GATA Switch Research Articles

The role of GATA switch in benzene metabolite hydroquinone inhibiting erythroid differentiation in K562 cells.

The phenolic metabolite of benzene, hydroquinone (HQ), has potential risks for hematological disorders and hematotoxicity in humans. Previous studies have revealed that reactive oxygen species, DNA methylation, and histone acetylation participate in benzene metabolites inhibiting erythroid differentiation in hemin-induced K562 cells. GATA1 and GATA2 are crucial erythroid-specific transcription factors that exhibit dynamic expression patterns during erythroid differentiation. We investigated the role of GATA factors in HQ-inhibited erythroid differentiation in K562 cells. When K562 cells were induced with 40μM hemin for 0-120h, the mRNA and protein levels of GATA1 and GATA2 changed dynamically. After exposure to 40μM HQ for 72h, K562 cells were induced with 40μM hemin for 48h. HQ considerably reduced the percentage of hemin-induced Hb-positive cells, decreased the GATA1 mRNA, protein, and occupancy levels at α-globin and β-globin gene clusters, and increased the GATA2 mRNA and protein levels significantly. ChIP-seq analysis revealed that HQ reduced GATA1 occupancy, and increased GATA2 occupancy at most gene loci in hemin-induced K562 cells. And GATA1 and GATA2 might play essential roles in the erythroid differentiation protein interaction network. These results elucidate that HQ decreases GATA1 occupancy and increases GATA2 occupancy at the erythroid gene loci, thereby downregulating GATA1 and upregulating GATA2 expression, which in turn modulates the expression of erythroid genes and inhibits erythroid differentiation. This partially explains the mechanism of benzene hematotoxicity.

Activation of lysophosphatidic acid receptors regulate differentiation of hematopoietic stem cells in myeloid lineage

Lysophosphatidic acid (LPA) is a membrane‐derived lysophospholipid that exists in plasma and platelet. It exerts its functions through activating various LPA receptors (LPARs), which belong to the family of G‐protein coupled receptors (GPCRs). Activation of LPARs has been implicated for important roles during stem cell differentiation. However, how LPA affects human hematopoietic stem cell (HSC) differentiation and underlying lineages remains elusive. In our previous studies, we have demonstrated that activation of LPA receptor 3 (LPA3) promotes erythropoiesis in human HSC and zebrafish. In the present study, pharmacological approach was adopted to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. Treatment of LPA2 agonist GRI977143 suppressed erythropoiesis, whereas activation of LPA3 by 2S‐OMPT promoted RBC differentiation both in vitro and in vivo. Furthermore, our results demonstrated a negative role of LPA3 during platelet differentiation both in vitro and in vivo. Moreover, the expression pattern of LPA receptors correlated to the hematopoietic transcriptional factors GATA1 and GATA2 in different stages of myeloid progenitors. In addition, manipulation of these GATA factors affected the expression level of LPA2 and LPA3. By luciferase assays, we demonstrated that the promoter region of LPAR2 and LPAR3 were regulated by these GATA factors. Mutation of identified GATA‐Binding‐Sites (GBS) in the region abrogate the luciferase activities, suggested that LPA2and LPA3 are regulated by GATA factors. In addition, chromatin immunoprecipitation assays confirmed the directly interaction between GATA factors and promoter region of LPARs. Taken together, our results suggested that the expression level of LPA2 and LPA3, which may be determined by GATA factors, is a regulatory switch for the lineage commitment in myeloid progenitors. The pharmacological activation of LPA receptor subtypes represents a novel strategyfor regulating erythropoiesis.LPA receptors are expressed at different stages during myeloid differentiation. GATA switch, which is an important molecular switch in hematopoiesis, cause the difference of expressional pattern of LPA receptor in different lineage.Figure 1

GATA Factor-Mediated Gene Regulation in Human Erythropoiesis.

SummaryErythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.

E2F and GATA switches turn off WD repeat domain 77 expression in differentiating cells.

WDR77 (WD repeat domain 77) is expressed during earlier lung development when cells are rapidly proliferating, but is absent from adult lung. It is re-activated during lung tumorigenesis and is essential for lung cancer cell proliferation. Signalling pathways/molecules that control WDR77 gene expression are unknown. Promoter mapping, gel shift assay and ChIP revealed that the WDR77 promoter contains bona fide response elements for E2F and GATA transcriptional factors as demonstrated in prostate cancer, lung cancer and erythroid cells, as well as in mouse lung tissues. The WDR77 promoter is transactivated by E2F1, E2F3, GATA1 and GATA6, but suppressed by E2F6, GATA1 and GATA3in prostate cancer PC3 cells. WDR77 expression is associated with E2F1, E2F3, GATA2 and GATA6 occupancy on the WDR77 gene, whereas, in contrast, E2F6, GATA1 and GATA3 occupancy is associated with the loss of WDR77 expression during erythroid maturation and lung development. More importantly, the loss of WDR77 expression that results from E2F and GATA switches is required for cellular differentiation of erythroid and lung epithelial cells. In contrast, lung cancer cells avoid post-mitotic differentiation by sustaining WDR77 expression. Altogether, the present study provides a novel molecular mechanism by which WDR77 is regulated during erythroid and lung development and lung tumorigenesis.

