associated histone methyltransferase (Suv39H1) and H3MeK9 binding (HP1alpha) activities.
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Du, Jian, Dharmesh Vyas, Qing Xi, and Steven J. Ackerman. "Functional Differences in GATA-1 Affinity for Double Versus Single GATA-1 Binding Sites Dictate Synergistic Versus Antagonistic Interactions of PU.1 and GATA-1 in Myeloid Gene Transcription." Blood 104, no. 11 (November 16, 2004): 1608. http://dx.doi.org/10.1182/blood.v104.11.1608.1608.
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Abstract Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.
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Rath, Nibedita, Zhishan Wang, Min Min Lu, and Edward E. Morrisey. "LMCD1/Dyxin Is a Novel Transcriptional Cofactor That Restricts GATA6 Function by Inhibiting DNA Binding." Molecular and Cellular Biology 25, no. 20 (October 15, 2005): 8864–73. http://dx.doi.org/10.1128/mcb.25.20.8864-8873.2005.
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ABSTRACT The activity of GATA factors is regulated, in part, at the level of protein-protein interactions. LIM domain proteins, first defined by the zinc finger motifs found in the Lin11, Isl-1, and Mec-3 proteins, act as coactivators of GATA function in both hematopoietic and cardiovascular tissues. We have identified a novel GATA-LIM interaction between GATA6 and LMCD1/dyxin. The LIM domains and cysteine-rich domains in LMCD1/dyxin and the carboxy-terminal zinc finger of GATA6 mediate this interaction. Expression of LMCD1/dyxin is remarkably similar to that of GATA6, with high-level expression observed in distal airway epithelium of the lung, vascular smooth muscle, and myocardium. In contrast to other GATA-LIM protein interactions, LMCD1/dyxin represses GATA6 activation of both lung and cardiac tissue-specific promoters. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that LMCD1/dyxin represses GATA6 function by inhibiting GATA6 DNA binding. These data reveal an interaction between GATA6 and LMCD1/dyxin and demonstrate a novel mechanism through which LIM proteins can assert their role as transcriptional cofactors of GATA proteins.
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Gurbuxani, Sandeep, Paresh Vyas, and John D. Crispino. "Recent insights into the mechanisms of myeloid leukemogenesis in Down syndrome." Blood 103, no. 2 (January 15, 2004): 399–406. http://dx.doi.org/10.1182/blood-2003-05-1556.
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Abstract GATA-1 is the founding member of a transcription factor family that regulates growth and maturation of a diverse set of tissues. GATA-1 is expressed primarily in hematopoietic cells and is essential for proper development of erythroid cells, megakaryocytes, eosinophils, and mast cells. Although loss of GATA-1 leads to differentiation arrest and apoptosis of erythroid progenitors, absence of GATA-1 promotes accumulation of immature megakaryocytes. Recently, we and others have reported that mutagenesis of GATA1 is an early event in Down syndrome (DS) leukemogenesis. Acquired mutations in GATA1 were detected in the vast majority of patients with acute megakaryoblastic leukemia (DS-AMKL) and in nearly every patient with transient myeloproliferative disorder (TMD), a “preleukemia” that may be present in as many as 10% of infants with DS. Although the precise pathway by which mutagenesis of GATA1 contributes to leukemia is unknown, these findings confirm that GATA1 plays an important role in both normal and malignant hematopoiesis. Future studies to define the mechanism that results in the high frequency of GATA1 mutations in DS and the role of altered GATA1 in TMD and DS-AMKL will shed light on the multistep pathway in human leukemia and may lead to an increased understanding of why children with DS are markedly predisposed to leukemia.
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Johnson, Kirby D., Myung-Jeom Ryu, Meghan E. Boyer, Sunduz Kelez, Jing Zhang, Youngsook Lee, and Emery H. Bresnick. "A “Master Regulatory Cis-element” Governs the Hematopoietic Stem/Progenitor Cell Compartment, Vascular Integrity, and Cardiovascular Development." Blood 118, no. 21 (November 18, 2011): 1304. http://dx.doi.org/10.1182/blood.v118.21.1304.1304.
