No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The Gata1 5' region harbors distinct cis-regulatory modules that direct gene activation in erythroid cells and gene inactivation in HSCs
Abstract
Key Points
The combination of 3 core cis-elements represents the lineage-specific regulatory function of the Gata1 gene regulatory region. Gata1 gene expression is inactivated in HSCs by the cis-repressive activity of a 3.2-kb element in the upstream Gata1 gene regulatory region.
... We utilized a 196 kb mouse Gata1 Bacterial Artificial Chromosome (abbreviated G1B) as the Cre delivery vehicle, which we had previously shown to faithfully recapitulate endogenous mouse Gata1 gene expression [41][42][43] . A tamoxifen (Tx)-inducible CreER T2 fusion gene 44 , encoding a Cre recombinase whose activity is dependent on the binding of the Tx ligand, was inserted at the ATG start codon of the Gata1 BAC by gene editing (Supplemental Fig. 2). ...
... In this study, we set out to investigate the effects of LSD1 inhibition on erythropoiesis in vivo; this required the generation of an erythroid lineage-specific, inducible Cre deleter line to investigate the effects of conditional Lsd1 loss-of-function in murine adult definitive erythroid cells. Previously, Takai et al. showed that a 196 kb mouse Gata1-GFP knock-in BAC exhibited prominent GFP expression in MEP and that this fluorescence continued to peak as erythropoiesis (at the CFU-E and proerythroblast stages) progressed, and then gradually weakened in basophilic and polychromatic erythroblasts 43 . Interestingly, they also reported that the first (weak) hematopoietic GFP fluorescence was detected in CMP; however, in R26T:G1BCreER T2 mice that were treated with tamoxifen, no CMP cells labeled with TdTomato. ...
... BAC recombination. BAC recombination and the 196 kbp mouse Gata1 BAC clone (RP23-443E19) have been described previously 43,64 . Briefly, the targeting DNA fragment was constructed by inserting a CreERT2-polyA-Neo cassette at the translational start site of mouse Gata1 (in the 2nd exon) between 5' and 3' homologous recombination arms. ...
Histone H3 lysine 4 methylation (H3K4Me) is proximally associated with chromatin activation, and therefore removing H3K4 methyl groups is normally coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a potential therapeutic target for multiple diseases, including for the treatment of the b-globinopathies (sickle cell disease and bthalassemia) since it is a component of multiple g-globin repressor complexes, and its inactivation leads to robust induction of the fetal globin genes. However, the effects of LSD1 inhibition in definitive erythroid cells are not well characterized. Here we examined the consequences of erythroid-specific conditional inactivation of Lsd1 in vivo using a new Gata1creERT2 bacterial artificial chromosome (BAC) transgene. Conditional loss of Lsd1 in adult mice led to a differentiation block in erythroid progenitor cells and the surprising expansion of a GMP-like cell pool, apparently converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human cells: inactivation of LSD1 in hematopoietic stem and progenitor cells (HSPC) also blocked erythroid differentiation, coincident with robust induction of myeloid transcription factor genes (e.g. Pu.1 and Cebpa). Remarkably, blocking the activity of PU.1 or RUNX1 (a transcriptional activator of Pu.1) at the same time as blocking LSD1 activity reverted the myeloid lineage conversion back to an erythroid phenotype. Taken together, the data show that LSD1 maintains erythropoiesis by reversibly repressing a myeloid cell fate in adult erythroid cell precursors, and that inhibition of the myeloid differentiation pathway can reverse the negative effects of LSD1 inactivation on erythroid differentiation.
... In this regard, we previously identified a cis-acting silencer element located between the G1HE and dbG elements that may play a pivotal role in Gata1 gene repression in HSPCs. We also found that DNA methyltransferase 1 (Dnmt1) is recruited to the element and participates in Gata1 gene methylation (29). We refer to this silencer element as the Gata1 methylation-determining region (G1MDR). ...
... In this regard, several lines of evidence are worthy of attention. First, Dnmt1 is strongly recruited to the G1MDR to maintain DNA methylation of the Gata1 locus in HSPCs, and deletion of the G1MDR selectively abrogates Gata1 gene repression in HSPCs, which is associated with an increase of GATA2 occupancy in the Gata1 gene enhancer (29). Second, demethylation of the Gata1 enhancer and promoter around the G1MDR is associated with the enhancement of gene expression during erythropoiesis (29). ...
... First, Dnmt1 is strongly recruited to the G1MDR to maintain DNA methylation of the Gata1 locus in HSPCs, and deletion of the G1MDR selectively abrogates Gata1 gene repression in HSPCs, which is associated with an increase of GATA2 occupancy in the Gata1 gene enhancer (29). Second, demethylation of the Gata1 enhancer and promoter around the G1MDR is associated with the enhancement of gene expression during erythropoiesis (29). Based on these observations, we hypothesized that derepression of Dnmt1-G1MDR-mediated repression is the key molecular mechanism that triggers the initial activation of Gata1 gene expression in HSPCs, which subsequently leads HSPCs to differentiate toward the erythroid lineage. ...
GATA1 is a critical regulator of erythropoiesis. While the mechanisms underlying the high-level expression of GATA1 in maturing erythroid cells have been studied extensively, the initial activation of the Gata1 gene in early hematopoietic progenitors remains to be elucidated. We previously identified a hematopoietic stem and progenitor cell (HSPC)-specific silencer element (the Gata1 methylation determining region; G1MDR) that recruits DNA methyltransferase 1 (Dnmt1) and provokes the methylation of the Gata1 gene enhancer. Here, we hypothesized that removal of the G1MDR-mediated silencing machinery is the molecular basis of the initial activation of the Gata1 gene and erythropoiesis. To address this hypothesis, we generated transgenic mouse lines harboring a Gata1 bacterial artificial chromosome in which G1MDR was deleted. The mice exhibited abundant GATA1 expression in HSPCs in a GATA2-dependent manner. The ectopic GATA1 expression repressed Gata2 transcription and induced erythropoiesis and apoptosis of HSPCs. Furthermore, genetic deletion of Dnmt1 in HSPCs activated Gata1 expression and depleted HSCPs, thus recapitulating the HSC-phenotype associated with GATA1 gain-of-function. These results demonstrate that G1MDR holds the key for HSPC maintenance and suggest that a release from this suppressive mechanism is a fundamental requirement for subsequent initiation of erythroid differentiation.
... The Gata3 B125 (wild-type) YAC transgenic lines (Tg B125-G3 ) have been described previously (9,12). The copy number and integrity of the transgenes were determined by genomic quantitative PCR as previously described (20). Primers used for genomic quantitative PCR are listed in Table 1. ...
... The fluorescence-activated cell sorter (FACS) Aria (BD Bioscience) was used to separate the GFP-positive cell population recovered from the kidneys of Gata3 g/g and Gata3 g/ϩ mice. Total RNA from GFPpositive cells was prepared with the RNeasy Micro kit (Qiagen) and subjected to quantitative reverse transcription-PCR (RT-qPCR) analysis, as described previously (20). Primers used for the RT-qPCR analysis are listed in Table 1. ...
... Total RNA was isolated using the RNeasy Micro kit (Qiagen). The RNA sample was subjected to RT-qPCR analysis, as described previously (20). Primers used for the RT-qPCR analysis are listed in Table 1. ...
GATA3 is a zinc-finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. A newly generated
Gata3
hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb
Gata3
YAC (yeast artificial chromosome), and these animals (G3YR) survive to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb
Gata3
YAC transgene recapitulated
Gata3
expression in the renal tubules, but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.
... This fragment elicits regulatory activity sufficient to direct Gata1 expression in yolk sac erythroid cells [26], and is referred to as Gata1 GdC minigene (Fig. 1c). The GdC-minigene fragment is indeed capable of functionally replacing the 3.7-kb 5 0upstream regulatory region of Gata1 gene by directing the hematopoietic lineage-specific gene expression in the context of a Gata1 bacterial artificial chromosome (BAC)-based transgenic mouse assay [35]. These three regulatory elements are thus crucial for hematopoietic Gata1 gene expression. ...
... The expression level of Gata1 gene is suppressed at a low level in HSC, with abundant GATA2 expression detected in this fraction [35,52]. It has been reported that forced GATA1 expression using retroviral transfection in HSC leads to the loss of self-renewal activity, suggesting that Gata1 gene inactivation is crucial for maintenance of HSC [53,54]. ...
... Recently, we demonstrated that the 3.2-kb genomic region intervening between G1HE core and dbGATA elements exerts DNA methylation-mediated silencing of Gata1 gene in HSC fraction (G1MDR; Gata1 methylationdetermining region; Fig. 6a) [35]. G1MDR sequences recruit DNA methyl transferase 1 (Dnmt1), which maintains high level of DNA methylation status throughout Gata1 locus in HSC fraction (Fig. 6b left panel). ...
