Epo Induces Phosphorylation of GATA-1 Transcription Factor Via a PI3-Kinase-Dependent Signaling Pathway.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 816-816
Author(s):  
Wei Zhao ◽  
Claire Kitidis ◽  
Mark D. Fleming ◽  
Harvey F. Lodish ◽  
Saghi Ghaffari
Keyword(s):  
Transcription Factor ◽  
Cell Differentiation ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Pi3 Kinase ◽  
Pcr Analysis ◽  
Red Cell ◽  
Wild Type ◽  
Fetal Liver Cells

Abstract Activation of both EpoR signaling and GATA-1 transcription factor is required for normal erythropoiesis. Whether any signal generated from Epo-stimulated EpoR regulates GATA-1 function is not known. In particular the function of the PI3-kinase-AKT signaling pathway downstream of EpoR is not clear. Retroviral (MSCV-IRES-GFP) transduction with a constitutively active but not wild type AKT induces red cell differentiation of both JAK2−/− and wild type fetal liver cells in the absence of Epo. The differentiation of fetal liver cells along the erythroid lineage was determined by the number of CFU-E-generated colonies in vitro, Realtime PCR analysis of red cell specific gene expression, FACS analysis of cell surface markers TER119 and CD71, morphological analysis and diaminobenzidine staining of hemoglobin of the transduced GFP+ cells. Consistent with a role for AKT serine threonine kinase in supporting erythroid differentiation, overexpression of a dominant negative AKT partially inhibited Epo-dependent erythroid differentiation of fetal liver cells and of cultured erythroid cells. Furthermore, the significant potential of the constitutively active AKT in inducing red cell differentiation of fetal liver cells could not be solely attributed to its survival signal. We have identified serine 310 (S310) within a putative AKT consensus phosphorylation sequence in GATA-1 transcription factor. This sequence is highly conserved among species and among hematopoietic GATA (1, 2, 3) members. Recombinant and immunocomplexes of activated AKT but not the related kinase SGK phosphorylated specifically GST-GATA-1 WT but not GST-GATA-1 S310A in vitro. The constitutively activate AKT transactivated wild type (WT) GATA-1 but not the mutated GATA-1 S310A in reporter gene assays. We raised an anti-GATA-1 pS310 antibody and analyzed by Western Blot GATA-1 phosphorylation in response to Epo in the Epo-starved erythroleukemic HCD57 cells. Phosphorylation of GATA-1 on S310 was detected within 30 minutes and up to several hours in nuclear extracts of Epo-stimulated HCD57 cells and was inhibited in the presence of the PI3-Kinase inhibitor LY294002 (10μM) but not the MAPkinase inhibitor. Interestingly, among the seven constitutively phosphorylated serines of GATA-1, serine 310 is the only residue that is hyperphosphorylated during DMSO-induced differentiation of murine erythroleukemia cells. To further investigate the role of GATA-1 phosphorylation, we retrovirally transduced GATA-1-deficient G1E cells with GATA-1 WT and mutants and assessed red cell differentiation by benzidine staining and Realtime RT-PCR analysis. G1E cells are arrested at a proerythroblast stage and differentiate to mature red cells when overexpressing WT GATA-1. Expression of GATA-1 Dephospho missing all seven phospho-serine residues in G1E cells induced only 40% of erythroid differentiation seen with WT GATA-1. Expression of GATA-1 DephosphoA310S mutant with S310 added back to GATA-1 Dephospho resulted in 70% of differentiation seen with GATA-1 WT. Similarly, retroviral expression of GATA-1 Dephospho blocked significantly erythroid differentiation of transduced GFP+ fetal liver cells in the presence of Epo whereas expression of GATA-1 DephosphoA310S did not have a significant inhibitory effect. Taken together, these data suggest that phosphorylation of GATA-1 is regulated by PI3-kinase downstream of EpoR and is important for red cell differentiation.