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Abstract Abstract 1304 The expression and activities of master regulatory proteins are exquisitely controlled to ensure the proper development of complex organisms. As a master regulator can orchestrate multiple developmental processes, presumably mechanisms involving a large ensemble of cis-elements and trans-acting factors are deployed to establish this control. GATA-2, a master regulator of hematopoiesis expressed in hematopoietic, endothelial, neuronal, and fat cells is likely subject to such complex regulation. GATA-2 occupies dispersed sites of the Gata2 locus, highlighting a positive autoregulatory mechanism. As GATA-1 levels rise during erythropoiesis, GATA-1 displaces GATA-2 from the Gata2 locus, instigating repression. “GATA switches” occur at five dispersed Gata2 sites (−77, −3.9, −2.8, −1.8 and +9.5 kb). Targeted deletion of GATA factor switch sites −2.8 and −1.8 revealed that these sites individually contribute to maximal Gata2 expression, but are otherwise dispensable for embryogenesis and steady-state hematopoiesis. Herein, we describe the targeted deletion of an intronic GATA switch site (+9.5) comprised of an E-box GATA factor composite cis-element. Strikingly, whereas the Gata2 gene knockout results in lethal anemia by E10.5, the +9.5 mutation yielded embryonic lethality around E13.5. The +9.5 mutation greatly reduced Gata2 expression in fetal livers, heart and endothelial cells, but not in the brain. Consequently, by E12.5 mutant mice exhibited severely reduced numbers of hematopoietic stem/progenitor cells (HSPCs) in fetal livers as assayed by cell sorting and colony assays. However, mutant mice were not overtly anemic and Ter119+ cells were abundant in the mutant fetal livers, accounting for the delay in lethality compared to the Gata2 knockout. Additionally, +9.5 mutant embryos displayed vascular/cardiovascular malformations and severe hemorrhaging. This work reveals that, unlike the −2.8 and −1.8 GATA switch sites, the +9.5 composite element functions as a “master regulatory cis-element” required for HSPC development, vascular integrity, and cardiovascular development. Genomic analyses identified a large cohort of loci containing conserved intronic composite elements resembling the +9.5 element, with heavy representation at genes critical for HSPC and vascular development/function, as well as novel genes of unknown function. Disclosures: No relevant conflicts of interest to declare.
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Thomas, Robin L., Natalie M. Crawford, Constance M. Grafer, Weiming Zheng, and Lisa M. Halvorson. "GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells." Journal of Molecular Endocrinology 51, no. 3 (September 9, 2013): 313–24. http://dx.doi.org/10.1530/jme-13-0089.
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Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.
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Yan, Bowen, Suming Huang, and Yi Qiu. "GATA-1 Deacetylation and Interaction with HDAC1 Is Critical for GATA-1 Mediated Gene Transcription." Blood 130, Suppl_1 (December 7, 2017): 945. http://dx.doi.org/10.1182/blood.v130.suppl_1.945.945.
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Abstract The transcription factor GATA-1 is an essential factor for globin gene transcrption and is required for erythroid and megakaryocytic cell differentiation and maturation. GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 chromatin binding activity. However, it is not clear whether GATA-1 acetylation can be reversed by a deacetylase. It is showed that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) associated NURD corepressor complexes through binding to FOG-1. However, we found that the NURD complex does not deacetylate GATA-1. We discovered that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for the interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) blocks the interaction of HDAC1, but doesn't affect its DNA binding in vitro. Importantly, the mutation does not affect the interaction with FOG-1, indicating that GATA-1 direct interaction and indirect association via FOG-1 with HDAC1 are separate events. To further understand the role of the HDAC1-GATA-1 direct interaction in gene transcription and erythropoiesis, we introduced the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Interestingly, upon estradiol induction, GATA-1 2RA does not promote b-globin gene transcription and erythroid differentiation of G1E cells, although GATA-1 2RA is highly acetylated. Chromatin immunoprecipitation assay (ChIP) shows that GATA-1 2RA binds poorly to HS3 and β-globin promoter. Similar binding defect is also detected on GATA-1 promoter, indicating defective GATA-1 recruitment on chromatin. Interestingly, HDAC1 binding to these regions are also significantly reduced, suggesting HDAC1-GATA-1 interaction may be important for GATA-1 deacetylation, as well as stabilizing GATA-1 binding. In order to investigate the effect of GATA-1 2RA in vivo, we generated a GATA-1 2RA knock in mice. The knock in mice are viable but suffered from anemia and thrombocytopenia. β-globin expression reduced at least 50% in knock in mice compare to wild type litter mate. To further identify and compare gene expression profiles regulated by HDAC1 direct or indirect associated GATA-1 during erythroid differentiation, we performed RNA sequencing assays to study the effects of GATA-1 2RA in gene expression in comparison with wild type GATA-1 or GATA-1-V205M (a mutation abolished binding with FOG-1). Expression of GATA-1 2RA largely affects gene expression profile in both GATA-1 activated and repressed genes compared to cells expressing wild type GATA-1. The gene expression pattern in 2RA cells also is largely different from cells expressing GATA-1 V205M, indicating the direct and indirect interaction with HDAC1 may mediate differential functions. Our results indicate that HDAC1 is required for GATA-1 recruitment and GATA-1 mediated transcription regulation. Thus, this study unveils a novel regulation of GATA-1 by its direct interaction with HDAC1. Disclosures No relevant conflicts of interest to declare.
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Shimizu, Ritsuko, Kinuko Ohneda, James Douglas Engel, Cecelia D. Trainor, and Masayuki Yamamoto. "Transgenic rescue of GATA-1–deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia." Blood 103, no. 7 (April 1, 2004): 2560–67. http://dx.doi.org/10.1182/blood-2003-07-2514.
