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Prem Ponka - One of the best experts on this subject based on the ideXlab platform.
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oxidative stress and increased destruction of red blood cells contribute to the pathophysiology of anemia caused by dmt1 deficiency
Blood, 2014Co-Authors: Zuzana Zidova, Prem Ponka, Vladimir Divoky, Katarina Kapralova, Pavla Koralkova, Renata Mojzikova, Dalibor Dolezal, Daniel Garciasantos, Monika HorvathovaAbstract:Inactivating mutations in divalent metal transporter 1 (DMT1) are associated with a severe defect in erythroid iron utilization and cause moderate to severe hypochromic microcytic anemia in human patients and two rodent models. We have previously shown that DMT1 deficiency impairs erythroid differentiation, induces apoptosis of erythroid precursors and causes the suppression of colony-forming capacity of erythroid progenitors. Using in vitro cultures of fetal liver cells we were able to recapitulate this in vivo defect. We confirmed abnormal pattern of erythroid differentiation and increased apoptosis (2.5-times) of DMT1-mutant Erythroblasts when compared to wild-type (wt) fetal liver erythroblats. Determination of 2’,7’-Dichlorofluorescein diacetate-dependent intensity of fluorescence, which is proportional to the concentration of reactive oxygen species (ROS), revealed elevated levels of ROS in DMT1-mutant erythroblats when compared to wt Erythroblast. This result suggests that oxidative stress contributes to the apoptosis in DMT1-mutant cells. We also observed that the defective erythroid differentiation of DMT1-mutant Erythroblasts is marked by a blunted induction of heme oxygenase-1, an enzyme that co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells (Garcia-Santos et al., Blood, 2014, 123 (14): 2269-77). In further studies we focused on mature red blood cells (RBC), because it is known that nutritional iron deficiency and certain types of congenital hypochromic anemia are associated with increased levels of ROS and shortened life span of RBC that can be at least partially attributed to a programmed cell death of erythrocytes, so called eryptosis (Lang et al., Int J Biochem Cell Biol, 2012, 44 (8): 1236-43). Using labeling with carboxyfluorescein diacetate succinimidyl ester, we observed an accelerated clearance of DMT1-mutant RBC from circulating blood when compared to wild-type RBC. In vitro, DMT1-mutant RBC exposed to hyperosmotic shock or glucose depletion showed significantly increased levels of phosphatidylserine on the membrane detected by Annexin V binding. Together, these results confirmed eryptosis of DMT1-mutant RBC. As eryptosis is proposed to be triggered via activation of Ca2+ cation channels, we next measured the concentration of cytosolic Ca2+ using Fluo3/AM fluorescent dye and found significantly elevated content of intracellular Ca2+ in DMT1-mutant RBC when compared to wt RBC. In addition, DMT1-mutant RBC had higher levels of ROS than wt RBC despite significantly increased activity of anti-oxidative defense enzymes; glutathione peroxidase (1.6-times), catalase (1.9-times) and methemoglobin reductase (1.9-times). This indicates that exaggerated anti-oxidative defense in DMT1-mutant RBC is not sufficient to eliminate ROS effectively. Furthermore, DMT1-mutant RBC also showed accelerated anaerobic glycolysis as detected by increased activities of hexokinase (2.5-times), pyruvate kinase (2.4-times), glucose-phosphate isomerase (3.2-times). This result together with reduced ATP/ADP (1.6-times) ratio in DMT1-mutant RBC when compared to wt RBC suggests an increased demand for ATP in DMT1-mutant erythrocytes. In conclusion we propose that increased oxidative stress and accelerated destruction of RBC contribute to the pathophysiology of anemia caused by DMT1-deficiency. Grant support: Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic, grant No. NT13587, Education for Competitiveness Operational Program, CZ.1.07/2.3.00/20.0164, Internal Grant of Palacky University Olomouc, LF_2014_011 and in part by the Canadian Institutes of Health Research (D.G-S., P.P.). Disclosures No relevant conflicts of interest to declare.