GATA transcription factor family members GATA1 and GATA2 play crucial roles in the regulation of lineage-restricted genes during erythroid differentiation. GATA1 is indispensable for survival and terminal differentiation of erythroid, megakaryocytic and eosinophilic progenitors, whereas GATA2 regulates proliferation and maintenance of hematopoietic stem and progenitor cells. Expression levels of GATA1 and GATA2 are primarily regulated at the transcriptional level through auto- and reciprocal regulatory networks formed by these GATA factors. The dynamic and strictly controlled change of expression from GATA2 to GATA1 during erythropoiesis has been referred to as GATA factor switching, which plays a crucial role in erythropoiesis. The regulatory network comprising GATA1 and GATA2 gives rise to the stage-specific changes in Gata1 and Gata2 gene expression during erythroid differentiation, which ensures specific expression of early and late erythroid genes at each stage. Recent studies have also shed light on the genome-wide binding profiles of GATA1 and GATA2, and the significance of epigenetic modification of Gata1 gene during erythroid differentiation. This review summarizes the current understanding of network regulation underlying stage-dependent Gata1 and Gata2 gene expressions and the functional contribution of these GATA factors in erythroid differentiation.
... data independently through real-time PCR assays ( Figures 1D and 7A). It has been unequivocally proven that Gata1 is an essential TF for erythrocyte differentiation and expressed at very low levels, and that Gata2 is expressed at higher levels in HSCs (Bresnick et al., 2010;Suzuki et al., 2013;Takai et al., 2013). However, our analysis indicated a significant downregulation of Gata2 ( Figure 7B) and upregulation of Gata1 ( Figure 7A) in CA/CA HSCs. ...
... Earlier studies have unequivocally demonstrated that Gata factors are essential for hematopoiesis; however, they need to be tightly controlled in HSPCs. In general, Gata2 is expressed in HSPCs and Gata1 is suppressed in HSPCs through Gata2-dependent mechanisms (Philipsen, 2013;Takai et al., 2013). The enforced expression of Gata1 in HSCs leads to the loss of self-renewal capacity (Ferreira et al., 2007). ...
Constitutive activation of the canonical NF-κB pathway has been associated with a variety of human pathologies. However, molecular mechanisms through which canonical NF-κB affects hematopoiesis remain elusive. Here, we demonstrate that deregulated canonical NF-κB signals in hematopoietic stem cells (HSCs) cause a complete depletion of HSC pool, pancytopenia, bone marrow failure, and premature death. Constitutive activation of IKK2 in HSCs leads to impaired quiescence and loss of function. Gene set enrichment analysis (GSEA) identified an induction of "erythroid signature" in HSCs with augmented NF-κB activity. Mechanistic studies indicated a reduction of thrombopoietin (TPO)-mediated signals and its downstream target p57 in HSCs, due to reduced c-Mpl expression in a cell-intrinsic manner. Molecular studies established Klf1 as a key suppressor of c-Mpl in HSPCs with increased NF-κB. In essence, these studies identified a previously unknown mechanism through which exaggerated canonical NF-κB signals affect HSCs and cause pathophysiology.
... These fragments were cloned into a vector containing a neomycin resistance-conferring gene (neo) cassette to generate the targeting construct. The copy number and the integrity of the transgenes were determined by quantitative genomic PCR (qGPCR) using primer pairs amplifying the hGATA1 and mGata1 gene loci (Table 1) (20). Induction of hemolytic anemia with phenylhydrazine (PHZ) was performed as previously described (21). ...
... Quantitative ChIP analysis. Chromatin immunoprecipitation (ChIP) analysis for detection of CTCF in the hGATA1 locus was performed using the K562 erythroid cell line and PHZ-treated splenic erythroblasts as previously described (20). Briefly, cells were fixed with 1.0% formaldehyde for 5 min at room temperature. ...
GATA1 is a key transcription factor for erythropoiesis. GATA1 gene expression is strictly regulated at the transcriptional level. While regulatory mechanisms governing the mouse Gata1 gene (mGata1) expression have been studied extensively, how expression of the human GATA1 gene (hGATA1) is regulated remains to be elucidated. To address this issue, we generated hGATA1-BAC (bacterial artificial chromosome) transgenic mouse lines harboring a 183-kb hGATA1 locus covering the hGATA1 exons and distal flanking sequences. The transgenic hGATA1 expression coincides with the endogenous mGata1 expression and fully rescues hematopoietic deficiency in mGata1 knockdown mice. The transgene exhibited copy number-dependent and integration position-independent expression of hGATA1, indicating the presence of chromatin insulator activity within the transgene. We found a novel insulator element at 29-kb 5' to the hGATA1 gene and referred to this element as 5' CTCF site. Substitution mutation of the 5' CTCF site in the hGATA1-BAC disrupted chromatin architecture and led to a reduction of hGATA1 expression in splenic erythroblasts under stress erythropoiesis. Our results demonstrate that expression of the hGATA1 gene is regulated through chromatin architecture organized by 5' CTCF site-mediated intrachromosomal interactions in the hGATA1 locus.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
... Modified BACs were subjected to microinjection, as described previously (5). The copy number and integrity of transgenes were determined by quantitative PCR (qPCR) as described previously (24). ...
... ChIP analysis. Chromatin immunoprecipitation (ChIP) analysis of the Gata1 locus was performed using Lin Ϫ c-Kit ϩ progenitor cells and Ter119 ϩ erythroblasts from male mice as described previously (24). Cells were fixed with 1.0% formaldehyde for 5 min at room temperature. ...
GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discrete cis-acting elements. In this study, we examine the activity of a promoter-proximal double-GATA (dbGATA) motif, using a Gata1 bacterial-artificial-chromosome-transgenic GFP reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA-motif led to significant reduction of GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in germ-line dbGATA-deleted mice (Gata1(ΔdbGATA)), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase of GATA2 in progenitors. When we crossed Gata1(ΔdbGATA) mice with Gata2 hypomorphic mutant mice (Gata2(fGN/fGN)), Gata1(ΔdbGATA)::Gata2(fGN/fGN) compound mutant mice succumbed to a significant decrease of progenitors, whereas either single mutant mice maintain progenitors and survive to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, effects of the dbGATA-site deletion on Gata1 expression are subtle in erythroblasts, which show increased GATA1 binding and enhanced accumulation of active histone marks around the 1st intron GATA-motif of the ΔdbGATA locus. These results thus reveal a novel role the dbGATA-motif plays for maintenance of Gata1 expression in the hematopoietic progenitors and a functional compensation between the dbGATA-site and the 1st intron GATA-motif in erythroblasts.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
... In this issue of Blood, Takai et al provide some tantalizing clues on how expression of the GATA1 transcription factor, a master regulator of erythroid/megakaryocytic differentiation, is suppressed in the hematopoietic stem/progenitor cell (HSPC) compartment. 1 S uppression is important because forced expression of GATA1 in the stem cell compartment leads to loss of self-renewal capacity. 2 Members of the GATA factor family of transcription factors have distinct and essential roles in hematopoiesis. 3 GATA2 is essential for maintenance of the HSPC compartment, but it is also involved in the initial activation of GATA1 expression at the first steps of erythroid/megakaryocytic differentiation. This is referred to as the GATA factor switch. 4 A conceptual problem of this mechanism is that GATA1 expression is suppressed in HSPCs, which are dependent on GATA2 activity. ...
... n In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities. 1 T here has been an explosion of interest in field of adoptive cell transfer due to the recent clinical success reported by several groups in the treatment of B cell malignancies. [2][3][4] Durable clinical responses have been documented in several patients, including those heavily pretreated with standard chemotherapy, using T cells genetically ...
In this issue of Blood, Takai et al provide some tantalizing clues on how expression of the GATA1 transcription factor, a master regulator of erythroid/megakaryocytic differentiation, is suppressed in the hematopoietic stem/progenitor cell (HSPC) compartment.
... 30 Furthermore, DNMT1 directly binds to the Gata-1 locus in murine hematopoietic stem cells, resulting in hypermethylation and downregulation of GATA-1, a transcription factor associated with megakaryopoiesis. 31 DENV infection was shown to reduce GATA-1 expression in megakaryocytes. 32 Our results further show that infection with DENV increased DNMT1 and decreased GATA-1 expression in Meg-01 cells ( Figure 4B). ...
Thrombocytopenia is the hallmark finding in dengue virus (DENV) infection. Prothymosin α (ProT) has both intracellular and extracellular functions involved in cell cycle progression, cell differentiation, gene regulation, oxidative stress response, and immunomodulation. In this study, we found that ProT levels were elevated in dengue patient sera as well as DENV-infected megakaryoblasts and their culture supernatants. ProT transgenic mice had reduced platelet counts with prolonged bleeding times. Upon treatment with DENV plus anti-CD41 antibody, they exhibited severe skin hemorrhage. Furthermore, overexpression of ProT suppressed megakaryocyte differentiation. Infection with DENV inhibited miR-126 expression, upregulated DNA (cytosine-5)-methyltransferase 1 (DNMT1), downregulated GATA-1, and increased ProT expression. Upregulation of ProT led to Nrf2 activation and reduced reactive oxygen species production, thereby suppressing megakaryopoiesis. We report the pathophysiological role of ProT in DENV infection and propose an involvement of the miR-126-DNMT1-GATA-1-ProT-Nrf2 signaling axis in DENV-induced thrombocytopenia.