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Abstract Association of GATA-1 and its cofactor Friend of GATA-1 (FOG-1) is essential for erythroid and megakaryocyte development. To assess functions of GATA-1–FOG-1 association during mouse development, we used the GATA-1 hematopoietic regulatory domain to generate transgenic mouse lines expressing a mutant GATA-1, which contains a substitution of glycine 205 for valine (V205G) that abrogates its association with FOG-1. We examined whether the transgenic expression of mutant GATA-1 rescues GATA-1 germ line mutants from embryonic lethality. In high-expressor lines we observed that the GATA-1V205G rescues GATA-1–deficient mice from embryonic lethality at the expected frequency, revealing that excess GATA-1V205G can eliminate the lethal anemia that is due to GATA-1 deficiency. In contrast, transgene expression comparable to the endogenous GATA-1 level resulted in much lower frequency of rescue, indicating that the GATA-1–FOG-1 association is critical for normal embryonic hematopoiesis. Rescued mice in these analyses exhibit thrombocytopenia and display dysregulated proliferation and impaired cytoplasmic maturation of megakaryocytes. Although anemia is not observed under steady-state conditions, stress erythropoiesis is attenuated in the rescued mice. Our findings reveal an indispensable role for the association of GATA-1 and FOG-1 during late-stage megakaryopoiesis and provide a unique model for X-linked thrombocytopenia with inherited GATA-1 mutation.
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Chou, Stella T., Charles L. Bailey, Ross C. Hardison, Gerd A. Blobel, Christopher R. Vakoc, and Mitchell J. Weiss. "GATA-1 Represses PU.1/Sfpi-1 Gene Transcription in Erythro-Megakaryocytic Progenitors." Blood 110, no. 11 (November 16, 2007): 1226. http://dx.doi.org/10.1182/blood.v110.11.1226.1226.
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Abstract Commitment of multipotential hematopoietic progenitors to unique cell fates is determined by the induction and interplay of specific nuclear factors that reinforce unilineage transcriptional programs. Previously, we reported that loss of transcription factor GATA-1 promotes the expansion of bipotential megakaryocyte erythroid progenitors (MEPs) from embryonic stem cell or fetal liver derived hematopoietic progenitors. These mutant cells, termed G1ME (for Gata-1− Megakaryocyte-Erythroid), proliferate in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. To evaluate gene regulation at the MEP stage of hematopoiesis, we performed microarray analysis of G1ME cells before and after GATA-1-induced differentiation. Expression of GATA-1 in G1ME cells induced numerous erythroid and megakaryocytic target genes. In addition, undifferentiated G1ME cells expressed numerous granulocyte and macrophage genes, suggesting that loss of GATA-1 derepresses a myeloid program in MEPs. Myeloid genes were rapidly inhibited upon expression of GATA-1. In particular, mRNA encoding the myeloid transcription factor PU.1 and more than thirty of its downstream targets were repressed by GATA-1. Chromatin immunoprecipitation (ChIP) showed that GATA-1 bound directly to the PU.1 gene (Sfpi1) at the promoter and at a −18kb upstream region coincident with repression. At the same regions, GATA-1 triggered release of both GATA-2, a related transcription factor expressed in multipotential progenitors, and PU.1, a positive autoregulator of its own gene. While GATA-1 is known to inhibit PU.1 functions through direct protein interactions, this work shows that antagonism also occurs at the level of Sfpi1 gene transcription. Together, our findings indicate that GATA-1 not only positively regulates an erythro-megakaryocytic program of gene expression, but also actively restrains myelopoiesis by inhibiting Sfpi1 expression directly. More generally, our work reveals a new mechanism through which cross-antagonism between transcription factors reinforces lineage commitment decisions in hematopoiesis.
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Lang, Wenjing, Fangyuan Chen, Jianyi Zhu, Lijing Shen, and Jihua Zhong. "Arsenic Trioxide Suppresses the Expression of EVI-1 Gene and Regulates Related Hematopoietic Transcription Factors in THP-1 Cells." Blood 128, no. 22 (December 2, 2016): 5120. http://dx.doi.org/10.1182/blood.v128.22.5120.5120.
