RNA Sequencing Of Adult Bone Marrow Erythroid Populations Reveals Significant Transcriptional Deregulation In Foxo3-/- Mutant Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3416-3416
Author(s):  
Genís Campreciós ◽  
Xin Zhang ◽  
Yan Kou ◽  
Avi Ma'ayan ◽  
Saghi Ghaffari
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Expression Profiles ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Wild Type ◽  
Adult Bone Marrow ◽  
Erythroid Maturation ◽  
Adult Bone

Abstract Transcriptional control of last stages of erythropoiesis is a complex and well orchestrated process controlled by lineage-specific transcription factors. The precise contribution of the different transcription factors to this multistep process has not been fully elucidated. Foxo3 is a transcription factor that is required for terminal erythroid maturation and Foxo3 mutant mice exhibit ineffective erythropoiesis. In order to gain further insight into the contribution of Foxo3 to the control of adult terminal erythroid maturation we analyzed the transcriptome of three adult bone marrow erythroid precursor populations: pro-, basophilic and polychromatophilic erythroblasts from wild type and Foxo3-/- mice. Populations were FACS sorted according to their TER119 and CD44 cell surface expression and FSC properties. RNA was then isolated and sequenced using the Illumina GaII platform. Genes were grouped into 3 categories according to their expression during erythroid cell maturation using the Short Time Series Expression Miner (STEM) program: no change (4577 genes), down-regulated (2868 genes) or up-regulated (2637) (Figure 1). Enrichment analysis of groups of genes using the ChEA database identified Myb, Meis1, Runx1, Fli1 and PU.1 as the main transcription factors regulating gene repression over erythroid maturation. In contrast, ChEA identified known erythroid transcription factors like Gata1, Eklf and Tal1 to drive the up-regulation of many of the erythroid-specific genes. This analysis also enabled the identification of putative novel transcription factors implicated in erythroid cell maturation. Interestingly, the difference between WT and Foxo3-/- cells increased gradually from pro- to polychromatophilic erythroblasts in correlation with increased Foxo3 expression during these steps of maturation. Strikingly, pathway enrichment analysis detected several immune-related pathways such as Toll-like receptors, TGF-β and IL-1 signaling as expressed in maturing wild type erythroid cells and significantly deregulated in Foxo3-/- cells. The expression of a number of these immune genes in erythroid cells has been validated by qRT-PCR. In addition, among others, a cluster of genes from the autophagy pathway was noted to be significantly down-regulated in Foxo3 mutant erythroid cells. In order to better dissect Foxo3 transcriptional control during erythroid maturation, STEM analysis of Foxo3-/- samples revealed an unexpected number of differences compared to WT. Most remarkably the STEM analysis identified that 90% of the 1198 genes that are continuously up-regulated during erythroid maturation from pro- to polychromatophilic are highly compromised in their level of expression during erythroid maturation in the absence of Foxo3. Interestingly, this group was also enriched for Foxo3 direct target genes as determined by ChIP-seq studies. We also identified a subset of genes whose expression increased from pro- to basophilic erythroblasts but decreased thereafter in the absence of Foxo3 in contrast to wild type cells. Interestingly, ChEA analysis on this group identified a subset of genes that are targets of Gata1, Eklf and Tal1 that may require Foxo3 for their full expression at the last stages of erythroid cell maturation. In conclusion, we present an unbiased genome-wide approach using RNA sequencing of adult bone marrow erythroid cells to study the contribution of Foxo3 to the regulation of gene expression at the last stages of erythroid cell maturation. This analysis enabled us to identify novel genes and pathways whose function in the control of red cell generation requires further investigations.Fig. 1Genes with FPKM>2 from WT and Foxo3-/- samples analyzed with the STEM software, divided into 6 different categories according to their expression profiles during terminal erythroid cell maturation from pro- to polychromatophillic erythroblasts. Genes were then further grouped in 3 subsets: down-regulated, up-regulated and no change. The number of genes in each profile is indicated at the bottom for wild type and Foxo3-/- samples.Fig. 1. Genes with FPKM>2 from WT and Foxo3-/- samples analyzed with the STEM software, divided into 6 different categories according to their expression profiles during terminal erythroid cell maturation from pro- to polychromatophillic erythroblasts. Genes were then further grouped in 3 subsets: down-regulated, up-regulated and no change. The number of genes in each profile is indicated at the bottom for wild type and Foxo3-/- samples. Disclosures: No relevant conflicts of interest to declare.