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new insights into erythropoiesis the roles of folate vitamin b12 and iron
Annual Review of Nutrition, 2004Co-Authors: Mark J. Koury, Prem PonkaAbstract:▪ Abstract Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B12, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B12 for proliferation during their differentiation. Deficiency of folate or vitamin B12 inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes Erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in Erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded product...
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new insights into erythropoiesis the roles of folate vitamin b12 and iron
Annual Review of Nutrition, 2004Co-Authors: Mark J. Koury, Prem PonkaAbstract:Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B12, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B12 for proliferation during their differentiation. Deficiency of folate or vitamin B12 inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes Erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in Erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded production rates with smaller, less hemoglobinized erythrocytes.
John G Conboy - One of the best experts on this subject based on the ideXlab platform.
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an important class of intron retention events in human Erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
RNA, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, the splicing machinery in differentiating Erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ∼50% of its transcripts in late Erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1 kb) retained introns than they are in small retained introns or in nonretained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, thereby blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the Erythroblast IR program.
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an important class of intron retention events in human Erythroblasts is regulated by cryptic exons proposed to function as splicing decoys
bioRxiv, 2018Co-Authors: Marilyn Parra, Ben W Booth, Richard Weiszmann, Brian A Yee, Gene W Yeo, James B Brown, Susan E Celniker, John G ConboyAbstract:During terminal erythropoiesis, differentiating Erythroblasts execute a robust program of intron retention (IR). We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late Erythroblasts as a nuclear isoform that retains intron 4. RNA-seq splice junction reads from nonsense-mediated decay (NMD)-inhibited cells revealed that highly conserved intron sequences encode cryptic cassette exons, and minigene splicing reporter assays showed that these cassettes function as decoys that promote IR. Novel decoy exons were common in large (>1kb) retained introns, and heterologous decoys promoted retention of intron 4. Although most decoys were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that decoy exons engage intron-terminal splice sites, blocking cross-intron interactions required for excision, and that developmental regulation of decoy function underlies a major component of the Erythroblast IR program.
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the erythroid intron retention program encompasses developmentally stable and dynamic networks and regulates diverse gene classes
Blood, 2015Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Computational analysis of RNA-seq data from highly purified human Erythroblasts has been instrumental in revealing changes in pre-mRNA splicing during terminal erythropoiesis. Here we report updated studies of intron retention (IR), a type of alternative splicing in which specific introns are retained in otherwise efficiently-processed transcripts, allowing post-transcriptional modulation of cellular mRNA levels. Differences in differentiation stage-specificity, degree of retention, nuclear/cytoplasmic localization, and sensitivity to nonsense-mediated decay (NMD) suggest the existence of multiple classes of Erythroblast IR subject to distinct regulatory controls. Two clusters comprising ~470 "developmentally dynamic" introns in 354 genes exhibit more efficient splicing in proErythroblasts, but elevated intron retention in orthochromatic Erythroblasts prior to enucleation. Dynamic regulation of late Erythroblast IR parallels previously described splicing switches involving alternative exons. Gene ontology analysis revealed that the dynamic intron group is highly enriched in genes with RNA processing functions. Among these are several spliceosomal factors including SF3B1, a commonly mutated gene in myelodysplasia patients. We also identified several clusters of "developmentally stable" introns whose IR levels are not substantially modulated during erythropoiesis. Among this latter type are two clusters containing 294 introns that are enriched in functions related to metal ion binding. Key genes include mitoferrin-1 (SC25A37; IR~50%) and mitoferrin-2 (SLC25A28; IR~20-30%), mitochondrial iron importers essential for heme biosynthesis. We observed a correlation between splice site strength and percent IR among developmentally stable but not dynamic intron clusters, indicating that splicing regulatory mechanism(s) for the latter must require additional sequence features. A search for such features revealed that IR was significantly higher adjacent to alternative 'PTC' exons containing premature termination codons than it was adjacent to other exons; moreover, by direct RT-PCR analysis we discovered novel (unannotated) PTC exons in additional retained introns. The proposed role of PTC exons in IR is being studied experimentally using an array of minigene splicing reporter constructs. Finally, we noted that while specific IR events are erythroid specific, e.g., in the alpha spectrin gene SPTA1, computational analysis of public RNA-seq data demonstrated that most Erythroblast IR events were also observed in granulocytes and in 16 other tissues surveyed by the human BodyMap project. Intron retention is likely to play critical roles in gene regulation in both hematological and non-hematological tissues. Disclosures No relevant conflicts of interest to declare.
