An Anti-Apoptotic Molecule, Anamorsin, Is Essential for Erythropoiesis Through the Regulation of Cellular Labile Iron Pool

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 610-610
Author(s):  
Akira Tanimura ◽  
Yuri Hamanaka ◽  
Natsuko Fujita ◽  
Yukiko Doi ◽  
Tomohiko Ishibashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Iron Homeostasis ◽  
Fetal Liver ◽  
Liver Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Labile Iron Pool ◽  
Cellular Iron ◽  
Fetal Liver Cells ◽  
Induction Of Apoptosis

Abstract Abstract 610 Introduction: Iron has crucial roles in many cellular biological processes. Cellular iron uptake and export must be tightly regulated. Insufficient iron concentrations impair the function of numerous iron proteins, whereas excess free iron can oxidize and damage the contents of cells. Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. It is thought that AM plays a crucial role in hematopoiesis, however its precise biological mechanisms remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569–5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the maturation of Fe/S cluster and the cellular iron homeostasis, especially, the regulation of labile iron pool (LIP) and that AM may affect the accumulation of reactive oxygen species (ROS), leading to impaired erythropoiesis. Methods and Results: To analyze the function of Fe/S protein, we established wild-type cell lines (AMWT) and AM-deficient cell lines (AMKO) from wild-type and AM-deficient fetal liver (14.5dpc) respectively by using SV40 large T antigen. Iron regulatory protein 1 (IRP1) is a well-known Fe/S protein with dual functions. In the presence of Fe/S cluster, IRP1 functions as a cytosolic aconitase. While, in the absence of Fe/S cluster, IRP1 stabilizes the transferrin receptor (TfR) mRNA by binding to the iron responsive element (IRE). We compared the aconitase activity and the IRE binding activity of IRP1 between AMWT and AMKO. The results showed that the cytosolic aconitase activity in AMKO decreased approximately 30% compared to AMWT and the IRE binding activity of IRP1 in AMKO increased 3-fold compared to AMWT. Furthermore, we compared the iron homeostasis. In the presence of iron chelator, desferrioxamine, the expression of TfR in AMWT was markedly elevated, while it was hardly elevated in AMKO. The LIP is a pool of chelatable and redox-active iron, which serves as a crossroad of cell iron metabolism. The measurement of LIP with the metal-sensitive sensor calcein acetoxymethyl ester showed that AMKO had 5-fold higher cellular LIP than AMWT. Moreover we evaluated the accumulation of ROS and the induction of apoptosis by extracellular iron uptake between AMWT and AMKO. The results showed the accumulation of ROS and the induction of apoptosis in AMKO were enhanced about twice as much as in AMWT. These enhancements could be restored by transduction of AM expressing retrovirus vector to AMKO. We also evaluated the effects of AM-deficiency on erythroid differentiation. Fetal liver cells from wild-type or AM-deficient embryos (14.5dpc) were divided into primitive and more matured erythroid populations based on their expression of CD71 and Ter119 by FACS analysis. AM-deficient fetal liver cells had a significant increase in the CD71low TER119low population, containing primitive erythroid progenitors, compared to wild-type (9.4±2.1% vs. 5.2±1.1%, P<0.05). Conversely, the CD71lowTER119highpopulation, comprised of late orthochromatophilic erythroblasts and reticulocytes, decreased in AM-deficient fetal liver cells compared to wild-type cells (2.3±0.8% vs. 7.4±1.3%, P < 0.05). Moreover we studied LIP in wild-type or AM-deficient embryo fetal liver cells. In accordance with the cell lines, the LIP in AM-deficient fetal liver cells increased 3 to 5-fold more than in wild-type fetal liver cells. The accumulation of ROS and the number of apoptotic cells also increased 2 to 5-fold in AM- deficient fetal liver cells compared to wild-type fetal liver cells. Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration, resulting in the increase of LIP, the accumulation of ROS and the induction of apoptosis. Furthermore, dysregulation of cellular iron homeostasis was thought to be the cause of the embryonic lethal due to AM deficiency. Conclusion: Our current findings indicate that AM functions in cytosolic Fe/S cluster biogenesis and iron homeostasis and is essential for erythropoiesis. Disclosures: Kanakura: Shire: Consultancy.