... 104 New findings have challenged this paradigm, where high DNA methylation by DNMT1, not GATA2, is responsible for an inactive GATA1 locus. 102,105 Detailed study is required to determine the role of the GATA switch in the commitment of HSCs to the NK lineage. ...
Natural killer (NK) cells are major innate lymphocytes. NK cells do not require prior antigen exposure to mediate antitumor cytotoxicity or proinflammatory cytokine production. Since they use only nonclonotypic receptors, they possess high clinical value in treatment against a broad spectrum of malignancies. Irrespective of this potential, however , the transcriptional regulation that governs human NK cell development remains far from fully defined. Various environmental cues initiate a complex network of transcription factors (TFs) during their early development, one of which is GATA2, a master regulator that drives the commitment of common lymphoid progenitors (CLPs) into immature NK progenitors (NKPs). GATA2 forms a core heptad complex with six other TFs (TAL1, FLI1, RUNX1, LYL1, LMO2, and ERG) to mediate its transcriptional regulation in various cell types. Patients with GATA2 haploinsufficiency specifically lose CD56 bright NK cells, with or without a reduced number of CD56 dim NK cells. Here, we review the recent progress in understanding GATA2 and its role in human NK cell development and functions.
... Both GATA1 and GATA2 are capable of autoactivating their own expression 112 . New findings have challenged this paradigm where a high DNA methylation by DNMT1, and not GATA2, is responsible for inactive GATA1 locus 110,113 . A detailed study is required to determine the role of the 'GATA switch' in the commitment of HSCs to NK lineage. ...
Natural killer (NK) cells are major innate lymphocytes. NK cells do not require prior antigen exposure to mediate antitumor cytotoxicity or proinflammatory cytokine production. Since they use only nonclonotypic receptors, they possess high clinical value in treatment against a broad spectrum of malignancies. Irrespective of this potential, however, the transcriptional regulation that governs human NK cell development remains far from fully defined. Various environmental cues initiate a complex network of transcription factors (TFs) during their early development, one of which is GATA2, a master regulator that drives the commitment of common lymphoid progenitors (CLPs) into immature NK progenitors (NKPs). GATA2 forms a core heptad complex with six other TFs (TAL1, FLI1, RUNX1, LYL1, LMO2, and ERG) to mediate its transcriptional regulation in various cell types. Patients with GATA2 haploinsufficiency specifically lose CD56bright NK cells, with or without a reduced number of CD56dlm NK cells. Here, we review the recent progress in understanding GATA2 and its role in human NK cell development and functions.
... While the expression of GATA2 is mandatory for the selfrenewal capacity of HSPCs, high GATA1 levels are needed for the transition to MEPs and the subsequent differentiation of the megakaryocyte-erythroid lineage (84,85). This switch between GATA transcription factor expression is realized through the direct transcriptional regulation of GATA1 by GATA2 and vice versa, as well as through epigenetic mechanisms, such as DNA methylation (85,86). It was already shown that, as a consequence of the loss of full length Gata1, the "GATA switch" is impaired, causing perturbation of erythropoiesis (87). ...
Individuals with Down syndrome are genetically predisposed to developing acute megakaryoblastic leukemia. This myeloid leukemia associated with Down syndrome (ML–DS) demonstrates a model of step-wise leukemogenesis with perturbed hematopoiesis already presenting in utero, facilitating the acquisition of additional driver mutations such as truncating GATA1 variants, which are pathognomonic to the disease. Consequently, the affected individuals suffer from a transient abnormal myelopoiesis (TAM)—a pre-leukemic state preceding the progression to ML–DS. In our review, we focus on the molecular mechanisms of the different steps of clonal evolution in Down syndrome leukemogenesis, and aim to provide a comprehensive view on the complex interplay between gene dosage imbalances, GATA1 mutations and somatic mutations affecting JAK-STAT signaling, the cohesin complex and epigenetic regulators.
... Previously, we reported that transgenic reporter mice generated using a bacterial artificial chromosome (BAC) faithfully recapitulated the endogenous expression pattern of the gene carried in the BAC DNA [19][20][21] . To explore the histamine-producing cells and the regulatory mechanism of Hdc gene expression, we generated a histidine decarboxylase BAC DNA-directed GFP reporter transgenic mouse using a 293-kb BAC clone containing the all Hdc exons and extended flanking sequences (referred to as Hdc-GFP, hereafter). ...
Histamine is a biogenic amine that is chiefly produced in mast cells and basophils and elicits an allergic response upon stimulation. Histidine decarboxylase (HDC) is a unique enzyme that catalyzes the synthesis of histamine. Therefore, the spatiotemporally specific Hdc gene expression profile could represent the localization of histamine-producing cells under various pathophysiological conditions. Although the bioactivity of histamine is well defined, the regulatory mechanism of Hdc gene expression and the distribution of histamine-producing cell populations in various disease contexts remains unexplored. To address these issues, we generated a histidine decarboxylase BAC (bacterial artificial chromosome) DNA-directed GFP reporter transgenic mouse employing a 293-kb BAC clone containing the entire Hdc gene locus and extended flanking sequences (Hdc-GFP). We found that the GFP expression pattern in the Hdc-GFP mice faithfully recapitulated that of conventional histamine-producing cells and that the GFP expression level mirrored the increased Hdc expression in lipopolysaccharide (LPS)-induced septic lungs. Notably, a CD11b⁺Ly6G⁺Ly6Clow myeloid cell population accumulated in the lung during sepsis, and most of these cells expressed high levels of GFP and indeed contain histamine. This study reveals the accumulation of a histamine-producing myeloid cell population during sepsis, which likely participates in the immune process of sepsis.
... This might subsequently lead to the potential loss of essential regulatory elements located in the intronic regions of mutated genes, causing an alteration in the dynamic expression of the genetic lesion in targeted cells and thus phenotypic differences. For example, the dynamic expression of the Gata1 gene in erythroid cells versus HSC was shown to be depended on untranslated regulatory elements located at its 5' region [109,110]. ...
Acute myeloid leukaemia (AML) is a rare but severe form of human cancer that results from a limited number of functionally cooperating genetic abnormalities leading to uncontrolled proliferation and impaired differentiation of hematopoietic stem and progenitor cells. Before the identification of genetic driver lesions, chemically, irradiation or viral infection-induced mouse leukaemia models provided platforms to test novel chemotherapeutics. Later, transgenic mouse models were established to test the in vivo transforming potential of newly cloned fusion genes and genetic aberrations detected in patients’ genomes. Hereby researchers constitutively or conditionally expressed the respective gene in the germline of the mouse or reconstituted the hematopoietic system of lethally irradiated mice with bone marrow virally expressing the mutation of interest. More recently, immune deficient mice have been explored to study patient-derived human AML cells in vivo. Unfortunately, although complementary to each other, none of the currently available strategies faithfully model the initiation and progression of the human disease. Nevertheless, fast advances in the fields of next generation sequencing, molecular technology and bioengineering are continuously contributing to the generation of better mouse models. Here we review the most important AML mouse models of each category, briefly describe their advantages and limitations and show how they have contributed to our understanding of the biology and to the development of novel therapies.
... We could demonstrate that decitabine treatment regulated many of the genes also regulated by hemin (including mRNA for erythroid-specific transcription factors and beta-like-globin genes), and induced GATA1 at the protein level, con-comitantly with demethylation at several cis-regulatory regions known to be important for the regulation of this gene. 36,37 Notably, the overlap between the transcriptome changes induced by decitabine versus hemin was more marked in the downregulated genes compared to the upregulated ones. Despite demonstrating GATA1 gene demethylation following decitabine treatment, we are unable to conclude that GATA1 induction is a direct consequence of demethylation or is occurring during erythroid differentiation triggered via other factors. ...
Hematologic responses to hypomethylating agents in myelodysplastic syndrome and acute myeloid leukemia patients are often delayed. Fetal hemoglobin is a potential novel biomarker for response: recently, we could demonstrate that its elevation prior to decitabine treatment was associated with superior outcome. We now asked whether early fetal hemoglobin induction during decitabine treatment also had prognostic value, and investigated the potential of decitabine for in vitro induction of erythroid differentiation and fetal hemoglobin expression. Fetal hemoglobin levels were measured by high-performance liquid chromatography in patients with higher-risk myelodysplastic syndrome (n=16) and acute myeloid leukemia (n=37) before treatment and after each decitabine course. Levels above 1.0% were considered induced. Patients achieving complete or partial remission as best response attained a median fetal hemoglobin of 1.9% after two courses, versus 0.8% in patients without complete or partial remission (p=0.015). Fetal hemoglobin induction after two courses was associated with early platelet doubling (p=0.006), and a subsequent decrease with hematologic relapse. In myelodysplastic syndrome patients, induced fetal hemoglobin after course 2 was associated with longer overall survival compared to non-induced levels: median of 22.9 versus 7.3 months (hazard ratio 0.2 [95% confidence interval 0.1-0.9], p=0.03). In vitro decitabine treatment of 2 bipotential myeloid leukemia cell lines (K562 and HEL) resulted in induction of an erythroid (not megakaryocytic) differentiation program, and of fetal hemoglobin mRNA and protein, associated with GATA1 gene demethylation and upregulation.