Metabolic Pathways Control Normal and Beta-Thalassemic Erythroid Cell Maturation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 369-369 ◽  
Author(s):  
Geníis Camprecióos ◽  
Xin Zhang ◽  
Valentina D'Escamard ◽  
Pauline Rimmele ◽  
Carolina L. Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Metabolic Pathways ◽  
Research Funding ◽  
Mtor Signaling ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Abstract 369 Erythroid cell maturation requires the integration of erythropoietin receptor (EpoR)-mediated signaling pathways and transcriptional programs of erythroid cell proliferation and differentiation largely orchestrated by GATA-1 and its transcriptional partners. Although red blood cells (RBC) purely rely on glycolysis for energy production, and metabolic processes specifically autophagy have been implicated in erythroid maturation, the potential involvement of metabolic pathways in the control of erythroid cell production is not known. Foxo3 transcription factor is a direct target of GATA-1, functionally regulated by EpoR signaling and essential for the redox regulation of erythropoiesis. Mammalian target of rapamycin (mTOR) kinase is a critical regulator of metabolic processes. We have found that generation and cycling of early erythroid precursors is controlled by a redox-mediated Foxo3-mTOR signaling. Terminal erythroid maturation is specifically compromised in Foxo3-deficient mice. Terminal maturation involves nuclear condensation, enucleation, mitochondrial clearance and complete adoption (or conversion) of glycolytic pathway by erythroid cells. We found RBC to be significantly decreased in Foxo3 mutant bone marrow and peripheral blood. This was in contrast to the increase of polychromatophilic erythroblasts associated with an increase in the total TER119+ population in Foxo3 mutant bone marrow likely reflecting a compensatory mechanism. Notably, using DRAQ5, a DNA-binding fluorescent dye, we found the rate of erythroblasts' enucleation in Foxo3 mutant mice to be significantly compromised. During erythroid maturation Riok3 and Mxi1 transcripts encoding for two important regulators of fetal liver erythroid enucleation are highly upregulated in the bone marrow and robustly expressed in the adult normoblasts and reticulocytes. In agreement with defective enucleation expression of both Riok3 and Mxi1 is highly reduced in Foxo3 mutant erythroblasts and reticulocytes. Interestingly, a two-week in vivo treatment of wild type (WT) and Foxo3−/− mice with rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1) activity, increased significantly the rate of enucleation in a Foxo3-dependent manner, suggesting that mTOR requires Foxo3 activity in supporting erythroid cell maturation. Importantly, targeting mTOR ameliorates beta-thalassemia as inhibition of mTOR signaling by rapamycin treatment improved erythroid cell maturation in the bone marrow, resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase) as well as significant reduction in reticulocyte production of beta-thalassemic intermedia (th3/+) mice. Combination of thiazole orange (an RNA and DNA probe) with DRAQ5 determined that in addition to enucleation, the relative production of reticulocytes is also decreased significantly in Foxo3 mutant bone marrow. Strikingly, 6,1% of RBC (CD71 negative) in the peripheral blood of Foxo3−/− animals contained mitochondria (CD71−Mito+) as compared to 0.7 % of wild type RBC. Autophagy is strongly implicated in late stage erythroid cell maturation and mitochondrial removal from reticulocytes. In agreement with a function for Foxo3 in control of mitochondrial removal, expression of Ulk1 (Atg1) and Nix (Bnip3l) both regulators of mitochondrial clearance via autophagy was highly downregulated in Foxo3 mutant normoblasts and reticulocytes. The expression of Nix was notable since Nix was upregulated over 40 fold in wild type but not in Foxo3 mutant reticulocytes as evaluated by the Fluidigm™ microfluidics array technology. These results are consistent with the expression pattern of Foxo3 that is highly upregulated with erythroid maturation and is the most highly expressed in normoblasts. Collectively our results indicate that Foxo3 has a key function in the regulation of terminal erythroid cell maturation. They also suggest that rapamycin may be considered for the treatment of beta-thalassemia. These results are consistent with the model of FOXO3a induction during late human erythroid cell maturation. Based on these studies we propose that Foxo3 coordinates metabolic pathways with the transcriptional program of terminal erythroid cell maturation. Understanding this metabolic program is likely to impact efficient RBC production in culture. Disclosures: Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

scholarly journals Chromatin structure and developmental expression of the human alpha-globin cluster.