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Abstract Introduction Acute myeloid leukemia (AML) is a malignant clonal hematopoietic stem cell disease. Ecotropic viral integration site-1(EVI-1) has been recognized as one of the most dominant oncogenes associated with murine and human myeloid leukemia. EVI-1 harbors several hallmark functions that are normally associated with leukemogenesis. Overexpression EVI-1 upregulates the transcription of the transcription factor gene GATA-2, which plays a critical role in the maintenance of hematopoietic stem cells.EVI-1 also binds to the transcription factors GATA-1,PU.1,RUNX-1,SCL and LMO2 thereby inhibiting their activity and blocking the differentiation of hematopoietic progenitors. Arsenic trioxide (ATO), which was used as a traditional Chinese medicine, has shown excellent therapeutic efficiency for acutepromyelocyticleukemia. A previous study has demonstrated that ATO targets EVI-1 protein to induce apoptosis . However, the molecular mechanisms underlying this regulation of AML cells by ATO have not been fully elucidated. We aim to expand our understanding of the cellular regulation and effect of ATO on EVI-1 and several related transcription factors (such as GATA1, GATA2, RUNX-1, LMO2, PU-1) in human acute monocytic leukemia THP-1 cells. Methods The endogenous expression of EVI-1 is higher in THP-1 cells, which was confirmed through gene analysis sequencing and comparison with Genbank.THP-1 cells were treated with various concentrations of ATO( 0,1,3,5¦ÌM) for 24h, 48h and 72h. Expressions of EVI-1,and related transcription factor genes(GATA-2 ,GATA-1, RUNX-1,MPO,LMO,PU.1,SCL2) were determined by qRT-PCR. Results Compared to controls, THP-1 cells with high expression of EVI-1 gene were selected to confirm that overexpression of EVI-1 can promote the expression of GATA-2 , and reduce the level of GATA-1, RUNX-1, MPO, LMO, PU.1, SCL2 simultaneously in vitro(Fig.1).To determine whether ATO is able to directly regulate EVI-1 and related transcription factor genes expression in THP-1 cells, we examined their mRNA expression by qRT-PCR after treatment with different concentrations of ATO. We found that ATO has the ability to down regulate EVI-1 gene in a dose-dependent and time-dependent manner( Fig.2a).Moreover, GATA-2 gene expression decreased when THP-1 cells were treated with ATO(Fig.2b). We examined the related transcription factors expression and found that ATO suppressed the expression levels of PU.1, RUNX-1, SCL and LMO2 (Fig.2c, 2d, 2e) in THP-1 cells. Interestingly, exposure to increasing concentrations of ATO for increasing lengths of time was associated with gradual promotion in the expression of GATA-1 mRNA( Fig 2f). Summary We verified that the endogenous expression of EVI-1 is higher in THP-1 cells, which confirmed THP-1 cells can be a vitro model to investigate EVI-1 gene functions. Meanwhile, related transcription factor gene GATA-2, which is regarded as EVI-1 regulatory element, is upregulated in THP-1 cells. Moreover, GATA-1, which is essential for erythroid differentiation, and other related transcription factor genes (PU.1, RUNX-1, SCL and LMO2) decreased in THP-1 cells. ATO dose-and time-dependently decreased expressions of EVI-1 and GATA-2 gene and regulated those related transcription factors in EVI-1-overexpressing cells compared with control cells. Disclosure: No relevant conflicts of interest to declare. Disclosures Lang: National Natural Science Fundation of China (no.81470312),: Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding. Chen:National Natural Science Fundation of China (no.81470312): Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding. Zhu:Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding; National Natural Science Fundation of China (no.81470312): Research Funding. Zhong:National Natural Science Fundation of China (no.81470312): Research Funding; Foundation of Shanghai Committee of Science and Technology (no. 14411950704): Research Funding.
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Johnson, Kirby D., Saumen Pal, Jeffrey A. Grass, and Emery H. Bresnick. "Differential GATA-1 Sensitivities of Target Loci." Blood 106, no. 11 (November 16, 2005): 1748. http://dx.doi.org/10.1182/blood.v106.11.1748.1748.
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Abstract The transcription factor GATA-1 is a key regulator of red cell differentiation, activating numerous erythroid-specific genes while downregulating genes that permit proliferation of erythroid precursors. We have demonstrated that GATA-1 only occupies a limited number of the high affinity WGATAR motifs present within target gene loci, FOG-1 is required for GATA-1 to occupy a subset of chromatin sites, and GATA-1 occupancy often coincides with GATA-2 displacement or a “GATA switch”. Once bound, GATA-1 elicits changes in the histone modification patterns and the three dimensional structure of target gene loci via recruitment of cofactors such as FOG-1 and CBP. As detailed mechanistic studies have not been conducted with most GATA-1 target genes, it is unclear whether these genes are equally sensitive to GATA-1 or if they respond differently to the rising GATA-1 levels during erythropoiesis. Using GATA-1 fusions to the estrogen receptor ligand binding domain (ER-GATA-1) in the GATA-1-null erythroid precursor cell line, G1E, we analyzed the responses of endogenous GATA-1 target genes to varied levels of GATA-1 activity. We found that transcriptional activation of Tac-2 required higher concentrations of ER-GATA-1 than is required for other GATA-1 target genes. Previously, we showed that Tac-2, which encodes the neurokinin-B precursor protein preprotachykinin B, is regulated by GATA-1 in erythroid cell lines and is induced upon ex vivo differentiation of human CD34+ peripheral blood cells. Importantly, whereas regulation of many GATA-1 target genes is only partially disrupted by removal of the N-terminal 193 amino acids from ER-GATA-1 (ER-GATA-1 ΔN), Tac-2 expression was very sensitive to this truncation. Whereas NK-B signals through G-protein-coupled receptors to modulate neuronal function, its functions beyond the nervous system are poorly understood. Although erythroid cells do not express NK-B receptors, the receptors, but not NK-B, are expressed in certain endothelial cell subtypes, and elevated levels of NK-B are implicated in the pregnancy-associated disorder pre-eclampsia. Tac-2 represents the first GATA-1 target gene that critically requires the N-terminus. Studies are underway to elucidate mechanisms underlying the exquisite sensitivity of Tac-2 to deletion of the GATA-1 N-terminus, the relationship between Tac-2 deregulation and GATA-1 N-terminal deletions in megakaryoblastic leukemia, and the function of erythroid cell-derived NK-B.