Dynamic Control of Enhancer Repertoires Drives Lineage and Stage-Specific Transcription during Hematopoiesis

Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.

Characterization of hematopoietic GATA transcription factor expression in mouse and human dendritic cells

Dendritic cells (DCs) are key initiators and regulators of the immune response. The development of the DC lineage and their subsets requires an orchestrated regulation of their transcriptional program. Gata1, a transcription factor expressed in several hematopoietic cell lineages, has been recently reported to be required for mouse DC development and function. In humans, GATA1 is involved in the lineage separation between monocyte-derived DCs and Langerhans cells (LC) and loss of GATA1 results in differentiation arrest at the monocyte stage. The hematopoietic GATA factors (i.e. Gata1, Gata2, Gata3) are known to regulate each other's expression and to function consecutively throughout lineage commitment (so-called GATA switch). In humans, mutations in GATA2 are causative of MonoMAC disease, a human immunodeficiency syndrome characterized by loss of DCs, monocytes, B and NK cells. However, additional data on the expression of hematopoietic GATA factors in the DC lineage is missing.In this study, we have characterized the expression of hematopoietic GATA factors in murine and human DCs and their expression dynamics upon TLR stimulation. We found that all hematopoietic GATA factors are expressed in DCs, but identified species-specific differences in the relative expression of each GATA factor, and how their expression fluctuates upon stimulation.

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

Thalassemia Modular Stratification System for Personalized Therapy of Beta-Thalassemia (THALAMOSS)

Mathematical modeling of GATA-switching for regulating the differentiation of hematopoietic stem cell

BackgroundHematopoiesis is a highly orchestrated developmental process that comprises various developmental stages of the hematopoietic stem cells (HSCs). During development, the decision to leave the self-renewing state and selection of a differentiation pathway is regulated by a number of transcription factors. Among them, genes GATA-1 and PU.1 form a core negative feedback module to regulate the genetic switching between the cell fate choices of HSCs. Although extensive experimental studies have revealed the mechanisms to regulate the expression of these two genes, it is still unclear how this simple module regulates the genetic switching.MethodsIn this work we proposed a mathematical model to study the mechanisms of the GATA-PU.1 gene network in the determination of HSC differentiation pathways. We incorporated the mechanisms of GATA switch into the module, and developed a mathematical model that comprises three genes GATA-1, GATA-2 and PU.1. In addition, a novel multiple-objective optimization method was designed to infer unknown parameters in the proposed model by realizing different experimental observations. A stochastic model was also designed to describe the critical function of noise, due to the small copy numbers of molecular species, in determining the differentiation pathways.ResultsThe proposed deterministic model has successfully realized three stable steady states representing the priming and different progenitor cells as well as genetic switching between the genetic states under various experimental conditions. Using different values of GATA-1 synthesis rate for the GATA-1 protein availability in the chromatin sites during the time period of GATA switch, stochastic simulations for the first time have realized different proportions of cells leading to different developmental pathways under various experimental conditions.ConclusionsMathematical models provide testable predictions regarding the mechanisms and conditions for realizing different differentiation pathways of hematopoietic stem cells. This work represents the first attempt at using a discrete stochastic model to realize the decision of HSC differentiation pathways showing a multimodal distribution.

LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated.In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival.Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures:No relevant conflicts of interest to declare.

A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

Transcriptional networks orchestrate complex developmental processes, and such networks are commonly instigated by master regulators for development. By now, considerable progress has been made in elucidating GATA factor-dependent genetic networks that control red blood cell development. Here we reported that GATA-1 and GATA-2 co-regulated the expression of two microRNA genes, microRNA-27a and microRNA-24, with critical roles in regulating erythroid differentiation. In general, GATA-2 occupied the miR-27a∼24 promoter and repressed their transcription in immature erythroid progenitor cells. As erythropoiesis proceeded, GATA-1 directly activated miR-27a∼24 transcription, and this involved a GATA-1-mediated displacement of GATA-2 from chromatin, a process termed ‘GATA switch’. Furthermore, the mature miR-27a and miR-24 cooperatively inhibited GATA-2 translation and favoured the occupancy switch from GATA-2 to GATA-1, thus completing a positive feedback loop to promote erythroid maturation. In line with the essential role of GATA factors, ectopic expression of miR-27a or miR-24 promoted erythropoiesis in human primary CD34+ haematopoietic progenitor cells and mice, whereas attenuated miR-27 or miR-24 level led to impaired erythroid phenotypes in haematopoietic progenitor cells and zebrafish. Taken together, these data integrated micro RNA expression and function into GATA factor coordinated networks and provided mechanistic insight into a regulatory circuit that comprised GATA1/2 switch and miR-27a/24 in erythropoiesis.