Conclusion:
Fetal hemoglobin may provide a useful dynamic biomarker during hypomethylating agent therapy of patients with myelodysplastic syndrome and acute myeloid leukemia.
... Dnmt1 is strongly recruited to the Gata1 methylation determining region (G1MDR) to maintain DNA methylation of the Gata1 locus in HSPCs, while deletion of G1MDR selectively promotes Gata1 repression associated with an increase of GATA2 occupancy in the GATA1 gene enhancer. That interplay between DNMT1-G1DMR-GATA1 enhancer has an essential role in HSPC maintenance, while demethylation promotes activation critical for subsequent GATA2-dependent Gata1 transcription initiation for erythroid commitment and differentiation [89,90]. An increased variability of dynamic DNA methylation at enhancers especially near leukocyte differentiation-promoting genes transitioning between silenced and expressed state in neonatal cord blood HSPCs suggests that methylation is a regulatory mechanism in granulopoiesis in a fashion similar to erythropoiesis. ...
DNA methylation (CpG methylation) exerts an important role in normal differentiation and proliferation of hematopoietic stem cells and their differentiated progeny, while it has also the ability to regulate myeloid versus lymphoid fate. Mutations of the epigenetic machinery are observed in hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) resulting in hyper- or hypo-methylation affecting several different pathways. Enhancers are cis-regulatory elements which promote transcription activation and are characterized by histone marks including H3K27ac and H3K4me1/2. These gene subunits are target gene expression ‘fine-tuners’, are differentially used during the hematopoietic differentiation, and, in contrast to promoters, are not shared by the different hematopoietic cell types. Although the interaction between gene promoters and DNA methylation has extensively been studied, much less is known about the interplay between enhancers and DNA methylation. In hematopoiesis, DNA methylation at enhancers has the potential to discriminate between fetal and adult erythropoiesis, and also is a regulatory mechanism in granulopoiesis through repression of neutrophil-specific enhancers in progenitor cells during maturation. The interplay between DNA methylation at enhancers is disrupted in AML and MDS and mainly hyper-methylation at enhancers raising early during myeloid lineage commitment is acquired during malignant transformation. Interactions between mutated epigenetic drivers and other oncogenic mutations also affect enhancers’ activity with final result, myeloid differentiation block. In this review, we have assembled recent data regarding DNA methylation and enhancers’ activity in normal and mainly myeloid malignancies.
... Expression of the Gata1 gene in hematopoietic stem cells and early progenitor cells is repressed by an epigenetic mechanism that precludes access of GATA2 (9). Along with differentiation, GATA2 primes Gata1 gene expression and results in Gata1 gene self-activation and Gata2 gene repression by GATA1. ...
The transcription factor GATA1 regulates the expression of essential erythroid and megakaryocytic differentiation genes through binding to the DNA consensus sequence WGATAR. The GATA1 protein has four functional domains, including two centrally located zinc-finger domains and two transactivation domains at the N- and C-termini. These functional domains play characteristic roles in the elaborate regulation of diversified GATA1 target genes, each of which exhibits a unique expression profile. Three types of GATA1-related hematological malignancies have been reported. One is a structural mutation in the GATA1 gene, resulting in the production of a short form of GATA1 that lacks the N-terminal transactivation domain and is found in Down syndrome-related acute megakaryocytic leukemia. The other two are cis-acting regulatory mutations affecting expression of the Gata1 gene, which have been shown to cause acute erythroblastic leukemia and myelofibrosis in mice. Therefore, imbalanced gene regulation caused by qualitative and quantitative changes in GATA1 is thought to be involved in specific hematological disease pathogenesis. In the present review, we discuss recent advances in understanding the mechanisms of differential transcriptional regulation by GATA1 during erythroid differentiation, with special reference to the binding kinetics of GATA1 at conformation-specific binding sites.
... MicroRNA-126 is highly expressed in HSCs but quickly downregulated during the early stages of hematopoietic differentiation and this strategy has successfully suppressed ectopic gene expression in HSCs, while maintaining expression in differentiated cells [Gentner et al., 2010]. Furthermore, erythroid-specific regulatory elements for GATA1 expression have already been identified in mice and provide a framework for lineage-restricted expression of GATA1 in hematopoietic cells [Shimizu et al., 2013;Takai et al., 2013]. In the future, unbiased screening approaches, such as massively parallel reporter assays, may have value for identifying regulatory elements with desired expression properties to allow for regulated delivery of such transgenes . ...
Congenital anemias comprise a group of blood disorders characterized by a reduction in the number of peripherally circulating erythrocytes. Various genetic etiologies have been identified that affect diverse aspects of erythroid physiology and broadly fall into two main categories: impaired production or increased destruction of mature erythrocytes. Current therapies are largely focused on symptomatic treatment and are often based on transfusion of donor-derived erythrocytes and management of complications. Hematopoietic stem cell transplantation represents the only curative option currently available for the majority of congenital anemias. Recent advances in gene therapy and genome editing hold promise for the development of additional curative strategies for these blood disorders. The relative ease of access to the hematopoietic stem cell compartment, as well as the possibility of genetic manipulation ex vivo and subsequent transplantation in an autologous manner, make blood disorders among the most amenable to cellular therapies. Here we review cell-based and gene therapy approaches, and discuss the limitations and prospects of emerging avenues, including genome editing tools and the use of pluripotent stem cells, for the treatment of congenital forms of anemia. © 2016 Wiley Periodicals, Inc.
... The dynamic expression of GATA1 is crucial for proper erythroid differentiation, and is tightly regulated by interactions among multiple transcription factors, epigenetic regulators and cisregulatory elements in the Gata1 gene locus. Gata1 locus contains multiple cis-regulatory elements involved in the regulation of Gata1 gene expression, including a Gata1 hematopoietic enhancer (G1HE), a palindromic GATA-binding site (dbG) and two adjacent CACCC boxes [11][12][13][14][15]. Upon erythroid differentiation, demethylation of GATA1 gene regulatory regions allows increased GATA2 binding to the GATA binding sites, which promotes the GATA1 gene expression in early hematopoietic progenitor cells [16]. ...
The dynamic and reversed expression of GATA1 and GATA2 are essential for proper erythroid differentiation. Our previous work demonstrates that LSD1, a histone H3K4 demethylase, represses GATA2 expression at late stage of erythroid differentiation. K562 and MEL cells were used and cultured in Roswell Park Memorial Institute-1640 medium (RPMI) and Dulbecco's modified Eagle's medium (DMEM), respectively. Western blot assay was used to examine the GATA1, GATA2, TAL1, HDAC1, HDAC2, CoREST and β-actin protein. The immunoprecipitation assay and GST pull-down assay were employed to detect the precipitated protein complexes and investigate the interaction between the proteins. The small interfering RNA (siRNA) and nonspecific control siRNA were synthesized to silence the target genes. Double fluorescence immunostaining was used to observe the association of LSD1 with GATA2 in K562 cells. The results indicated that knockdown of LSD1 in K562 cell causes increased H3K4 di-methylation at GATA1 locus and activates GATA1 expression, demonstrating that LSD1 represses GATA1 expression through LSD1-mediated histone demethylation. Upon induced erythroid differentiation of K562 cells, the interaction between GATA2 and LSD1 is decreased, consistent with a de-repression of GATA1 expression. Meanwhile, the interaction between TAL1 and LSD1 is increased, which forms a complex that efficiently suppresses GATA2 expression. In conclusion, these observations reveal an elegant mechanism to modulate GATA1 and GATA2 expression during erythroid differentiation. While LSD1 mainly forms complex with GATA2 to repress GATA1 expression in hematopoietic progenitor cells, it mostly forms complex with TAL1 to repress GATA2 expression in differentiated cells.
... According to these study results, Takai et al. proposed a mechanism in which GATA1 hypomethylation results in an accessible locus for GATA2 binding which enables transactivation of GATA1 gene expression to initiate erythropoiesis in megakaryo-erythroid progenitors (Ref. 15). Loss of GATA1 results in a marked increase of GATA2 expression, indicating not only that GATA2 partially compensates for GATA1 but also that GATA1 suppresses GATA2 transcription during normal erythropoiesis (Ref. ...
The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.
... The copy number and integrity of the transgene were determined by genomic quantitative PCR as previously described (21). Primers used for genomic quantitative PCR are listed in Table 1. ...