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an erythroid specific intron retention program regulates expression of selected genes during terminal erythropoiesis
Blood, 2014Co-Authors: Harold Pimentel, Narla Mohandas, Marilyn Parra, Lior Pachter, Sherry L Gee, John G ConboyAbstract:Erythroid RNAs, like their nonerythroid counterparts, are subject to post-transcriptional processing events that critically impact their coding capacity for the erythroid proteome. Previous studies have shown that differentiating human and mouse Erythroblasts execute an extensive and dynamic alternative splicing program involving regulation of numerous alternative exons. Here we report that controlled excision of selected introns is also an important component of the Erythroblast alternative splicing program. Intron retention (IR) patterns in differentiating human Erythroblasts were determined via RNA-seq analysis of FACS-purified Erythroblast populations. Comparison of IR among Erythroblast populations and between Erythroblasts and other hematopoietic cells suggests that regulation of IR occurs in a differentiation stage- and tissue-specific manner. For example, there was little overlap of intron retention events in Erythroblasts with those reported in differentiating granulocytes. Moreover, the IR profile of proErythroblasts differed substantially from that in orthochromatic Erythroblasts, with IR generally increasing in the more mature cells that are preparing for enucleation. IR in Erythroblasts affected numerous genes functioning in RNA processing, iron homeostasis and heme biosynthesis, protein translation, and membrane properties. Mature Erythroblasts exhibited retention of introns in several human disease genes including SF3B1, a splicing factor often mutated in myelodysplasia; TFR2, encoding transferrin receptor 2 that is mutated in a form of hemochromatosis; and FUS, an RNA binding protein implicated in ALS. Inspection of intronic RNA-seq reads in >60 genes with IR revealed that single or multiple introns can be retained within a transcript; however, other introns within the same genes, and indeed the great majority of introns in Erythroblast-expressed genes, are efficiently spliced with minimal or no IR. Retained introns may be flanked by either constitutively or alternatively spliced exons, suggesting different regulatory mechanisms. Ongoing studies will explore whether IR in some transcripts might function to down-regulate gene expression by introduction of premature termination codons that induce nonsense-mediated decay, or alternatively, whether IR transcripts could represent a reserve of nearly-completed mRNAs that can be processed in response to appropriate physiological stimuli. In sum, these results suggest that a highly regulated IR program plays an important role in erythroid differentiation. Disclosures No relevant conflicts of interest to declare.
Mark J. Koury - One of the best experts on this subject based on the ideXlab platform.