An Anti-Apoptotic Molecule, Anamorsin, Functions in Both Iron-Sulfur Protein Assembly and Cellular Iron Homeostasis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2106-2106 ◽  
Author(s):  
Akira Tanimura ◽  
Yuri Kondo ◽  
Hirokazu Tanaka ◽  
Itaru Matsumura ◽  
Tomohiko Ishibashi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Lines ◽  
Iron Homeostasis ◽  
Binding Activity ◽  
Wild Type ◽  
S Protein ◽  
Aconitase Activity ◽  
Cellular Iron ◽  
Iron Sulfur ◽  
Retrovirus Vector

Abstract Abstract 2106 Introduction: Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells (J.Exp.Med. 199:581–592, 2004). AM has no structural homology to any known anti-apoptosis molecules such as Bcl-2 and IAP family members. AM-deficient (AM−/−) mice are embryonic lethal at late gestation. Its embryos are anemic and the size of embryos is very small. It is thought that AM plays a crucial role in hematopoiesis, however the precise biological mechanisms of AM remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569-5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the cellular iron regulation. Methods and Results: At first, in order to analyze molecular and cellular events, we established cell lines from wild-type or AM-deficient embryonic fetal liver (14.5dpc) by using SV40 large T antigen. We isolated 5 wild-type cell lines (AM WTs) and 2 AM-deficient cell lines (AM KOs) respectively. Next, we compared the cell growth and apoptosis in both cell lines and found that the growth rate of AM KOs were slightly lower than that of AM WTs although these cell lines were immortalized. AM KOs showed more significant apoptosis induced by oxidative stress; the percents of Annexin V positive fraction were 12 ± 4 and 36 ± 6 in AM WTs and AM KOs respectively under the condition of 0.1mM H2O2 for 16hr. In addition to oxidative stress, AM KOs were more sensitive to UV irradiation. These differences were cancelled by transduction of AM-expression retrovirus vector in AM KOs. It was reported that Dre2 functions in cytosolic Fe/S protein biogenesis. We examined whether AM might be involved in the maturation of cytosolic Fe/S proteins. Iron regulatory protein 1 (IRP1) is a well-known cytosolic Fe/S protein with dual functions; in the presence of an [4Fe-4S] cluster it functions as a cytosolic aconitase, while IRP1 binds to mRNA stem-loop structures called iron responsive elements (IREs) and confer the mRNA stability when the [4Fe-4S] cluster is missing. In the iron-deficient cells, IRP1 binds to IREs located at the mRNA of iron transferrin receptor (TfR), ferritin and other iron metabolism transcripts, thereby enhancing iron uptake. In this way, it is thought that IRP1 plays important roles in iron homeostasis. We therefore compared the aconitase activity and IRE binding activities of IRP1 between AM WTs and AM KOs and found that AM deficiency resulted in the decrease of cytosolic aconitase activity (approximately 30% compared to AM WTs). In contrast to cytosolic aconitase activity, the mitochondrial aconitase activity showed little change regardless of AM deficiency. In order to analyze whether AM deficiency might increase IRE binding activity of IRP1, cytoplasmic extracts of AM WTs and AM KOs were compared by RNA precipitation assay. In AM KOs, the expression level of IRP1 decreased approximately one third compared to AM WTs. However, the binding activity of IRP1 to biotin-labeled IRE increased in the extract of AM KOs approximately three-fold in comparison to AM WTs. These differences was cancelled by transduction of AM-expression retrovirus vector to AM KOs. All these findings demonstrated the involvement of AM in the maturation of the cytosolic Fe/S protein, IRP1. Furthermore, we examined the expression of TfR, which is known to be modulated by IRP1-mediated posttranscriptional regulation. In the presence of iron chelator, desferrioxamine, the expression of TfR in AM WTs was markedly elevated. On the other hand, in AM KOs, the expression of TfR was hardly elevated. Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration due to the decreased function of IRP1. Conclusion: Our current findings indicate that AM is essential for cytosolic Fe/S cluster biogenesis and iron homeostasis. Now the influence of the AM-mediated iron homeostasis on hematopoiesis is under investigation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1004-1004
Author(s):  
Daphne Dupere-Richer ◽  
Jianping Li ◽  
Sayantan Maji ◽  
Crissandra Pipe ◽  
Sharon Norton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Fetal Liver ◽  
Liver Cells ◽  
Wild Type ◽  
Post Transplantation ◽  
Demethylase Activity ◽  
Fetal Liver Cells

Abstract In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM. We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM. Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. Disclosures Licht: Celgene: Research Funding.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 1990-1998 ◽  
Author(s):  
Wolfgang E. Kaminski ◽  
Per Lindahl ◽  
Nancy L. Lin ◽  
Virginia C. Broudy ◽  
Jeffrey R. Crosby ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Liver Growth ◽  
Hematologic Disorders ◽  
Fetal Liver Cells ◽  
Β Receptor