Chronic inflammation underlies the pathological progression of various diseases, and thus many efforts have been made to quantitatively
evaluate the inflammatory status of the diseases. In this study, we generated a highly sensitive inflammation-monitoring mouse
system using a bacterial artificial chromosome (BAC) clone containing extended flanking sequences of the human interleukin
6 gene (hIL6) locus, in which the luciferase (Luc) reporter gene is integrated (hIL6-BAC-Luc). We successfully monitored lipopolysaccharide-induced systemic inflammation in various tissues of the hIL6-BAC-Luc mice using an in vivo bioluminescence imaging system. When two chronic inflammatory disease models, i.e., a genetic model of atopic dermatitis
and a model of experimental autoimmune encephalomyelitis (EAE), were applied to the hIL6-BAC-Luc mice, luciferase bioluminescence was specifically detected in the atopic skin lesion and central nervous system, respectively.
Moreover, the Luc activities correlated well with the disease severity. Nrf2 is a master transcription factor that regulates
antioxidative and detoxification enzyme genes. Upon EAE induction, the Nrf2-deficient mice crossed with the hIL6-BAC-Luc mice exhibited enhanced neurological symptoms concomitantly with robust luciferase luminescence in the neuronal tissue. Thus,
whole-body in vivo monitoring using the hIL6-BAC-Luc transgenic system (WIM-6 system) provides a new and powerful diagnostic tool for real-time in vivo monitoring of inflammatory status in multiple different disease models.
... Gata-1 plays a role in erythroid and megakaryocyte differentiation but not in HSCs. However, recent analyses of cis-regulatory elements in the Gata-1 locus identified 3.2-kb interspacing sequences in its upstream region which inactivates Gata-1 expression in HSCs (Takai et al., 2013). Removal of this interspacing sequence in Gata-1-GFP BAC Tg mouse led to a significant increase in frequency of GFP + cells in CD150 + CD48 -KSL fraction. ...
There has been considerable interest in identifying a cis-regulatory element that targets gene expression to stem cells. Such an element, termed stem cell enhancer, holds the promise of providing important insights into the transcriptional programs responsible for inherent stem cell-specific properties such as self-renewal capacity. The element also serves as a molecular handle for stem cell-specific marking, transgenesis and gene targeting, thereby becoming invaluable to stem cell research. A series of candidate enhancers have been identified for hematopoietic stem cells (HSCs). This review summarizes currently known HSC enhancers with emphasis on an intronic enhancer in the Runx1 gene which is essential for the generation and maintenance of HSCs. The element, named eR1 (+24m), is active specifically in HSCs, but not in progenitors, and is hence the most definitive HSC enhancer.
... In the 'GATA switch' process, GATA2 (which maintains the HSC state) is displaced by GATA1 (which promotes erythroid commitment) [216]. This process is best characterized at autoregulatory enhancers targeting the GATA1 and GATA2 genes ( Figure 9B), where the switch can have a neutral regulatory effect or can change the direction of an enhancer's activity (e.g., activator to repressor) [216,[222][223][224]. Importantly, several ChIP-seq studies have demonstrated substantial overlap in the regions targeted by GATA1 and GATA2 at different stages, suggesting that the GATA switch might be part of a global mechanism during erythroid commitment [194,218,221,[225][226][227]. ...
Transcription factors (TFs) influence cell fate by interpreting the regulatory DNA within a genome. TFs recognize DNA in a specific manner; the mechanisms underlying this specificity have been identified for many TFs based on 3D structures of protein-DNA complexes. More recently, structural views have been complemented with data from high-throughput in vitro and in vivo explorations of the DNA-binding preferences of many TFs. Together, these approaches have greatly expanded our understanding of TF-DNA interactions. However, the mechanisms by which TFs select in vivo binding sites and alter gene expression remain unclear. Recent work has highlighted the many variables that influence TF-DNA binding, while demonstrating that a biophysical understanding of these many factors will be central to understanding TF function.
... After 4 weeks of continuous Dox feeding (at 8 weeks old), mice were subjected to analysis (see Fig. 3A). Genomic quantitative real-time PCR was performed to determine the recombination efficiency of the Gata2 flox allele after knockout induction as described earlier (25). Primers used are listed in Table 1. ...
The transcription factor GATA2 plays pivotal roles in early renal development, but its distribution and physiological functions
in adult kidney are largely unknown. We examined the GATA2 expression pattern in the adult kidney by tracing green fluorescent
protein (GFP) fluorescence in Gata2GFP/+ mice that recapitulate endogenous GATA2 expression and found a robust GFP expression specifically in the renal medulla. Upon
purification of the GFP-positive cells, we found that collecting duct (CD)-specific markers, including aquaporin 2 (Aqp2),
an important channel for water reabsorption from urine, were abundantly expressed. To address the physiological function of
GATA2 in the CD cells, we generated renal tubular cell-specific Gata2-deficient mice (Gata2-CKO) by crossing Gata2 floxed mice with inducible Pax8-Cre mice. We found that the Gata2-CKO mice showed a significant decrease in Aqp2 expression. The Gata2-CKO mice exhibited high 24-h urine volume and low urine osmolality, two important signs of diabetes insipidus. We introduced
biotin-tagged GATA2 into a mouse CD-derived cell line and conducted chromatin pulldown assays, which revealed direct GATA2
binding to conserved GATA motifs in the Aqp2 promoter region. A luciferase reporter assay using an Aqp2 promoter-reporter showed that GATA2 trans activates Aqp2 through the GATA motifs. These results demonstrate that GATA2 regulates the Aqp2 gene expression in CD cells and contributes to the maintenance of the body water homeostasis.
Our current understanding of the kinetics and dynamics of erythroid differentiation is based almost entirely on the ex vivo expansion of cultured hematopoietic progenitor cells. In this study, we used an erythroid-specific, inducible transgenic mouse line to investigate for the first time, the in vivo erythroid differentiation kinetics under steady-state conditions. We demonstrated that bipotent premegakaroycyte/erythroid (PreMegE) progenitor cells differentiate into erythroid–committed proerythroblast/basophilic erythroblasts (ProBasoE) after 6.6 days under steady-state conditions. During this process, each differentiation phase (from PreMegE to precolony forming unit-erythroid [PreCFU-E], PreCFU-E to CFU-E, and CFU-E to ProBasoE) took ∼2 days in vivo. Upon challenge with 5-flurouracil (5-FU), which leads to the induction of stress erythropoiesis, erythroid maturation time was reduced from 6.6 to 4.7 days. Furthermore, anemia induced in 5-FU-treated mice was shown to be due not only to depleted bone marrow erythroid progenitor stores but also to a block in reticulocyte exit from the bone marrow into the circulation, which differed from the mechanism induced by acute blood loss.
Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.
Mast cells serve as a first-line defense of innate immunity. IL-6 induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1 clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.
Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of b-globinopathies (sickle cell disease and b-thalassemia) since it is a component of g-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 BAC transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of GMP-like cells, converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells (HSPC), coincident with induction of myeloid transcription factors (e.g. PU.1 and CEBPa). Finally, blocking the activity of transcription factors PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors, and that inhibition of the myeloid differentiation pathway reverses the lineage switch induced by LSD1 inactivation.
Sideroblastic anemia is characterized by the presence of ring sideroblasts, which are caused by iron accumulation in the mitochondria of erythroblasts and are present in both acquired and congenital forms of sideroblastic anemia. However, the mechanism leading to ring sideroblast formation remains elusive. Acquired sideroblastic anemia is usually observed in myelodysplastic syndrome. Because a subset of myelodysplastic syndrome harbors a somatic mutation of TET2, it may be involved in iron metabolism and/or heme biosynthesis in erythroblasts. Tet2 knockdown (Tet2trap) induced exhibited mild normocytic anemia and elevated serum ferritin levels in 4-month-old mice. Although typical ring sideroblasts were not observed, increased mitochondrial ferritin amounts were observed in the erythroblasts of Tet2-knockdown mice. Quantitative real-time–polymerase chain reaction demonstrated significant dysregulation of genes involved in iron and heme metabolism, including Hmox1, Fech, Abcb7, and Sf3b1 downregulation. Following the identification of a CpG island in the promoters of Fech, Abcb7, and Sf3b1, we evaluated DNA methylation status and found significantly high methylation levels at the CpG sites in erythroblasts of Tet2-knockdown mice. Furthermore, Tet2 knockdown in erythroblasts resulted in decreased heme concentration as well as accumulation of mitochondrial ferritin. Thus, TET2 plays a role in the iron and heme metabolism in erythroblasts.
The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and show overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching of the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice in vivo have shown that GATA2 and GATA1 are functionally non-redundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, demonstrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematological analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematological diseases, focusing on the regulation of GATA factor gene expression.
GATA1 organizes erythroid and megakaryocytic differentiation by orchestrating the expression of multiple genes that show diversified
expression profiles. Here, we demonstrate that GATA1 monovalently binds to a single GATA motif (Single-GATA), while a monomeric
and a homodimeric GATA1 bivalently bind to two GATA motifs in palindromic (Pal-GATA) and direct-repeat (Tandem-GATA) arrangements,
respectively, and form higher stoichiometric complexes on respective elements. The amino-terminal zinc (N)-finger of GATA1
critically contributes to high-occupancy of GATA1 on Pal-GATA. GATA1 lacking the N-finger-DNA association fails to trigger
a comparable rate of target gene expression seen with the wild type GATA1, especially when expressed at low-level. This study
revealed that Pal-GATA and Tandem-GATA generate distinct transcriptional responses from GATA1 target genes than Single-GATA.
