Nuclear Opening and Histone Release Are Essential for Nuclear but Not Chromatin Condensation during Terminal Erythropoiesis

Abstract Nuclear condensation and enucleation are characteristic processes in mammalian terminal erythropoiesis. These processes are associated with the transient nuclear opening formation that mediates partial histone release to the cytoplasm. Our previous report showed that caspases are involved in the cleavage of nuclear lamina to enable histone release. However, it remains unclear whether nuclear opening formation and histone release regulate the genomic three-dimensional organization during nuclear condensation. To answer this question, we cultured E13.5 mouse fetal liver Ter119 negative erythroid progenitor cells in erythropoietin (EPO) containing medium for 48 h with or without the presence of caspase inhibitor. As expected, caspase inhibitor blocked nuclear opening formation and histone release, and significantly reduced nuclear condensation and enucleation. We next performed a Hi-C sequencing to investigate chromatin structural change during terminal differentiation and nuclear condensation. To this end, the cultured fetal liver erythroid cells with or without caspase inhibitor were harvested at 30 h right before enucleation for Hi-C sequencing. The sequencing results showed that cells at 30 h contain significantly more interactions than freshly isolated erythroid progenitors, which is consistent with chromatin condensation during terminal erythropoiesis. Further analysis showed that increased interactions mainly accumulate as inter-chromosomal interactions, suggesting inter-chromosome interaction is the dominant structural force driving erythrocyte chromatin condensation. Surprisingly, there were no significant chromatin structural changes between caspase inhibitor treated and mock-treated cells when compared at 30 h. We also performed ATAC-seq and RNA-seq with the same experiment settings, both corresponded to Hi-C sequencing and showed little difference under caspase inhibitor treatment. These results indicate that although histone release and nuclear condensation are compromised with the inhibition of caspases, chromatin stays condensed with well-organized three-dimensional structure and appropriate gene expression regulations. To further confirm this phenomenon, we generated caspase-3 and -7 double knock out (cas3cas7-/-) mice. Cas3cas7-/- mice are embryonically lethal due to defective cardiac development. The hematopoietic tissues in these mice have not been well studied. We harvested fetal liver Ter119 negative erythroid progenitor cells from E13.5 cas3cas7-/- mice and the cells from the littermate (cas7-/-, cas3+/-cas7-/-) mice were used as controls. We first cultured Ter119 negative fetal liver erythroid progenitors in EPO containing medium for 48 h. Immunofluorescence analysis showed that the nuclear opening was significantly inhibited, and the nuclear size significantly increased in the erythroid cells from cas3cas7-/- mice due to failure of histone release into cytoplasm. Flow cytometry analysis showed that enucleation was significantly impaired in cas3cas7-/- cells, but the cells could still differentiate although with lower efficiency. We further performed an in vivo assay in which E13.5 cas3cas7-/- fetal liver cells were transplanted into wild type lethally irradiated recipient mice. EPO medium cultured bone marrow lineage negative cells from these transplanted mice showed significant reduction in nuclear opening and histone release, and enlargement of nuclear size. However, these mice survived well despite anemia. These results indicate a portion of orthochromatic erythroblasts managed to enucleate even with the less condensed nuclei. Overall, our study demonstrates that nuclear opening and histone release are essential for nuclear condensation but have minimal effects on chromatin condensation or the regulation of gene expression in terminal erythropoiesis. Appropriate nuclear condensation is important for efficient enucleation. However, orthochromatic erythroblasts could still manage to enucleate although with low efficacies. Disclosures No relevant conflicts of interest to declare.

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α4β1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development

The Journal of Cell Biology ◽

10.1083/jcb.200702080 ◽

2007 ◽

Vol 177 (5) ◽

pp. 871-880 ◽

Cited By ~ 61

Author(s):

Shawdee Eshghi ◽

Mariette G. Vogelezang ◽

Richard O. Hynes ◽

Linda G. Griffith ◽

Harvey F. Lodish

Keyword(s):

Extracellular Matrix ◽

Fetal Liver ◽

Red Cell ◽

Erythroid Progenitors ◽

Two Phase ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fibronectin Fragments ◽

Proliferation And Differentiation ◽

Precise Role

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that α4, α5, and β1 are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage α4β1-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, α4β1-integrin–dependent phase.

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TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽

10.1182/blood.v124.21.2912.2912 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2912-2912

Author(s):

Petros Papadopoulos ◽

Laura Gutierrez ◽

Jeroen Demmers ◽

Dimitris Papageorgiou ◽

Elena Karkoulia ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fetal Liver Cells ◽

Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

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A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2266 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2266-2277 ◽

Cited By ~ 33

Author(s):

G D Longmore ◽

P N Pharr ◽

H F Lodish

Keyword(s):

Cell Line ◽

Progenitor Cells ◽

Cell Lines ◽

Leukemia Virus ◽

Active Form ◽

Erythropoietin Receptor ◽

Mutant Form ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.