Abstract Platelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

Stress Erythropoiesis Elicits a Program of Gene Regulation That Overlaps with Interferon-γ.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2161-2161
Author(s):  
Kai Huang ◽  
Monica L. Bailey ◽  
Dwayne L. Barber
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Early Gene ◽  
Deubiquitinating Enzyme ◽  
Wild Type ◽  
Data Set ◽  
Time Points ◽  
Stress Erythropoiesis ◽  
Adult Mice ◽  
Fetal Liver Cells

Abstract Erythropoietin (EPO), the primary cytokine regulator of red blood cell production, acts through binding to its cognate receptor (EPO-R), which is primarily expressed on erythroid precursors. Knockout studies have illustrated a critical role for EPO, EPO-R and the downstream tyrosine kinase JAK2 in embryogenesis as mice lacking any of these components die from a fatal anemia at E13.5. These data suggest that EPO-R and/or JAK2 are required to promote erythropoiesis in vivo. EPO provides mitogenic, differentiative and cell survival signals to erythroid progenitors. We have performed microarray studies to identify target genes regulated by EPO in cell lines and primary cells. We utilized an erythroid cell line (HCD-57), a myeloid cell line stably expressing the EPO-R (Ba/F3-EPO-R), fetal liver cells isolated from E13.5 mice as well as splenocytes isolated from Phenylhydrazine (PHZ)-primed adult mice. Fetal liver cells permit the study of normal erythropoiesis in a fetal setting whereas the PHZ-primed erythroblasts permit analysis of stress erythropoiesis in adult mice. We harvested cells at 1, 8, 12 and 24 hr after EPO stimulation which correspond to immediate early gene induction (1 hr), S phase entry (8 hr) and G2/M (24 hr) time points. RNA was prepared and hybridized to the Affymetrix U74A mouse chip. Data was analyzed and only those genes with statistical significance (p < 0.05) were considered for further characterization. Analysis of the 1 hr time points has revealed that six genes are co-regulated by EPO in all four cellular environments. Included within this co-hort are the Suppressor of Cytokine Signaling genes (Cis, SOCS-1 and SOCS-3) and Myc, as well as two novel genes. We compared our datasets with other published analyses. The Williams laboratory has identified an Interferon-Stimulated Gene “ISG” data set corresponding to genes induced by Type I or Type II Interferon’s. We queried our PHZ-primed erythroblast data set against the Williams ISG database. Of the 305 human genes in the ISG database, 218 are expressed on the Affymetrix chip. We searched our dataset for genes that are induced 1.5-fold or greater at 2 of 4, 3 of 4 or 4 of 4 time points. Thirty-four genes are also stimulated by EPO in PHZ-primed erythroblasts including classical IFN-regulated genes such as Interferon-regulator factor-1 (IRF-1), Interferon-stimulated gene-15 (ISG-15), Interferon-induced transmembrane protein 3-like (IFITM-3l), Protein Kinase R (PKR) and Signal Transducer and Activator of Transcription-1 (STAT1). We have previously demonstrated that STAT1 is a negative regulator of murine erythropoiesis utilizing STAT1-deficient mice. We also analyzed immediate early gene regulation in fetal liver cells and PHZ-primed erythroblasts isolated from STAT1-deficient mice stimulated with EPO for 1 hr. These data were compared with the relevant wild type data sets. EPO stimulates the induction of the ubiquitin-like protein, ISG-15 in both wild type and STAT1−/− erythroblasts. Several signaling proteins have been shown to be covalently modified by ISG-15 including STAT1. ISG-15 is removed from ISGylated products by the deubiquitinating enzyme, Ubp43. EPO stimulates a rapid accumulation of Ubp43 in wild type cells, however, EPO fails to induce Ubp43 mRNA in STAT1-deficient fetal liver and PHZ-primed erythroblasts. Experiments are underway to confirm that the mechanism by which STAT1 exerts negative regulation of erythropoiesis is via upregulation of the deubiquitinating enzyme, Ubp43.

scholarly journals Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1269-1269 ◽  
Author(s):  
Lynn S. White ◽  
Deepti Soodgupta ◽  
Rachel L. Johnston ◽  
Jeffrey A. Magee ◽  
Jeffrey J. Bednarski
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Lower Percentage ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