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Galloway, Jenna L., Christine Thisse, Yi Zhou, Rosanna Beltre, Bernard Thisse, and Leonard I. Zon. "Conversion of Erythropoiesis to Myelopoiesis in Gata1-Deficient Zebrafish Embryos." Blood 104, no. 11 (November 16, 2004): 2774. http://dx.doi.org/10.1182/blood.v104.11.2774.2774.
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Abstract Primitive hematopoiesis in vertebrates initiates in the embryonic yolk sac and yields nucleated erythrocytes and macrophages that later enter circulation. In zebrafish, a group of blood cells contained within the axial vein make up the intermediate cell mass (ICM), the teleost equivalent of the mammalian yolk sac. To identify novel genes involved in hematopoiesis, a high-throughput whole embryo in situ hybridization screen was performed. Examination of the expression pattern of 3700 clones from an adult zebrafish hematopoietic cDNA library discovered 24 genes with expression in the blood during development. Each stage of hematopoiesis was defined by a subset of genes, providing a molecular signature of blood cell maturation, from hematopoietic progenitors to terminally differentiated erythrocytes. By using antisense morpholinos to the transcription factors gata1 and gata2, we were able to dissect the regulation of these 24 genes. Examination of gene expression in Gata1, Gata2, and Gata1/Gata2-deficient animals revealed that most erythroid genes are dependent upon Gata factors for expression. Surprisingly, three novel genes, expressed in hematopoietic progenitors, do not require Gata factors for their expression demonstrating that some erythroid genes are regulated in a Gata-independent manner. During our analysis, we also found persistent ectopic expression of the myeloid transcription factor, PU.1, in the ICM cells and a subsequent expansion of mpo expressing granulocytes and L-plastin expressing macrophages. By utilizing gata1-GFP transgenic zebrafish, we were able to isolate blood cells by flow cytometry and examine their morphology. We discovered that blood cells from the Gata1-deficient animals exhibited features characteristic of myeloid cells when compared to wild-type blood cells. By confocal microscopy, we detected some blood cells in the ICM of Gata1-deficient embryos that co-express globin and PU.1, while blood cells of wild-type embryos never co-express these markers at this stage. These observations demonstrate that in the absence of Gata1 the presumptive erythroid progenitors have transformed into the myeloid lineage, and that a major cell fate alteration has occurred. Ultimately, our studies have molecularly defined blood development by gene expression, and illustrated that Gata1 governs lineage fate decisions of hematopoietic progenitors in the developing embryo.
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Nerlov, Claus, Erich Querfurth, Holger Kulessa, and Thomas Graf. "GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription." Blood 95, no. 8 (April 15, 2000): 2543–51. http://dx.doi.org/10.1182/blood.v95.8.2543.
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Abstract The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein-protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1–mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function.
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Nerlov, Claus, Erich Querfurth, Holger Kulessa, and Thomas Graf. "GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription." Blood 95, no. 8 (April 15, 2000): 2543–51. http://dx.doi.org/10.1182/blood.v95.8.2543.008k19_2543_2551.
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The GATA-1 transcription factor is capable of suppressing the myeloid gene expression program when ectopically expressed in myeloid cells. We examined the ability of GATA-1 to repress the expression and function of the PU.1 transcription factor, a central regulator of myeloid differentiation. We found that GATA-1 is capable of suppressing the myeloid phenotype without interfering with PU.1 gene expression, but instead was capable of inhibiting the activity of the PU.1 protein in a dose-dependent manner. This inhibition was independent of the ability of GATA-1 to bind DNA, suggesting that it is mediated by protein-protein interaction. We examined the ability of PU.1 to interact with GATA-1 and found a direct interaction between the PU.1 ETS domain and the C-terminal finger region of GATA-1. Replacing the PU.1 ETS domain with the GAL4 DNA-binding domain removed the ability of GATA-1 to inhibit PU.1 activity, indicating that the PU.1 DNA-binding domain, rather than the transactivation domain, is the target for GATA-1–mediated repression. We therefore propose that GATA-1 represses myeloid gene expression, at least in part, through its ability to directly interact with the PU.1 ETS domain and thereby interfere with PU.1 function.
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Yan, Bowen, Tao Yang, Suming Huang, and Yi Qiu. "HDAC1 Can Deacetylate GATA-1 and Regulates Its Activity through a FOG-1 Independent Manner." Blood 124, no. 21 (December 6, 2014): 5125. http://dx.doi.org/10.1182/blood.v124.21.5125.5125.