Genetic Determinants of the Definitive Hematopoietic Stem/Progenitor Cell Compartment

Abstract 1226How transcriptional and post-transcriptional mechanisms control the levels/activities of master developmental regulators has fundamental importance for understanding complex developmental processes such as hematopoiesis and associated pathological disorders. GATA-2 is an essential regulator of hematopoiesis, and GATA-2 mutations characterize heritable disease associated with myelodysplastic syndrome and acute myeloid leukemia, including MonoMAC (syndrome of monocytopendia, B and NK cell lymphopenia, and mycobacterial, fungal and viral infection). However, many questions remain unanswered regarding mechanisms underlying GATA-2 regulation and function. We demonstrated that a MonoMAC patient harbors a 28 bp deletion within GATA2 intron 5 that eliminates a conserved E-box and 5 base pairs of an 8 base pair spacer between the E-box and a conserved GATA motif, which constitutes an E-box-GATA composite element. This composite element resides within the +9.5 kb “GATA switch site” that binds GATA-2 and GATA-1 in the transcriptionally active and repressed states, respectively, and confers hematopoietic and vascular endothelial enhancer activities in transgenic mouse embryos. Importantly, this patient lacked mutations in the GATA2 coding sequence characteristic of other MonoMAC patients, but exhibited prototypical MonoMAC. To elucidate the mechanism underlying the function of the +9.5 composite element, we generated a targeted deletion of the murine element, which yielded embryonic lethality at E13 to E14. Prior to death, +9.5−/− mice exhibit reduced liver size, hemorrhaging, and edema. Nucleated primitive red cells are abundant in the +9.5−/− embryos, in contrast to Gata2 knockout mice, which die at approximately E10.5 from anemia due to failure of primitive and definitive hematopoiesis. Furthermore, primitive erythroid (EryP) colony assays conducted with yolk sacs revealed that the mutation does not affect primitive erythroid precursor functionality. However, the +9.5 deletion strongly reduced Gata2 expression at sites of definitive hematopoiesis, including the fetal liver (8.1 fold, P < 0.004) and cultured explants of the hematopoietic stem cell-generating Aortic Gonadal Mesonephric (AGM) region (4.0 fold, P < 0.001). The homozygous mutant animals exhibited a nearly complete loss of hematopoietic stem cells as determined by flow cytometry (20-fold reduction of Lin-Mac1+CD41-CD48-CD150+Sca+Kit+ cells, P < 0.005) and competitive repopulation (complete loss, P < 0.02) assays, as well as progenitors as determined by colony assays (BFU-E, 60-fold reduction, P < 0.002; CFU-GM, 8.8-fold reduction, P < 0.0001; CFU-GEMM, 19-fold reduction, P < 0.001). To investigate the underlying mechanisms, we developed an allele-specific Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay with heterozygous fetal liver cells to test whether the deletion influences Gata2 chromatin accessibility at the +9.5 region. The deletion significantly reduced (8.4 fold reduction, P < 0.001) chromatin accessibility at this region within the mutant allele, while the wild type allele was unaffected. Thus, any potential remaining cis-elements are insufficient to confer chromatin accessibility, supporting a model in which the transcription factors that normally occupy this GATA switch site lose the capacity to access their respective cis-elements in the context of the mutant allele. Our human and murine studies have therefore revealed a cis-element indispensable for the regulation of Gata2 expression in multiple developmental contexts and necessary for the generation of the definitive hematopoietic stem/progenitor cell compartment. As additional elements are likely to confer Gata2 expression in distinct contexts, including primitive erythropoiesis, we have implemented a multi-faceted effort to identify such elements and to compare their mechanisms with that of the +9.5 site, which will provide fundamental insights into genetic mechanisms controlling normal and malignant hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Chromatin occupancy analysis reveals genome-wide GATA factor switching during hematopoiesis

AbstractThere 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.

A “Master Regulatory Cis-element” Governs the Hematopoietic Stem/Progenitor Cell Compartment, Vascular Integrity, and Cardiovascular Development

Abstract 1304The 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.

Identification of a GATA Switch In Megakaryocytic Development.