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In vitro maturation of nascent reticulocytes to erythrocytes
2016Co-Authors: Red Cells, Mark J. Koury, Prapaporn Kopsombut, Stephen T. Koury, Maurice C BondurantAbstract:Most studies of mammalian reticulocyte maturation have used blood reticulo-cytes. Nascent reticulocytes, as found in bone marrow, have not been available in developmentally synchronized popula-tions. Nascent murine reticulocytes formed in vitro by enucleation of Friend virus–infected Erythroblasts were puri-fied and recultured for 110 hours. At 0 hours, all recultured cells were lobulated and contained dense, centralized reticu-lin. By 110 hours, about 20 % to 25 % of the cells became biconcave erythrocytes. Most ribosomes and cellular RNAs were degraded within 20 hours, and during that period, heme synthesis declined from a rate equal to that of late Erythroblasts to less than 10 % of that rate. Many mitochon-dria appeared normal until they showed outer membrane swelling, degradation, and apparent fusion with intracellular vacuoles at 40 hours of culture. During the period of mitochondrial loss, Bcl-XL, an antiapoptotic protein that accumu-lates during Erythroblast differentiation and maintains mitochondrial membrane integrity, demonstrated progressive de-creases and changes consistent with de-amidation. Nevertheless, the reticulo-cytes did not undergo apoptosis, because their apoptotic machinery was degraded. This experimental system that provides a developmentally synchronized popula-tion of nascent murine reticulocytes that mature into biconcave erythrocytes in vitro should be useful in further investiga-tions of the cellular events involved in reticulocyte maturation. (Blood. 2005
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CHROMATIN CONDENSATION IN TERMINALLY DIFFERENTIATING MOUSE ErythroblastS DOES NOT INVOLVE SPECIAL ARCHITECTURAL PROTEINS BUT DEPENDS ON HISTONE DEACETYLATION
Chromosome research : an international journal on the molecular supramolecular and evolutionary aspects of chromosome biology, 2009Co-Authors: Evgenya Y. Popova, Sharon Wald Krauss, Sarah A. Short, Gloria Lee, Jonathan Villalobos, Joan Etzell, Mark J. Koury, Paul A. Ney, Joel Anne Chasis, Sergei A. GrigoryevAbstract:Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation and chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian Erythroblast chromatin condensation, we used Friend virus-infected murine spleen Erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late-stage Erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected Erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate Erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.
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direct interaction with macrophages increases proErythroblast proliferation while preserving erythropoietin epo dependence
Blood, 2006Co-Authors: Melissa Rhodes, Prapaporn Kopsombut, Maurice C Bondurant, James O Price, Mark J. KouryAbstract:INTRODUCTION: EPO regulates erythropoiesis by preventing apoptosis at the relatively late developmental stages of CFU-E and proErythroblasts. Cells in these EPO-dependent stages are actively dividing, but after several divisions they enter a G0 state from which they enucleate. In vivo these erythroid progenitor cells associate physically with macrophages in the bone marrow, forming Erythroblastic islands. Erythroblastic islands appear to be necessary for proper development of Erythroblasts into erythrocytes, but our current knowledge about erythroid progenitor-macrophage interactions in the Erythroblastic islands is limited. METHODS: Spleens of mice in the acute Erythroblastic phase of Friend virus disease were used to reconstitute Erythroblastic islands in a co-culture system that enabled study of interactions between macrophages and developmentally synchronized EPO-dependent erythroid progenitors. Proliferation and differentiation of these erythroid progenitors in macrophage co-cultures was compared to controls in which the same erythroid progenitors were cultured alone. Erythroblasts adherent to macrophages and non-adherent Erythroblasts from co-cultures, as well as control Erythroblasts cultured without macrophages were collected at 6, 20, 32, and 44 hrs after initial culture for cell counts, cytospin preparations for morphology, flow cytometry analyses for apoptosis (TUNEL), cell cycle phases, and expression of two surface molecules known to be expressed on differentiating Erythroblasts, phosphatidylserine (PS) and α 4 integrin. Experiments were also done with Erythroblasts cultured in macrophage-conditioned media. RESULTS: Splenic Erythroblasts cultured alone proliferated 4.6 ± 0.7 fold over 44 h, while Erythroblasts co-cultured with splenic macrophages proliferated 14.2 ± 2 fold (n=12). Control Erythroblasts had the same proliferation in macrophage-conditioned medium as they did in normal medium. In EPO dose-response experiments, percentages of apoptosis were the same among adherent and non-adherent co-cultured Erythroblasts and control Erythroblasts. Cytospin preparations revealed no differences in morphology among non-adherent and adherent Erythroblasts in co-cultures and control Erythroblasts. No differences were found in enucleation percentages, extruded nuclei, or reticulocyte formation at 44 h. Likewise no differences were found in percentages of apoptotic cells, distribution of cell cycle phases, or surface expressions of PS or α 4 integrin during the 44 h of differentiation. CONCLUSIONS: Co-culture with macrophages in reconstituted Erythroblastic islands dramatically increases the Erythroblast proliferation, without affecting differentiation. The increase in proliferation is not due to decreased apoptosis, increased EPO responsiveness, or soluble factors released by the macrophages. Preservation of EPO-dependence during this expansion of Erythroblasts mediated by direct interaction with macrophages indicates that erythropoietic regulation by EPO affects a larger population of erythroid progenitor cells in later stages of erythropoiesis and, thereby, accounts for relatively rapid increases or decreases in erythrocyte production following changes in EPO levels in vivo.