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Abstract The transcription factor GATA-1 is essential for erythroid and megakaryocytic cell differentiation and maturation. It has been well documented that GATA-1 can indirectly interact with histone deacetylase 1 (HDAC1) containing NuRD corepressor complexes through an association with FOG-1. Our previous work also shows that HDAC1 acetylation modulates the activity of FOG-1 associated NuRD complexes. Earlier studies show GATA-1 can be acetylated by p300/CBP, and the acetylation modulates GATA-1 binding activity to chromatin. However, it is not clear whether the acetylation can be reversed by a deacetylase. In this study, we found that GATA-1 can directly interact with HDAC1 in a FOG-1 independent manner. The interaction results in the deacetylation of GATA-1. We have identified two arginine sites within GATA-1 that are important for its interaction with HDAC1. The arginine to alanine mutation on these sites (2RA) can largely decrease the interaction of these two proteins, but doesn't affect its interaction with FOG-1, indicating that the direct interactions with HDAC1 and FOG-1 dependent association of NuRD complexes are separate events. The mutations also do not affect GATA-1 DNA binding activity in vitro. To further investigate the function of this interaction in erythropoiesis, we introduced wild type or the 2RA mutant of GATA-1 fused with estrogen receptor ligand binding domains into G1E cells, a GATA-1-null erythroid progenitor cell line. Upon estradiol induction, GATA-1 2RA inhibits the differentiation in G1E cells. We performed RNA sequencing to study the effect of GATA-1 2RA in gene expression in comparison with wild type GATA-1 and GATA-1-V205M (a mutation abolished binding with FOG-1) on a genome wild scale. GATA-1 2RA affects GATA-1 function in both GATA-1 activated and repressed genes. Although there is some overlap between 2RA and V205M mutations in both activated and repressed genes, more genes affected by these two mutations are different. Thus, this study unveils a novel regulation for GATA-1 by its direct interaction with HDAC1 during hematopoiesis. Disclosures No relevant conflicts of interest to declare.
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Inoue, AI, Tohru Fujiwara, Yoko Okitsu, Noriko Fukuhara, Yasushi Onishi, Kenichi Ishizawa, and Hideo Harigae. "Exploring The Mechanisms To Reveal The Contribution Of LMO2 To The Transcriptional Regulation In Human Erythroblasts." Blood 122, no. 21 (November 15, 2013): 2178. http://dx.doi.org/10.1182/blood.v122.21.2178.2178.
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Abstract Background Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA1 and SCL/TAL1), chromatin looping factor LDB1 (referred as GATA-SCL/TAL1 complex) (Wadman et al. EMBO J. 1997). Recently, we have demonstrated that LMO2 contributes to the expression of GATA-1 target genes such as HBB and SLC4A1, through modulating the assembly of GATA-1 as well as the components of SCL/TAL1 complex at the endogeneous loci (ASH 2012). To gain new mechanistic insights, we have extended our study to reveal the contribution of LMO2 to the GATA-1 activity in human erythroblasts. Methods For LMO2, TAL1 and LDB1 knockdown, anti-LMO2, anti-TAL1, anti-LDB1 siRNA (Thermo Scientific Dharmacon) were used. Western blotting and quantitative ChIP analyses were performed using antibodies for GATA-1 (CST and abcam), LMO2, 6×His tag (abcam), TAL1 and LDB1 (Santa Cruz). Human induced pluripotent stem cell (iPS)-derived erythroid progenitor cells (HiDEP), which have a capacity to differentiate into enucleated red blood cells (Kurita et al. PLOS ONE. 2013), were included for the analysis. For the exogeneous expression of 6×His tagged wild-type GATA-1 and mutant GATA-1 in K562 cells, pBABEpuro retroviral vector and PLAT-GP packaging cell line were used (Fujiwara et al. Exp Hematol. 2013). Results We previously demonstrated that transient LMO2 knockdown in K562 cells, which did not affect the expression of GATA-1, SCL/TAL1 and LDB1, resulted in the significantly decreased chromatin occupancy of GATA-1 and the components of SCL/TAL1 complex at beta-globin locus control region (LCR) and SLC4A1 loci (ASH 2012). Based on iPS-derived erythroblasts (HiDEP), we further confirmed the significant downregulation of GATA-1-target genes (HBB, HBA and SLC4A1), and concomitant decrease in GATA-1 chromatin occupancy at the target gene loci, by siRNA-mediated LMO2 knockdown. To reveal the molecular mechanism linking LMO2 and GATA-1, we first expressed 6×His tagged wild-type GATA-1 or mutated GATA-1, including R202Q and R217D, which impaired direct binding with LMO2 (Wilkinson-White et al. PNAS. 2011), in K562 cells. Quantitative ChIP analysis anti-6×His tag antibody revealed significantly diminished occupancy of the mutated GATA-1 (R202Q and R217D) at the beta-globin LCR, HBA and SLC4A1 loci. Next, in addition to the direct interaction between GATA-1 and LMO2, we examined whether the knockdown of each individual component of the SCL/TAL1 complex, such as SCL/TAL1 and LDB1, could affect GATA-1 chromatin occupancy. The expression of GATA-1 target genes, such as HBB, HBA, and SLC4A1, were downregulated by either SCL/TAL1 or LDB1 transient knockdown, whereas the expression of GATA-1 was unaffected. Under the condition, GATA-1 chromatin occupancy was significantly reduced, suggesting that impaired assembly of the individual component of SCL/TAL1 complex may also affect GATA-1 chromatin occupancy. Conclusion LMO2 contributes to the assembly of components of the GATA-SCL/TAL1 complex at endogenous loci in erythroblasts, which may lead to dysregulation of a subset of GATA-1 target genes. Our results may lead to the identification of novel disease mechanisms involving anemia as well as leukemia. Disclosures: No relevant conflicts of interest to declare.