Roles of MOZ in Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2269-2269
Author(s):  
Takuo Katsumoto ◽  
Yukiko Aikawa ◽  
Takahiro Ochiya ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Liver Cells ◽  
Recipient Mouse ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Homozygous Mutant ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract The AML1-CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene, which encodes a MYST-type histone acetyltransferase (HAT), is also involved in leukemia-associated translocations such as t(8;16), t(8;22) and inv(8), which are associated with acute myeloid leukemia with M4/5 subtypes. We previously found that MOZ functions as a potent coactivator for AML1. To investigate roles of MOZ in normal hematopoiesis, we generated MOZ-deficient mice using gene-targeting method. MOZ homozygous mutant is embryonic lethal and it died between days 14 and 15 of gestation. In fetal liver of MOZ-deficient E14.5 embryos, the total cell numbers and the colony-forming cells (CFCs) in a methylcellulose medium were remarkably reduced when compared with wild-type littermates. Flow cytometry analysis indicated that hematopoietic stem cells (HSCs) and progenitors of both myeloid and lymphoid lineages were severely reduced in MOZ-deficient embryos. Especially, the levels of c-kit expression were strongly reduced in lineage-negative cells. Differentiation arrest of erythroid progenitors at a terminal stage and increase in the numbers of Mac-1 and Gr-1 positive cells suggest that MOZ also plays roles in cell differentiation of erythroid, monocytic and granulocytic lineages. In E12.5 MOZ deficient fetal liver cells, expression profile analysis revealed decreases in expressions of thrombopoietin receptor c-mpl, Wnt related ligand dkk2 and HoxA9 and increase in HoxA5 expression. To further determine roles of MOZ in HSCs functions and their progenitors differentiation ability, competitive reconstitution assays were performed. Ly5.2+ fetal liver cells from wild-type, heterozygous or homozygous mutant embryos together with Ly5.1+ competitor fetal liver cells were transplanted into γ-irradiated Ly5.1+/Ly5.2+ recipient mouse. Ly5.2+ wild-type cells were observed in recipient mice after transplantation. However, cells derived from MOZ homozygous mutant embryos were not detected in peripheral blood, bone marrow, spleen and thymus. Reduced population of cells derived from heterozygous mutant embryos were observed. These data suggest that MOZ is required for lymphoid and myeloid development and for self-renewal of HSCs.

Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2525-2525
Author(s):  
Takuo Katsumoto ◽  
Issay Kitabayashi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals What Links Neutropenia to Immature Cardiolipin in Patients with Barth Syndrome (tafazzin-deficiency)?

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3579-3579
Author(s):  
Jihee Sohn ◽  
Thomas Brouse ◽  
Najihah Aziz ◽  
David B Sykes
Keyword(s):  
Er Stress ◽  
Knockout Mouse ◽  
Mitochondrial Membrane ◽  
Fetal Liver ◽  
Liver Cells ◽  
Barth Syndrome ◽  
Developmental Defect ◽  
Wild Type ◽  
Inner Mitochondrial Membrane ◽  
Fetal Liver Cells