Our results support the notion that the distinct alignments in binding motifs are part of a critical regulatory strategy that
diversifies and modulates transcriptional regulation by GATA1.
In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities.
During renal development, the proper emergence of the ureteric bud (UB) from the Wolffian duct is essential for formation
of the urinary system. Previously, we showed that expression of transcription factor GATA-2 in the urogenital primordium was
demarcated anteroposteriorly into two domains that were regulated by separate enhancers. While GATA-2 expression in the caudal
urogenital mesenchyme is controlled by the UG4 enhancer, its more-rostral expression is regulated by UG2. We found that anteriorly
displaced budding led to obstructed megaureters in Gata2 hypomorphic mutant mice, possibly due to reduced expression of the downstream effector bone morphogenetic protein 4 (BMP4).
Here, we report that UG4-driven, but not UG2-driven, GATA-2 expression in the urogenital mesenchyme significantly reverts
the uropathy observed in the Gata2 hypomorphic mutant mice. Furthermore, the data show that transgenic rescue by GATA-2 reverses the rostral outgrowth of the
UB. We also provide evidence for a GATA-2–BMP4 epistatic relationship by demonstrating that reporter gene expression from
a Bmp4 bacterial artificial chromosome (BAC) transgene is altered in Gata2 hypomorphs; furthermore, UG4-directed BMP4 expression in the mutants leads to reduced incidence of megaureters. These results
demonstrate that GATA-2 expression in the caudal urogenital mesenchyme as directed by the UG4 enhancer is crucial for proper
development of the urinary tract and that its regulation of BMP4 expression is a critical aspect of this function.
Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.
Leukemia stem cells (LSCs) might originate from malignant transformation of normal hematopoietic stem cells (HSCs), or alternatively, of progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. LSCs are on top of a hierarchy and generate leukemia cells with more differentiated characteristics. While most leukemia cells are initially sensitive to chemo- and radiotherapy, LSCs are resistant and are considered to be the basis for disease relapse after initial response. Albeit important knowledge on LSC biology has been gained from xenogeneic transplantation models introducing human leukemia cells into immune deficient mouse models, the prospective identification and isolation of human LSC candidates has remained elusive and their prognostic and therapeutic significance controversial. This review focuses on the identification, enrichment and characterization of human LSC derived from patients with acute myeloid leukemia (AML). Experimental data demonstrating the clinical significance of estimating LSC burden and strategies to eliminate LSC will be summarized. For long-term cure of AML, it is of importance to define LSC candidates and to understand their tumor biology compared to normal HSCs. Such comparative studies might provide novel markers for the identification of LSC and for the development of treatment strategies that might be able to eradicate them.
A region located at kbp −3.9 to −2.6 5′ to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection
analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent
manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent
and also required the presence of the endogenousGATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in
transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus
additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed
that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL−/− embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls
the expression of this gene.
DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer.
GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5′ region of the enhancer. However, the
importance of the GC-rich region adjacent to the 3′-end of this GATA site has been also suggested. In this study, we show
that this GC-rich region contains five contiguous deoxyguanosine residues (G5 string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G5 string (G4 mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding
factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89
to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89
activates the Gata1 enhancer depending on the G5 string sequence. Finally, transgenic mouse studies reveal that the G4 mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G5 string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.
GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP)
reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully
recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification
protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and
substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage
expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression
of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results
demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.
The transcription factor GATA-1 is expressed in a subset of hemopoietic cells, where it mediates the celltype specific expression
of several genes. We have cloned the mouse and human GATA-1 genes. A region upstream to the first exon, and highly conserved
between mouse and man, acts as an erythroid specific enhancer in transient assays, if linked to the GATA-1 or to the SV40
promoter. The activity of the enhancer is almost completely dependent on the integrity of a dimeric GATA-1 binding site.
Transcription factor GATA-1 is required for the terminal differentiation of both the primitive and definitive erythroid cell
lineages, and yet the regulatory mechanisms of GATA-1 itself are not well understood. To clarify how the GATA-1 gene is transcriptionally
controlled in vivo, presumptive regulatory regions of the gene were tested by fusion to a reporter gene and then examined in transgenic mice.
We found that a transcriptional control element located between −3.9 and −2.6 kb 5′ to the erythroid first exon serves as
an activating element and that this sequence alone is sufficient to recapitulate the expression of GATA-1 (but uniquely in
primitive erythroid cells). Addition of sequences from the GATA-1 first intron to this upstream element provides a necessary
and sufficient condition for complete recapitulation of GATA-1 expression in both primitive and definitive erythroid cells.
The first intron element does not possess intrinsic transcriptional activation potential when linked to the GATA-1 gene promoter
but rather requires the upstream activating element for its activity. These experiments show that GATA-1 gene expression is
regulated by discrete transcriptional control elements during definitive and primitive erythropoiesis: The 5′ element displays
properties anticipated for a primitive erythroid cell-specific activating element, and the novel element within the GATA-1
first intron specifically augments this activity in definitive erythroid cells.
To elucidate the in vivo function of GATA-1 during hematopoiesis, we specifically disrupted the erythroid promoter of the GATA-1 gene in embryonic
stem cells and generated germ line chimeras. Male offspring of chimeras bearing the targeted mutation were found to die by
12.5 days post coitus due to severe anemia while heterozygous females displayed characteristics ranging from severe anemia
to normal erythropoiesis. When female heterozygotes were crossed with transgenic males carrying a reporter gene, which specifically
marks primitive erythroid progenitors, massive accumulation of undifferentiated erythroid cells were observed in the yolk
sacs of the GATA-1-mutant embryos, demonstrating that GATA-1 is required for the terminal differentiation of primitive erythroid
cells in vivo.
Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.
Interplay among GATA transcription factors is an important determinant of cell fate during hematopoiesis. Although GATA-2 regulates hematopoietic stem cell function, mechanisms controlling GATA-2 expression are undefined. Of particular interest is the repression of GATA-2, because sustained GATA-2 expression in hematopoietic stem and progenitor cells alters hematopoiesis. GATA-2 transcription is derepressed in erythroid precursors lacking GATA-1, but the underlying mechanisms are unknown. Using chromatin immunoprecipitation analysis, we show that GATA-1 binds a highly restricted upstream region of the approximately 70-kb GATA-2 domain, despite >80 GATA sites throughout the domain. GATA-2 also binds this region in the absence of GATA-1. Genetic complementation studies in GATA-1-null cells showed that GATA-1 rapidly displaces GATA-2, which is coupled to transcriptional repression. GATA-1 also displaces CREB-binding protein (CBP), despite the fact that GATA-1 binds CBP in other contexts. Repression correlates with reduced histone acetylation domain-wide, but not altered methylation of histone H3 at lysine 4. The GATA factor-binding region exhibited cell-type-specific enhancer activity in transient transfection assays. We propose that GATA-1 instigates GATA-2 repression by means of disruption of positive autoregulation, followed by establishment of a domain-wide repressive chromatin structure. Such a mechanism is predicted to be critical for the control of hematopoiesis.
The erythrocyte colony-forming unit (CFU-E) is a rare bone marrow (BM) progenitor that generates erythrocyte colonies in 48 hours. The existence of CFU-Es is based on these colonies, but CFU-Es have not been purified prospectively by phenotype. We have separated the "nonstem," "nonlymphoid" compartment (lineage marker [lin]-c-Kit+Sca-1-IL-7Ralpha-) into interleukin 3 receptor alpha negative (IL-3Ralpha-) and IL-3Ralpha+ subsets. Within IL-3Ralpha- but not IL-3Ralpha+ cells we have identified TER119-CD41-CD71+ erythrocyte-committed progenitors (EPs). EPs generate CFU-E colonies at about 70% efficiency and generate reticulocytes in vivo. Depletion of EPs from BM strongly reduces CFU-E frequencies. EPs lack potential for erythrocyte burst-forming unit, megakaryocyte, granulocyte (G), and monocyte (M) colonies, and for spleen colony-forming units. Chronically suppressed erythropoiesis in interferon consensus sequence-binding protein (ICSBP)-deficient BM is associated with reduced frequencies of both the EP population and CFU-E colonies. During phenylhydrazine-induced acute anemia, numbers of both the EP population and CFU-E colonies increase. Collectively, EPs (lin-c-Kit+Sca-1-IL-7Ralpha-IL-3Ralpha-CD41-CD71+) account for most, if not all, CFU-E activity in BM. As a first molecular characterization, we have compared global gene expression in EPs and nonerythroid GM progenitors. These analyses define an erythroid progenitor-specific gene expression pattern. The prospective isolation of EPs is an important step to analyze physiologic and pathologic erythropoiesis.