AbstractAbstract 2605GATA 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.

Ikaros Is a GATA Switch Target Gene That Restrains Megakaryopoiesis In Part by Blocking Notch Signaling.

AbstractAbstract 1602The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures:No relevant conflicts of interest to declare.

GATA Switches as Developmental Drivers

Transcriptional networks orchestrate complex developmental processes. Such networks are commonly instigated by master regulators of development. Considerable progress has been made in elucidating GATA factor-dependent genetic networks that control blood cell development. GATA-2 is required for the genesis and/or function of hematopoietic stem cells, whereas GATA-1 drives the differentiation of hematopoietic progenitors into a subset of the blood cell lineages. GATA-1 directly represses Gata2 transcription, and this involves GATA-1-mediated displacement of GATA-2 from chromatin, a process termed a GATA switch. GATA switches occur at numerous loci with critical functions, indicating that they are widely utilized developmental control tools.

FOG-1-mediated recruitment of NuRD is required for cell lineage re-enforcement during haematopoiesis

The transcriptional co-factor Friend of GATA1 (FOG-1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N-terminus. To test the importance of FOG-1/NuRD interaction for haematopoiesis in vivo, we generated mice with a mutation that specifically disrupts FOG-1/NuRD interaction (FOG-1(R3K5A)). Homozygous FOG-1(R3K5A) mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG-1(R3K5A/R3K5A) megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG-1/NuRD interaction is required for the earlier described 'GATA Switch'. In addition, ablation of FOG-1/NuRD interaction led to inappropriate expression of mast cell and eosinophil-specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG-1(R3K5A/R3K5A) megakaryocytes, suggesting that FOG-1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG-1/NuRD interaction for the re-enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo.

Distinct Functions of Dispersed GATA Factor Complexes at an Endogenous Gene Locus

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.

GATA-1-mediated Transcriptional Repression Yields Persistent Transcription Factor IIB-Chromatin Complexes

The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream -77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.

Transcriptional Regulation Via GATA Switches: Proteasome-Mediated Exchange of Chromatin-Bound GATA Factors.

The transcription factors GATA-1 and GATA-2 confer both shared and unique activities to regulate hematopoiesis. GATA-2 is essential for the survival and proliferation of hematopoietic stem cells and multipotent hematopoietic precursors, whereas GATA-1 is critical for erythropoiesis. GATA-1 and GATA-2 are expressed reciprocally during erythropoiesis, with GATA-2 expression preceding that of GATA-1. Erythroid maturation appears to require GATA-2 repression. We have shown that chromatin-bound GATA-2 is displaced by GATA-1, which instigates a distinct transcriptional output at the respective locus. We hypothesize that such “GATA switches” are an important transcriptional mechanism that drives erythropoiesis. Their distinct transcriptional outputs suggest that GATA-1 and GATA-2 have unique biochemical properties. However, biochemical analyses have primarily identified similarities between the factors. For instance, the two zinc fingers of GATA-1 are highly homologous to those of GATA-2, and therefore both factors interact with the cofactor FOG-1 and bind an identical WGATAR sequence with high affinity. We found that GATA-2 is considerably less stable than GATA-1. Studies using cycloheximide to block protein synthesis in erythroid precursor cell lines derived from embryonic stem cells indicate that the half-life of endogenous GATA-2 is ~45–90 min, whereas that of endogenous GATA-1 is >4 h. Biosynthetic labeling studies to measure the stabilities of endogenous GATA-1 and GATA-2 expressed in the same cell confirmed that GATA-2 is considerably less stable than GATA-1. GATA-2 levels increase in cells treated with the proteasome inhibitors MG132 or clasto-lactacystin β-lactone. Importantly, we have found that rapid GATA-2 proteolysis is essential for the proper temporal control of erythroid-specific gene transcription. Quantitative chromatin immunoprecipitation analysis revealed that proteasomal inhibition attenuated the ability of GATA-1 to displace GATA-2 from target genes, which are regulated by GATA switches. Proteasomal inhibition resulted in impaired GATA-2 displacement from GATA-2, and consequently a significantly lower degree of GATA-2 repression. Proteasomal inhibition also diminished GATA-1-mediated displacement of GATA-2 from GATA-1, α-globin, and ALAS-2, genes at which GATA switches precede transcriptional activation. We have therefore identified the 26S proteasome as a key regulator of GATA factor exchange on chromatin. Our studies indicate that rapid proteasomal degradation of GATA-2 is required for GATA-1 and GATA-2 to establish unique patterns of gene expression during erythropoiesis. Studies are underway to determine whether GATA-2 is similarly unstable in multipotent hematopoietic precursors, or if its instability is established upon commitment to the erythroid lineage.