Barth syndrome is an inherited X-linked disorder characterized by cardiomyopathy, skeletal muscle myopathy, and neutropenia. The syndrome arises because of inherited mutations in the gene TAZ, resulting in a loss of function of the protein tafazzin. Of note, a group of investigators recently described how tafazzin can regulate 'stemness' in models of acute myeloid leukemia (Cell Stem Cell, 2019). Tafazzin is an enzyme that processes the final step of cardiolipin maturation, replacing saturated with unsaturated acyl chains. Cardiolipin is a 4-tailed phospholipid that is almost-exclusively found in the inner membrane of the mitochondria. The lack of tafazzin activity results in a cardiolipin pool that contains more highly saturated lipid tails and it is this lack of unsaturated cardiolipins that contributes to a disorganized inner mitochondrial membrane. The link between tafazzin-deficiency and myopathy is generally explained by the dependence of muscle cells on mitochondrial function as well as oxidative respiration. The components of the electron transport chain are co-localized with cardiolipin in the inner mitochondrial membrane, and it is felt that their appropriate organization within the membrane lipid bilayer is dependent on the presence of mature cardiolipin which is lacking in those individuals with Barth syndrome. The link between tafazzin-deficiency and neutropenia is less clear. Neutrophils are terminally-differentiated effector cells of the innate immune system. They are critical for protection against bacterial and fungal pathogens and patients without sufficient neutrophils are among the most immunocompromised and at risk of lethal infection. Neutrophils have few mitochondria at baseline and are generally believed to rely primarily on glycolysis for energy production. It is not known if the mechanism of neutropenia in Barth syndrome is due to a lack of production or due to increased clearance (e.g. more prone to apoptosis). We undertook the study of tafazzin-deficient neutrophils to try to elucidate the mechanism of neutropenia in patients with Barth syndrome. We took advantage of an existing tafazzin-knockout mouse and a system of conditional immortalization of granulocyte-monocyte progenitors (GMP) using the ER-Hoxb8 system pioneered in our laboratory. This ER-Hoxb8 system allows for the unlimited ex vivo expansion of myeloid progenitors in the presence of estradiol and active Hoxb8. Once estradiol is removed from culture media, the Hoxb8 protein is inactive and the cells undergo normal, synchronous and terminal neutrophilic differentiation. In this manner, we were able to generate tafazzin-wild-type and knockout GMP lines from murine fetal liver cells. Analysis of the myeloid progenitor compartment in fetal liver cells (d14.5-d16.5) showed no difference between wild-type and knockout mice, arguing against a developmental defect (E15 results shown in PANEL A). Furthermore, the tafazzin-deficient ER-Hoxb8 GMPs and neutrophils were remarkably normal when tested across a variety of assays including phagocytosis, cytokine production and ROS generation (ROS by H2DCFDA shown in PANEL B). We hypothesized that the unpredictable neutropenia in patients with Barth Syndrome might be due to an increased proclivity to apoptosis because of the mitochondrial membrane defect. Indeed, the tafazzin-deficient GMPs showed an increased sensitivity to Bcl2-inhibition following treatment with ABT199 (PANEL C). Two lines of evidence have suggested that the increased tendency towards apoptosis may be due to endoplasmic-reticulum (ER) stress. (1) Transmission electron microscopy demonstrated 'swollen' ER in the tafazzin-deficient cells (not shown) and (2) a comparison of gene expression patterns demonstrated an increased expression of ATF4 and CHOP (DDIT3) in the tafazzin-deficient cells (PANEL D). We are now focused on validating these findings and in establishing models to confirm the ER-stress phenotype in vivo in the TAZ-knockout mouse model as well as primary samples from patients with Barth Syndrome. We hope that this line of work will confirm the mechanism of neutropenia and shed light on potential targets for therapeutic intervention. In addition, this very rare disorder has provided insight into a previously-unexpected link between neutrophil survival and the membrane integrity of the inner mitochondrial membrane. Figure Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder.

Impaired Repopulating Activity of Fus-Deficient Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2222-2222
Author(s):  
Takeaki Sugawara ◽  
Atsushi Iwama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Chromosomal Translocation ◽  
Liver Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Fetal Liver Cells

Abstract RNA-binding protein FUS (also known as TLS) was originally identified in chromosomal translocation in human myxoid liposarcoma. The FUS gene is also translocated with the transcription factor gene ERG in human myeloid leukemia with recurrent chromosomal translocation t(16;21). Multiple data suggest that wild-type FUS is also involved in the development of leukemia as one of the downstream targets for oncoproteins including BCR-ABL. However, little is known about the role of FUS in the normal hematopoiesis. The previous report demonstrated that Fus-deficient (Fus−/−) newborn mice, which die shortly after birth because they cannot suckle, have a non-cell-autonomous defect in B lymphocyte development. No cell-autonomous defect of Fus−/− hematopoietic cells has been documented. Here we report the detailed analyses of the Fus−/− fetal liver hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 were smaller in size and exhibited a significant reduction in hematopoietic cell numbers by 60% compared with the wild type (WT). Nonetheless, no significant difference was observed in the proportion of stem/progenitor cell fraction (lineage-marker-c-Kit+Sca-1+; KSL) as well as colony-forming cells between WT and Fus−/− fetal livers. Fus−/− KSL cells proliferated and differentiated almost normally in vitro. To examine in vivo repopulating activity, we transplanted fetal liver cells to lethally irradiated CD45.1 recipients with competitor bone marrow cells. Fus−/− fetal liver donor cells reconstituted recipients’ hematopoiesis for the long term and contributed to all cell lineages including B lymphocytes. In contrast to the in vitro results, however, the chimerism of donor-derived cells was significantly lower in recipients receiving Fus−/− fetal liver cells compared with WT controls (approximately 2-fold reduction). This trend was reproducible with both unfractionated and purified KSL fetal liver test cells. Our data demonstrated that the proto-oncogene Fus is involved in the maintenance of normal HSC functions. Detailed analyses on the underlying mechanisms are in progress.

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