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A Minimal Cytoplasmic Subdomain of the Erythropoietin Receptor Mediates Erythroid and Megakaryocytic Cell Development

Blood ◽

10.1182/blood.v94.10.3381.422k25_3381_3387 ◽

1999 ◽

Vol 94 (10) ◽

pp. 3381-3387 ◽

Cited By ~ 28

Author(s):

Chris P. Miller ◽

Zi Y. Liu ◽

Constance T. Noguchi ◽

Don M. Wojchowski

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Fetal Liver ◽

Cell Development ◽

Erythropoietin Receptor ◽

Erythroid Progenitor ◽

Epo Receptor ◽

Receptor Complexes ◽

Erythroid Progenitor Cells ◽

Fetal Liver Cells

Signals provided by the erythropoietin (Epo) receptor are essential for the development of red blood cells, and at least 15 distinct signaling factors are now known to assemble within activated Epo receptor complexes. Despite this intriguing complexity, recent investigations in cell lines and retrovirally transduced murine fetal liver cells suggest that most of these factors and signals may be functionally nonessential. To test this hypothesis in erythroid progenitor cells derived from adult tissues, a truncated Epo receptor chimera (EE372) was expressed in transgenic mice using a GATA-1 gene-derived vector, and its capacity to support colony-forming unit-erythroid proliferation and development was analyzed. Expression at physiological levels was confirmed in erythroid progenitor cells expanded ex vivo, and this EE372 chimera was observed to support mitogenesis and red blood cell development at wild-type efficiencies both independently and in synergy with c-Kit. In addition, the activity of this minimal chimera in supporting megakaryocyte development was tested and, remarkably, was observed to approximate that of the endogenous receptor for thrombopoietin. Thus, the box 1 and 2 cytoplasmic subdomains of the Epo receptor, together with a tyrosine 343 site (each retained within EE372), appear to provide all of the signals necessary for the development of committed progenitor cells within both the erythroid and megakaryocytic lineages.

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Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽

10.1182/blood.v88.5.1576.1576 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1576-1582 ◽

Cited By ~ 243

Author(s):

M Silva ◽

D Grillot ◽

A Benito ◽

C Richard ◽

G Nunez ◽

...

Keyword(s):

Progenitor Cells ◽

Apoptotic Cell ◽

Ectopic Expression ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Survival Factor ◽

Erythroid Progenitor Cells ◽

Survival Signal ◽

Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

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Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Blood ◽

10.1182/blood.v90.2.651 ◽

1997 ◽

Vol 90 (2) ◽

pp. 651-657 ◽

Cited By ~ 41

Author(s):

Xingwei Sui ◽

Sanford B. Krantz ◽

Zhizhuang Zhao

Keyword(s):

Polycythemia Vera ◽

Progenitor Cells ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Gel Filtration ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Normal Cells ◽

Protein Tyrosine ◽

Erythroid Progenitor Cells

Abstract Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

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Pregnancy-Secreted Acid Phosphatase, Uteroferrin, Enhances Fetal Erythropoiesis

Endocrinology ◽

10.1210/en.2014-1397 ◽

2014 ◽

Vol 155 (11) ◽

pp. 4521-4530 ◽

Cited By ~ 3

Author(s):

Wei Ying ◽

Haiqing Wang ◽

Fuller W. Bazer ◽

Beiyan Zhou

Keyword(s):

Acid Phosphatase ◽

Progenitor Cells ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Erythroid Progenitor ◽

Colony Forming Unit ◽

Erythroid Progenitor Cells ◽

Uterine Glands ◽

Fetal Erythropoiesis

Abstract Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage.

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PU.1 determines the self-renewal capacity of erythroid progenitor cells

Blood ◽

10.1182/blood-2003-11-4089 ◽

2004 ◽

Vol 103 (10) ◽

pp. 3615-3623 ◽

Cited By ~ 79

Author(s):

Jonathan Back ◽

Andrée Dierich ◽

Corinne Bronn ◽

Philippe Kastner ◽

Susan Chan

Keyword(s):

Progenitor Cells ◽

Fluorescent Protein ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Self Renewal ◽

Adult Mice ◽

Erythroid Progenitor Cells ◽

Low Levels ◽

Green Fluorescent ◽

Fluorescent Protein Reporter

Abstract PU.1 is a hematopoietic-specific transcriptional activator that is absolutely required for the differentiation of B lymphocytes and myeloid-lineage cells. Although PU.1 is also expressed by early erythroid progenitor cells, its role in erythropoiesis, if any, is unknown. To investigate the relevance of PU.1 in erythropoiesis, we produced a line of PU.1-deficient mice carrying a green fluorescent protein reporter at this locus. We report here that PU.1 is tightly regulated during differentiation—it is expressed at low levels in erythroid progenitor cells and down-regulated upon terminal differentiation. Strikingly, PU.1-deficient fetal erythroid progenitors lose their self-renewal capacity and undergo proliferation arrest, premature differentiation, and apoptosis. In adult mice lacking one PU.1 allele, similar defects are detected following stress-induced erythropoiesis. These studies identify PU.1 as a novel and critical regulator of erythropoiesis and highlight the versatility of this transcription factor in promoting or preventing differentiation depending on the hematopoietic lineage.

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UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis

Blood ◽

10.1182/blood.v112.11.5372.5372 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5372-5372

Author(s):

Alvaro A Elorza ◽

Brigham B Hyde ◽

Hanna Mikkola ◽

Sheila Collins ◽

Orian S Shirihai

Keyword(s):

Cell Proliferation ◽

Progenitor Cells ◽

Fetal Liver ◽

Mitochondrial Protein ◽

Erythroid Progenitor ◽

Heme Synthesis ◽

Erythroid Progenitor Cells ◽

Deficient Mice

Abstract UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.

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