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new insights into erythropoiesis the roles of folate vitamin b12 and iron
Annual Review of Nutrition, 2004Co-Authors: Mark J. Koury, Prem PonkaAbstract:▪ Abstract Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B12, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B12 for proliferation during their differentiation. Deficiency of folate or vitamin B12 inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes Erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in Erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded product...
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new insights into erythropoiesis the roles of folate vitamin b12 and iron
Annual Review of Nutrition, 2004Co-Authors: Mark J. Koury, Prem PonkaAbstract:Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B12, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B12 for proliferation during their differentiation. Deficiency of folate or vitamin B12 inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes Erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in Erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded production rates with smaller, less hemoglobinized erythrocytes.
Harvey F Lodish - One of the best experts on this subject based on the ideXlab platform.
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Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal Erythroblasts
Haematologica, 2010Co-Authors: Victor Yeh, Maki Murata-hori, Tzutzuy Ramirez, Harvey F LodishAbstract:Background During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver Erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Design and Methods Mouse fetal liver Erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short harirpin RNA. Results We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver Erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal Erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect Erythroblast proliferation, differentiation, or apoptosis. Conclusions These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.
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Histone Deacetylases Are Essential for Chromatin Condensation and Enucleation in Mammalian Erythroblasts
Blood, 2008Co-Authors: Francisco J. Sánchez-rivera, Harvey F LodishAbstract:In the last steps of differentiation mammalian erythroid cells undergo chromatin condensation and enucleation; the latter process does not occur in other vertebrates. Enucleation was critical for the evolution of mammals, as it permits an enhanced hemoglobin concentration – and thus oxygen- carrying capacity – in mammalian red blood cells. We previously reported that Rac GTPases and their downstream forming target mDia2 are required for mouse fetal Erythroblast enucleation. We also found that the nucleus undergoes a gradual ~10- fold decrease in volume during erythropoiesis. Since histone deacetylases (HDACs) play important roles in chromatin condensation, we hypothesized that HDACs are involved in mammalian Erythroblast enucleation. To test this hypothesis, we purified E13.5 mouse fetal TER119 negative Erythroblasts and cultured them in fibronectin-coated plates. Cells were then treated with Trichostatin A (TSA), a pan-HDAC inhibitor, at different times during erythropoiesis. TSA completely blocked enucleation and kinetic studies showed this inhibitory effect occurred earlier than the step catalyzed by the Rac-mDia2 pathway. This indicates that chromatin condensation is required for the final extrusion of the nucleus. We further investigated the activity ofHDAC6 specifically, since HDAC6 is known to interact with mDia2. We found that mDia2is acetylated in vitro and in vivo and that HDAC6 interacts with and deacetylates mDia2. Treatment of purified TER119 negative mouse fetal Erythroblasts with a specific HDAC6inhibitor partially blocked enucleation. We conclude that histone deacetylase activities are essential for mammalian Erythroblasts to undergo enucleation and that HDACs act both on chromatin condensation and mDia2 deacetylation to promote enucleation.