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Muntean, Andrew G., Liyan Pang, Mortimer Poncz, Steve Dowdy, Gerd Blobel, and John Crispino. "Cyclin D: CDK4/6 Kinase Activity, Regulated by GATA-1, Is Required for Megakaryocyte Polyploidization." Blood 108, no. 11 (November 16, 2006): 780. http://dx.doi.org/10.1182/blood.v108.11.780.780.
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Abstract Megakaryocytes, which fragment to give rise to platelets, undergo a unique form of cell cycle, termed endomitosis, to become polyploid and terminally differentiate. During this process, cells transverse the cell cycle but the late stages of mitosis are bypassed to lead to accumulation of DNA up to 128N. While the mechanisms of polyploidization in megakaryocytes are poorly understood, a few cell cycle regulators, such as cyclin D3, have been implicated in this process. Hematopoietic transcription factors, including GATA-1 and RUNX1 are also essential for polyploidization, as both GATA1-deficient and RUNX1-null megakaryocytes undergo fewer rounds of endomitosis. Interestingly, GATA-1 deficient megakaryocytes are also smaller than their wild-type counterparts. However, the link between transcription factors and the growth and polyploidization of megakaryocytes has not been established. In our studies to identify key downstream targets of GATA-1 in the megakaryocyte lineage, we discovered that the cell cycle regulators cyclin D1 and p16 were aberrantly expressed in the absence of GATA-1: cyclin D1 expression was reduced nearly 10-fold, while that of p16ink4a was increased 10-fold. Luciferase reporter assays revealed that GATA-1, but not the leukemic isoform GATA-1s, promotes cyclinD1 expression. Consistent with these observations, megakaryocytes that express GATA-1s in place of full-length GATA-1 are smaller than their wild-type counterparts. Chromatin immunoprecipitation studies revealed that GATA-1 is bound to the cyclin D1 promoter in vivo, in primary fetal liver derived megakaryocytes. In contrast, GATA-1 is not associated with the cyclin D1 promoter in erythroid cells, which do not become polyploid. Thus, cyclin D1 is a bona fide GATA-1 target gene in megakaryocytes. To investigate whether restoration of cyclin D1 expression could rescue the polyploidization defect in GATA-1 deficient cells, we infected fetal liver progenitors isolated from GATA-1 knock-down mice with retroviruses harboring the cyclin D1 cDNA (and GFP via an IRES element) or GFP alone. Surprisingly, expression of cyclin D1 did not increase the extent of polyploidization of the GATA-1 deficient megakaryocytes. However, co-overexpression of cyclin D1 and Cdk4 resulted in a dramatic increase in polyploidization. Consistent with the model that cyclinD:Cdk4/6 also regulates cellular metabolism, we observed that the size of the doubly infected cells was also significantly increased. Finally, in support of our model that cyclin D:Cdk4/6 kinase activity is essential for endomitosis, we discovered that introduction of wild-type p16 TAT fusion protein, but not a mutant that fails to interact with Cdk4/6, significantly blocked polyploidization of primary fetal liver derived megakaryocytes. Taken together, our data reveal that the process of endomitosis and cell growth relies heavily on cyclinD:Cdk4/6 kinase activity and that the maturation defects in GATA-1 deficient megakaryocytes are due, in part, to reduced Cyclin D1 and increase p16 expression.
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Shimizu, Ritsuko, Takashi Kuroha, Osamu Ohneda, Xiaoqing Pan, Kinuko Ohneda, Satoru Takahashi, Sjaak Philipsen, and Masayuki Yamamoto. "Leukemogenesis Caused by Incapacitated GATA-1 Function." Molecular and Cellular Biology 24, no. 24 (December 15, 2004): 10814–25. http://dx.doi.org/10.1128/mcb.24.24.10814-10825.2004.
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ABSTRACT GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.