Complete Absence of the Lineage-Determining Transcription Factor C/EBPα Results in Loss of Myeloid Identity in Bcr/abl Induced Malignancy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 646-646
Author(s):  
Katharina Wagner ◽  
Pu Zhang ◽  
Frank Rosenbauer ◽  
Bettina Drescher ◽  
Susumu Kobayashi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Myeloproliferative Disease ◽  
Granulocytic Differentiation ◽  
Human Acute Myeloid Leukemia ◽  
Fetal Liver Cells ◽  
Factor C

Abstract The lineage-determining transcription factor C/EBPα is required for myeloid differentiation. Decreased function or expression of C/EBPα is often found in human acute myeloid leukemia. However, the precise impact of C/EBPα deficiency on the maturation arrest in leukemogenesis is not well understood. To address this question, we used a murine transplantation model of a bcr/abl induced myeloproliferative disease. The expression of bcr/abl in C/EBPα+/+ and C/EBPα+/− fetal liver cells lead to a chronic myeloid leukemia-like disease. Surprisingly, bcr/abl expressing C/EBPα−/− fetal liver cells fail to induce a myeloid disease in transplanted mice, but instead cause a fatal, transplantable erythroleukemia. Accordingly, increased expression of SCL and GATA-1 in hematopoietic precursor cells of C/EBPα−/− fetal livers was found. The mechanism for the lineage shift from myeloid to erythroid leukemia was studied in a bcr/abl positive cell line. Consistent with findings of the transplant model, expression of C/EBPα and GATA-1 was inversely correlated. Id1, an inhibitor of erythroid differentiation, was upregulated upon C/EBPα expression. Chromatin immunoprecipitation was done and C/EBPα binding to a 3 prime enhancer of the Id1 gene was observed. Downregulation of Id1 by RNA interference impaired C/EBPα induced granulocytic differentiation. Thus, Id1 is a direct and critical target of C/EBPα. Taken together, our study provides the first evidence that myeloid lineage identity of malignant hematopoietic progenitor cells requires the residual expression of C/EBPα.

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.

The Role of K-ras Signaling in Erythropoiesis In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3136-3136
Author(s):  
Jing Zhang ◽  
Yangang Liu ◽  
Caroline Beard ◽  
Rudolf Jaenisch ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Ras Signaling ◽  
Erythroid Progenitors ◽  
Akt Activation ◽  
Fetal Liver Cells ◽  
Terminal Erythroid Differentiation

Abstract K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

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.

scholarly journals TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽  
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.

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.

scholarly journals The mouse Klf1 Nan variant impairs nuclear condensation and erythroid maturation

2018 ◽  
Author(s):  
Ileana Cantú ◽  
Harmen J.G. van de Werken ◽  
Nynke Gillemans ◽  
Ralph Stadhouders ◽  
Steven Heshusius ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Single Amino Acid ◽  
Erythroid Cells ◽  
Nuclear Condensation ◽  
Similar Phenotype ◽  
Fetal Liver Cells ◽  
Erythroid Maturation

ABSTRACTKrüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >65 KLF1 variants, causing different erythroid phenotypes, have been described. The Klf1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the ~780 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We conclude that reduced expression of XPO7 is partially responsible for the erythroid defects observed in Nan erythroid cells.

scholarly journals Fetal liver cells produce both fetal and adult erythrocytes in semiallogeneic radiation chimeras: possible influence of adult environment

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 586-591 ◽  
Author(s):  
G Mouchiroud ◽  
JP Blanchet
Keyword(s):  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Parental Origin ◽  
Adult Type ◽  
Fetal Liver Cells ◽  
Kinetics Of ◽  
Hybrid Mice ◽  
Fetal Antigen

Abstract Two kinds of erythrocytes are released in the blood of irradiated adult hybrid mice grafted with parental fetal liver cells: fetal antigen- bearing erythrocytes (Ft+ cells) and adult-type Ft- erythrocytes. Both are of parental origin, as determined by immune lysis using histocompatibility alloantigens. The latter cells make up all the recipient's red blood cells 2 mo after receipt of the graft, Ft+ cells then being no longer detected. The transient duality of erythropoiesis in irradiated adults grafted with fetal liver cells has been confirmed by studying the kinetics of CFU-E populations, as characterized by their ability to give rise to Ft+ or Ft- erythrocytes. The results are discussed in terms of environmental factors that influenc erythroid differentiation.

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.

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