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Enucleation of cultured mouse fetal Erythroblasts requires Rac GTPases and mDia2
Nature cell biology, 2008Co-Authors: Senthil Raja Jayapal, Harvey F LodishAbstract:Mammalian erythroid cells undergo enucleation, an asymmetric cell division involving extrusion of a pycnotic nucleus enveloped by the plasma membrane. The mechanisms that power and regulate the enucleation process have remained obscure. Here, we show that deregulation of Rac GTPase during a late stage of erythropoiesis completely blocks enucleation of cultured mouse fetal Erythroblasts without affecting their proliferation or differentiation. Formation of the contractile actin ring (CAR) on the plasma membrane of enucleating Erythroblasts was disrupted by inhibition of Rac GTPases. Furthermore, we demonstrate that mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments, is also required for enucleation of mouse fetal Erythroblasts. We show that Rac1 and Rac2 bind to mDia2 in a GTP-dependent manner and that downregulation of mDia2, but not mDia1, by small interfering RNA (siRNA) during the late stages of erythropoiesis blocked both CAR formation and Erythroblast enucleation. Additionally, overexpression of a constitutively active mutant of mDia2 rescued the enucleation defects induced by the inhibition of Rac GTPases. These results reveal important roles for Rac GTPases and their effector mDia2 in enucleation of mammalian Erythroblasts.
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role of ras signaling in erythroid differentiation of mouse fetal liver cells functional analysis by a flow cytometry based novel culture system
Blood, 2003Co-Authors: Jing Zhang, Merav Socolovsky, Alec W Gross, Harvey F LodishAbstract:Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary Erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver Erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained Erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early Erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected Erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early Erythroblasts and supports their erythropoietin (Epo)–independent growth.
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ineffective erythropoiesis in stat5a 5b mice due to decreased survival of early Erythroblasts
Blood, 2001Co-Authors: Merav Socolovsky, Carlo Brugnara, Mark D Fleming, Volker H Haase, Harvey F LodishAbstract: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-xLand increased apoptosis in Stat5a−/−5b−/−early Erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early Erythroblast survival.
Nicola Foad - One of the best experts on this subject based on the ideXlab platform.
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transcription factor levels after forward programming of human pluripotent stem cells with gata1 fli1 and tal1 determine megakaryocyte versus erythroid cell fate decision
Stem cell reports, 2018Co-Authors: Amanda Dalby, Jose Ballesterbeltran, Chiara Lincetto, Annett Mueller, Nicola Foad, Amanda L EvansAbstract:Summary The production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or Erythroblasts. Using “rainbow” lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an Erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or Erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine.
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Transcription Factor Levels after Forward Programming of Human Pluripotent Stem Cells with GATA1, FLI1, and TAL1 Determine Megakaryocyte versus Erythroid Cell Fate Decision
'Elsevier BV', 2018Co-Authors: Amanda Dalby, Chiara Lincetto, Annett Mueller, Nicola Foad, Jose Ballester-beltrán, Amanda Evans, James Baye, Ernest Turro, Thomas Moreau, Marloes R. TijssenAbstract:Summary: The production of blood cells and their precursors from human pluripotent stem cells (hPSCs) in vitro has the potential to make a significant impact upon healthcare provision. We demonstrate that the forward programming of hPSCs through overexpression of GATA1, FLI1, and TAL1 leads to the production of a population of progenitors that can differentiate into megakaryocyte or Erythroblasts. Using “rainbow” lentiviral vectors to quantify individual transgene expression in single cells, we demonstrate that the cell fate decision toward an Erythroblast or megakaryocyte is dictated by the level of FLI1 expression and is independent of culture conditions. Early FLI1 expression is critical to confer proliferative potential to programmed cells while its subsequent silencing or maintenance dictates an erythroid or megakaryocytic fate, respectively. These committed progenitors subsequently expand and mature into megakaryocytes or Erythroblasts in response to thrombopoietin or erythropoietin. Our results reveal molecular mechanisms underlying hPSC forward programming and novel opportunities for application to transfusion medicine. : The authors show that overexpression of GATA1, TAL1, and FLI1 in hPSCs leads to the formation of progenitors that are committed early during differentiation to either the megakaryocytic or erythroid lineage. The cell fate decision is independent of the cytokines used (TPO or EPO) but is dictated by the level of the FLI1 transgene. Keywords: pluripotent stem cells, forward programming, megakaryocyte, Erythroblast, lineage fate decisio