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Katsumura, Koichi Ricardo, Chenxi Yang, Jing Zhang, Lingjun Li, Kirby D. Johnson, and Emery H. Bresnick. "Mechanistic Deficits Of a Leukemogenic GATA-2 Mutant." Blood 122, no. 21 (November 15, 2013): 3668. http://dx.doi.org/10.1182/blood.v122.21.3668.3668.
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Abstract Recent studies have demonstrated a role for the master regulator of hematopoiesis GATA-2 in MonoMAC Syndrome, a human immunodeficiency disorder associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Though GATA2 coding region and cis-regulatory element mutations underlie MonoMAC syndrome, many questions remain unanswered regarding how GATA-2 is controlled physiologically and how it is dysregulated in pathological contexts. We dissected how a T354M mutation in the GATA-2 DNA binding zinc finger, which is frequently detected in MonoMAC syndrome and familial MDS/AML, alters GATA-2 activity. The T354M mutation reduced GATA-2 chromatin occupancy, induced GATA-2 hyperphosphorylation, and disrupted GATA-2 subnuclear localization. These molecular phenotypes also characterized an additional familial MDS/AML-associated GATA-2 mutant (Δ355T). T354M hyperphosphorylation and ectopic subnuclear localization were detected in hematopoietic and non-hematopoietic cell lines. We developed a new model system in mouse aortic endothelial (MAE) cells to quantitate GATA-2 activity to regulate endogenous target genes. T354M exhibited significantly reduced activity in this assay (GATA-2: 200-fold activation; T354M: 7.7-fold activation). Mass spectrometric analysis of the phosphorylation states of GATA-2 and T354M revealed that the T354M mutation enhanced phosphorylation at several GATA-2 residues. Analysis of single phosphorylation site mutants indicated that only mutation of S192 (S192A) abolished T354M-induced hyperphosphorylation. The S192A mutation attenuated phosphorylation of sites within wild-type GATA-2 and reduced transactivation activity (50% decrease, p < 0.01). A distinct 60 amino acid (aa) region within the GATA-2 N-terminus was required for T354M hyperphosphorylation and ectopic subnuclear localization. Deletion of this sequence decreased GATA-2 transactivation activity (60 aa deletion: 85% decrease, p < 0.01; 10 aa deletion: 45% decrease, p < 0.05). GATA-1 lacks an analogous subnuclear targeting sequence, and accordingly, a GATA-1(T263M) mutant, which corresponds to the GATA-2(T354M) mutant, localized normally and was not hyperphosphorylated. However, a GATA-1 chimera containing the GATA-2 subnuclear targeting sequence localized to ectopic subnuclear foci in a T263M-dependent manner. The GATA-2 N-terminus endowed GATA-1 with the capacity to induce GATA-2 target genes. By contrast, a GATA-2 chimera containing the GATA-1 N-terminus exhibited normal subnuclear localization. Thus, the leukemogenic T354M mutation utilizes the GATA-2-specific subnuclear targeting sequence to disrupt the normal subnuclear localization pattern, and this disruption is associated with S192-dependent hyperphosphorylation. In addition to its involvement in AML, GATA-2 interfaces with RAS signaling to promote the development of non-small cell lung cancer. We discovered that RAS signaling promotes S192-dependent GATA-2 hyperphosphorylation and ectopic subnuclear localization and propose that GATA-2 is an important component in oncogenic RAS-dependent leukemogenesis, which is being formally tested using innovative mouse models. In summary, dissecting the mechanistic deficits of a leukemogenic GATA-2 mutant revealed unexpected insights into mechanisms underlying physiological GATA-2 function and GATA-2-dependent hematologic pathologies. Disclosures: No relevant conflicts of interest to declare.
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Lamonica, Janine M., Christopher R. Vakoc, and Gerd A. Blobel. "Acetylation of GATA-1 Is Required for Chromatin Occupancy." Blood 108, no. 11 (November 16, 2006): 1179. http://dx.doi.org/10.1182/blood.v108.11.1179.1179.
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Abstract All three hematopoietic GATA transcription factors GATA-1, GATA-2, and GATA-3 are acetylated, although the in vivo role of this modification remains unclear. It has been proposed that acetylation of GATA-1 increases its affinity for DNA in vitro, although this finding has not been observed by others. To study the role of GATA-1 acetylation, we examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 largely abrogates its biological activity but does not impair its nuclear localization, steady state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to its cellular target sites in vivo, including genes that are normally activated (α- and β-globin, EKLF, FOG-1, Band3, and AHSP) and repressed (GATA-2 and c-kit) by GATA-1. Together, these results suggest that acetylation is required for GATA-1 chromatin occupancy. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo. To identify proteins that interact with acetylated GATA-1, we performed peptide affinity chromatography using acetylated GATA-1 peptides. Using this technique coupled with mass spectrometry, several proteins that bind to GATA-1 peptides in an acetylation-dependent manner were identified. The identified proteins contain known acetyl-lysine binding modules (bromodomains) consistent with their binding properties. The in vivo role of these proteins with regard to GATA-1 function is being examined and will be discussed.