The interaction between stem cells and their supportive microenvironment is critical for their maintenance, function, and survival. Whereas hematopoietic stem cells (HSCs) are among the best characterized of tissue stem cells, their precise site of residence (referred to as the niche) in the adult bone marrow has not been precisely defined. In this study, we found that a Gata2 promoter directs activity in all HSCs. We show that HSCs can be isolated efficiently from bone marrow cells by following Gata2-directed GFP fluorescence, and that they can also be monitored in vivo. Each individual GFP-positive cell lay in a G0/G1 cell cycle state, in intimate contact with osteoblasts beside the endosteum, at the edge of the bone marrow. We conclude that the HSC niche is composed of solitary cells and that adult bone marrow HSC are not clustered.
• GATA-2
• GFP knock-in mouse
• imaging
Transcription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFPhigh fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFPlow/transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burst-forming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues.
DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.
Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.
• hematopoiesis
• chromatin
• gene expression
One of the most challenging projects in the field of epigenetics is the generation of detailed functional maps of DNA methylation in different cell and tissue types in normal and disease-associated conditions. This information will help us not only understand the role of DNA methylation but also identify targets for therapeutic treatment. The completion of the various epigenome projects depends on the design of novel strategies to survey and generate detailed cartograms of the DNA methylome. Methyl-DNA immunoprecipitation (MeDIP) assays, in combination with hybridization on high-resolution microarrays or high-throughput sequencing (HTS) techniques, are excellent methods for identifying methylated CpG-rich sequences. We provide a critical overview of different genome-wide techniques for DNA methylation analysis and propose that MeDIP assays may constitute a key method for elucidating the hypermethylome of cancer cells.
The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre-mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre-mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.
The zinc-finger transcription factor GATA-2 plays a critical role in maintaining the pool of early hematopoietic cells. To define its specific functions in the proliferation, survival, and differentiation of hematopoietic cells, we analyzed the hematopoietic potential of GATA-2−/− cells in in vitro culture systems for proliferation and maintenance of uncommitted progenitors or differentiation of specific lineages. From a two-step in vitro differentiation assay of embryonic stem cells and in vitro culture of yolk sac cells, we demonstrate that GATA-2 is required for the expansion of multipotential hematopoietic progenitors and the formation of mast cells, but dispensable for the terminal differentiation of erythroid cells and macrophages. The rare GATA-2−/− multipotential progenitors that survive proliferate poorly and generate small colonies with extensive cell death, implying that GATA-2 may play a role in both the proliferation and survival of early hematopoietic cells. To explore possible mechanisms resulting in the hematopoietic defects of GATA-2−/− cells, we interbred mutant mouse strains to assess the effects of p53 loss on the behavior of GATA-2−/− hematopoietic cells. Analysis of GATA-2−/−/p53−/− compound-mutant embryos shows that the absence of p53 partially restores the number of total GATA-2−/− hematopoietic cells, and therefore suggests a potential link between GATA-2 and p53 pathways.
Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.
In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.
Transcription of erythroid-expressed genes and normal erythroid development in vivo are dependent on a regulatory protein (GATA-1) that recognizes a consensus GATA motif. GATA-1 expression is itself restricted to erythroid progenitors and to two related hematopoietic lineages, megakaryocytes and mast cells. During cellular maturation the levels of GATA-1 RNA and protein increase progressively. In an effort to delineate mechanisms by which this pivotal transcription factor is itself regulated we have characterized the mouse GATA-1 gene and cis-elements within its promoter. We find that the isolated promoter retains cell specificity exhibited by the intact gene. Full promoter activity requires the presence of proximal CACCC box sequences and an upstream, double GATA motif that binds a single GATA-1 molecule in an asymmetric fashion. Using in vivo footprinting of mouse erythroleukemic cells we detect protein binding in vivo to both cis-elements. On the basis of these findings we propose that a positive feedback loop mediated through GATA-1 serves two complementary functions: maintenance of the differentiated state by locking the promoter into an "on" state, and programming the progressive increase in protein content throughout cellular maturation.
Retrovirus-mediated gene transfer was used to study the effects of dysregulated expression of the zinc-finger transcription factor, GATA-1, which has been shown to be required for erythropoiesis. A retroviral vector (PGK-GATA-1) was constructed with the murine GATA-1 gene linked to the human phosphoglycerate kinase (PGK) promoter. Expression of GATA-1 was demonstrated by super-shift analysis with a monoclonal antibody against murine GATA-1 using extracts of nonerythroid cytotoxic T-lymphocyte line (CTLL) cells transduced with the PGK-GATA-1 virus. Mouse bone marrow cells were transduced in vitro and transplanted into recipient animals. Polymerase chain reaction (PCR) analysis performed on DNA extracted from peripheral blood 12 to 40 weeks posttransplantation demonstrated the presence of the PGK-GATA-1 provirus. Proviral integrity and copy number were demonstrated by Southern blot analysis of DNA from spleen, thymus, and bone marrow tissues from the long-term animals. At 16 weeks posttransplant, animals that received cells transduced by the GATA-1 virus maintained a lower white blood cell (WBC) count and absolute neutrophil count (ANC) and a higher red blood cell (RBC) count than control animals that received cells transduced with a virus containing a neor gene. Erythropoiesis was stimulated in GATA-1 and control animals by phlebotomy. GATA-1 animals required more extensive phlebotomy to reach a hematocrit less than 25 and their hematocrit returned to normal levels sooner than control animals. The effect of twice-daily injections of 10 U recombinant erythropoietin (epo) was also examined. The hematocrit of GATA-1 animals showed a more rapid and elevated response to epo than the hematocrit of control animals. These data suggest that dysregulated expression of GATA-1 in primitive hematopoietic cells enlarges the pool of epo-responsive erythroid progenitor cells.
Blood cell development relies on the expansion and maintenance of haematopoietic stem and progenitor cells in the embryo. By gene targeting in mouse embryonic stem cells, we demonstrate that the transcription factor GATA-2 plays a critical role in haematopoiesis, particularly of an adult type. We propose that GATA-2 regulates genes controlling growth factor responsiveness or the proliferative capacity of early haematopoietic cells.
The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.
In the hematopoietic lineage, the transcription factors GATA-1 and GATA-2 show restricted and largely overlapping expression profiles, but GATA-2 is uniquely expressed in early hematopoietic progenitors. GATA-3 is found exclusively in T cells of hematopoietic lineage. To clarify whether these expression profiles are preserved or changed during the development of malignancies, we analyzed the expression of GATA factors in the blasts from leukemic children. A total of 18 myelogenous leukemia and 24 lymphoblastic leukemia (ALL) cases were investigated. In the majority of the former cases, GATA-2 mRNA expression and the expression of CD34 and c-kit antigens on leukemic cells were demonstrated. In contrast, GATA-2 mRNA and c-kit antigen could not be detected in CD34-positive cells from ALL patients. GATA-3 mRNA was expressed in all T-ALL cases, but not in any precursor B-ALL. These findings suggest that down-regulation of GATA-2 and expression of GATA-3 are important events for the commitment of cells to lymphoid and T cell lineage, respectively. The expression profiles of GATA factors in leukemic cells are generally consistent with those in their normal counterparts, and thus provide a useful tool to determine the lineage commitment of unclassified leukemia.
The zinc-finger transcription factor GATA-2 plays a critical role in maintaining the pool of early hematopoietic cells. To define its specific functions in the proliferation, survival, and differentiation of hematopoietic cells, we analyzed the hematopoietic potential of GATA-2-/- cells in in vitro culture systems for proliferation and maintenance of uncommitted progenitors or differentiation of specific lineages. From a two-step in vitro differentiation assay of embryonic stem cells and in vitro culture of yolk sac cells, we demonstrate that GATA-2 is required for the expansion of multipotential hematopoietic progenitors and the formation of mast cells, but dispensable for the terminal differentiation of erythroid cells and macrophages. The rare GATA-2-/- multipotential progenitors that survive proliferate poorly and generate small colonies with extensive cell death, implying that GATA-2 may play a role in both the proliferation and survival of early hematopoietic cells. To explore possible mechanisms resulting in the hematopoietic defects of GATA-2-/- cells, we interbred mutant mouse strains to assess the effects of p53 loss on the behavior of GATA-2-/- hematopoietic cells. Analysis of GATA-2-/-/p53-/- compound-mutant embryos shows that the absence of p53 partially restores the number of total GATA-2-/- hematopoietic cells, and therefore suggests a potential link between GATA-2 and p53 pathways.
The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5' 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element.
Despite the accumulation of informat on on the origin of hematopoietic stem cells, it is still unclear how these cells are generated in ontogeny. Isolation of cell lines equivalent to early embryonic hematopoietic progenitor cells can be helpful. A multipotent hematopoietic progenitor cell line, A-6, was isolated from H-1 embryonic stem (ES) cells. The self-renewal of A-6 cells was supported by basic-fibroblast growth factor (b-FGF) and their differentiation into definitive erythroid cells, granulocytes and macrophages was induced after co-culture with ST-2 stromal cells. A-6 cells were positive for the surface markers of hematopoietic stem cell, c-kit, CD31, CD34, Flt3/Flk2, PgP-1, and HSA, but were negative for that of the differentiated cells. Reverse transcription-polymerase chain reaction analysis showed that A-6 cells produced mRNA from SCL/tal-1 and GATA-2 genes. Among various cytokines examined, on y stem cell factor (SCF) and Flt3/Flk2 ligand (FL) supported the proliferation of A-6 cells instead of b-FGF. The FL, as well as b-FGF, supported the self-renewal of A-6 cells, whereas SCF induced differentiation into myeloid cells. A-6 cells will be useful for the characterization of hematopoietic progenitor cells derived from ES cells and provide a model system to realize the control mechanisms between self-renewal and different ation of hematopoietic stem cells.
Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.
Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a(-/-)5b(-/-) mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a(-/-)5b(-/-) mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a(-/-)5b(-/-) mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a(-/-)5b(-/-) hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-x(L) and increased apoptosis in Stat5a(-/-)5b(-/-) early erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.
Aberrant methylation of promoter-associated CpG islands is an epigenetic oncogenic mechanism. The objective of this study was to define the methylation characteristics of patients with acute lymphocytic leukemia (ALL).
Using bisulfite-PCR followed by restriction enzyme digestion (COBRA), we have analyzed the methylation status of 10 promoter-associated CpG islands in 80 untreated adult patients with ALL.
Mean methylation density of MDR1, THBS2, MYF3, ER, p15, THBS1, CD10, C-ABL, and p16 was 24.5%, 20.8%, 17.6%, 16.1%, 11.3%, 8.9%, 4.5%, 3.7%, and 1.3% respectively. p73 was methylated in 17 of 80 cases (21.2%). A total of 86.2% of the cases had methylation of at least one gene, and 42.5% of the cases had methylation of three or more genes. MDR1 methylation was inversely correlated with age (P = 0.01). CD10 methylation inversely correlated with CD10 expression (P = 0.0001). Methylation of MDR1 and THBS1 was inversely associated with the presence of the Philadelphia chromosome, whereas C-ABL methylation correlated with the presence of the p210 variant of the Philadelphia chromosome. In univariate analysis, methylation of THBS1 was associated with a favorable outcome (P = 0.02), whereas methylation of p73, p15, and C-ABL was associated with a trend toward worse prognosis.
Aberrant DNA methylation of promoter-associated CpG islands is very common in adult ALL and potentially defines subgroups with distinct clinical and biological characteristics.
The transcription factors GATA-1 and GATA-2 play key roles in gene regulation during erythropoiesis. Gene ablation studies in mouse revealed that GATA-2 is crucial for the maintenance and proliferation of immature hematopoietic progenitors, whereas GATA-1 is essential for the survival of erythroid progenitors as well as the terminal differentiation of erythroid cells. Both GATA-1 and GATA-2 are regulated in a cell-type-specific manner, their expression being strictly controlled during the development and differentiation of erythroid cells. Closer examination revealed a cross-regulatory mechanism by which GATA-1 can control the expression of GATA-2 and vice versa, possibly via essential GATA binding sites in their cis-acting elements. In addition, recent studies identified several human inherited hematopoietic disorders that are caused by mutations in cis-acting GATA binding motifs or mutations in GATA-1 itself.
The GATA-1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5' to the haematopoietic first exon, is essential for GATA-1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA-1 gene in vivo, indicating that additional regulatory sequences are necessary.
We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis-acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type-specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA-1 cDNA under the control of the complete 979 bp minigene rescued GATA-1 germ line mutant mice from embryonic lethality.
A combination of four distinct sequence motifs co-operatively serve as a fundamental functional unit for GATA-1 erythroid transcription in vivo.
The erythroid transcription factor GATA-1 is also expressed in Sertoli cells of the testis. The testicular expression of GATA-1 is regulated in a developmental and spermatogenic stage-specific manner. To further clarify the regulatory mechanisms of testicular GATA-1 gene expression, we carried out transgenic reporter gene expression analyses.
We found that GATA-1 expression in Sertoli cells is markedly decreased concomitant with the emergence of elongated spermatids in the seminiferous tubules. Transgenic reporter mouse analyses revealed that a 15 kb GATA-1 genomic region is sufficient to recapitulate the gene expression profile in Sertoli cells. While the GATA-1 haematopoietic enhancer and the proximal first exon are included within the 15 kb genomic region, these regulatory elements are not essential for GATA-1 expression in Sertoli cells. Further analyses using deletion constructs revealed that a 1.5 kb region 5' to the GATA-1 haematopoietic enhancer is essential for gene expression in Sertoli cells and this region is referred to as the GATA-1 testis activation region.
These results thus demonstrated that the GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene. The 15 kb genomic region is applicable and useful for the expression vector system specific for adult Sertoli cells in stage VII to IX.
GATA-1 is an essential transcription factor for megakaryocyte and erythrocyte (MegE) development. Here we show that hematopoietic progenitors can be reprogrammed by the instructive action of GATA-1. Enforced expression of GATA-1 in hematopoietic stem cells led to loss of self-renewal activity and the exclusive generation of MegE lineages. Strikingly, ectopic GATA-1 reprogrammed common lymphoid progenitors as well as granulocyte/monocyte (GM) progenitors to differentiate into MegE lineages, while inhibiting normal lymphoid or GM differentiation. GATA-1 upregulated critical MegE-related transcription factors such as FOG-1 and GATA-2 in lymphoid and GM progenitors, and their MegE development did not require "permissive" erythropoietin signals. Furthermore, GATA-1 induced apoptosis of proB and myelomonocytic cells, which could not be prevented by enforced permissive Bcl-2 or myeloid cytokine signals. Thus, GATA-1 specifically instructs MegE commitment while excluding other fate outcomes in stem and progenitor cells, suggesting that regulation of GATA-1 is critical in maintaining multilineage homeostasis.
The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.
Pluripotent stem cells, similar to more restricted stem cells, are able to both self-renew and generate differentiated progeny. Although this dual functionality has been much studied, the search for molecular signatures of 'stemness' and pluripotency is only now beginning to gather momentum. While the focus of much of this work has been on the transcriptional features of embryonic stem cells, recent studies have indicated the importance of unique epigenetic profiles that keep key developmental genes 'poised' in a repressed but activatable state. Determining how these epigenetic features relate to the transcriptional signatures of ES cells, and whether they are also important in other types of stem cell, is a key challenge for the future.
Recent years have seen unprecedented characterization of mammalian chromatin thanks to advances in chromatin assays, antibody development, and genomics. Genome-wide maps of chromatin state can now be readily acquired using microarrays or next-generation sequencing technologies. These datasets reveal local and long-range chromatin patterns that offer insight into the locations and functions of underlying regulatory elements and genes. These patterns are dynamic across developmental stages and lineages. Global studies of chromatin in embryonic stem cells have led to intriguing hypotheses regarding Polycomb/trithorax and RNA polymerase roles in 'poising' transcription. Chromatin state maps thus provide a rich resource for understanding chromatin at a 'systems level', and a starting point for mechanistic studies aimed at defining epigenetic controls that underlie development.
GATA-12dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling
- J A Grass
- M E Boyer
- S Pal
- J Wu
- M J Weiss
- E H Bresnick
Grass JA, Boyer ME, Pal S, Wu J, Weiss MJ,
Bresnick EH. GATA-12dependent transcriptional
repression of GATA-2 via disruption of positive
autoregulation and domain-wide chromatin
remodeling. Proc Natl Acad Sci USA. 2003;
100(15):8811-8816.
Chromatin state maps: new technologies, new insights.
- Mendenhall
Mendenhall EM, Bernstein BE. Chromatin state
maps: new technologies, new insights. Curr Opin
Genet Dev. 2008;18(2):109-115.
Ineffective erythropoiesis in Stat5a(2/2)5b(2/2) mice due to decreased survival of early erythroblasts
- M Socolovsky
- H Nam
- M D Fleming
- V H Haase
- C Brugnara
- H F Lodish
Socolovsky M, Nam H, Fleming MD, Haase VH,
Brugnara C, Lodish HF. Ineffective erythropoiesis
in Stat5a(2/2)5b(2/2) mice due to decreased
survival of early erythroblasts. Blood. 2001;
98(12):3261-3273.
Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis
- J L Attema
- P Papathanasiou
- E C Forsberg
- J Xu
- S T Smale
- I L Weissman
Attema JL, Papathanasiou P, Forsberg EC,
Xu J, Smale ST, Weissman IL. Epigenetic
characterization of hematopoietic stem cell
differentiation using miniChIP and bisulfite
sequencing analysis. Proc Natl Acad Sci USA.
2007;104(30):12371-12376.
- Ec Buss
- Ad Ho
Buss EC, Ho AD. Leukemia stem cells. Int J
Cancer. 2011;129(10):2328-2336.
A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.
- Akashi
Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis.
- Attema
Ineffective erythropoiesis in Stat5a(−/−)5b(−/−) mice due to decreased survival of early erythroblasts.
- Socolovsky
GATA-1−dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling.
- Grass