1986 ◽  
Vol 6 (4) ◽  
pp. 1108-1116 ◽  
Author(s):  
M Yagi ◽  
R Gelinas ◽  
J T Elder ◽  
M Peretz ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromatin Structure ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Adult Bone Marrow ◽  
Hypersensitive Sites ◽  
Alpha Globin ◽  
Adult Bone

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

scholarly journals Development and differentiation of early innate lymphoid progenitors

2017 ◽  
Vol 215 (1) ◽  
pp. 249-262 ◽  
Author(s):  
Christelle Harly ◽  
Maggie Cam ◽  
Jonathan Kaye ◽  
Avinash Bhandoola
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Lymphoid Cell ◽  
Innate Lymphoid Cell ◽  
Adult Bone Marrow ◽  
Lymphoid Progenitors ◽  
Development And Differentiation ◽  
Adult Bone

Early innate lymphoid progenitors (EILPs) have recently been identified in mouse adult bone marrow as a multipotential progenitor population specified toward innate lymphoid cell (ILC) lineages, but their relationship with other described ILC progenitors is still unclear. In this study, we examine the progenitor–successor relationships between EILPs, all-lymphoid progenitors (ALPs), and ILC precursors (ILCps). Functional, bioinformatic, phenotypical, and genetic approaches collectively establish EILPs as an intermediate progenitor between ALPs and ILCps. Our work additionally provides new candidate regulators of ILC development and clearly defines the stage of requirement of transcription factors key for early ILC development.

Chromatin structure and developmental expression of the human alpha-globin cluster

1986 ◽  
Vol 6 (4) ◽  
pp. 1108-1116
Author(s):  
M Yagi ◽  
R Gelinas ◽  
J T Elder ◽  
M Peretz ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromatin Structure ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Globin Gene ◽  
Erythroid Cells ◽  
Adult Bone Marrow ◽  
Hypersensitive Sites ◽  
Alpha Globin ◽  
Adult Bone

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

Elevated P21 (CDKN1a) Mediates Apoptosis of Beta-Thalassemic Erythroid Cells in Mice but Its Ablation Doesn't Improve Erythroid Maturation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-19
Author(s):  
Miao Lin ◽  
Vijay Menon ◽  
Raymond Liang ◽  
Tasleem Arif ◽  
Laura Breda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Double Mutant ◽  
Complete Blood Count ◽  
Globin Gene ◽  
Mutant Mice ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Wild Type ◽  
The Status

Beta-thalassemias are caused by mutations in the β-globin gene leading to anemia. In β-thalassemia, excessive accumulation of unpaired α globin chains in erythroblasts, triggers redox-mediated reactions, which is associated with increased production of immature erythroid precursors that fail to mature. This impaired maturation is in part due to increased apoptosis of late maturing erythroblasts in β-thalassemic patients that aggravates anemia despite enhanced erythropoiesis leading to what is called ineffective erythropoiesis and ultimately resulting in extramedullary expansion of hematopoiesis. The mechanism of apoptosis in beta-thalassemia remains poorly understood. To investigate this, we examined the status of mediators of stress response during erythroid cell maturation ofHbbth3/+ (th3/+) mice, a model that mimics the beta-thalassemia intermedia phenotype in humans. We found that both Foxo3 and p53 were prematurely activated in th3/+ beta-thalassemic erythroblasts as compared to wild type controls. We crossed Hbbth3/+ (th3/+) and Foxo3-/- mice and found that red blood cell (RBC) count and hemoglobin content were improved (by 1g/L, n=10), and erythroblast apoptosis was decreased to similar levels as in the WT during erythroblast maturation of double mutant mice. However, loss of Foxo3 did not ameliorate the splenomegaly of th3/+mice. We also found that p53 direct target, p21 the cyclin-dependent kinase inhibitor was greatly upregulated in th3/+erythroblasts as well as in beta-thalassemic patients' erythroblasts. To address the contribution of p21, we crossed p21-/- and Th3/+. It showed a significant decrease of apoptosis in CD45- TER119+ erythroblasts both in the bone marrow and spleen of double mutant mice (30% and 23% reduction respectively, n=6 mice each genotype). Although, as in beta-thalassemic patients, serum erythropoietin (Epo) was elevated in the peripheral blood of th3/+mice, the double mutant mice had significantly lower level of Epo than th3/+ (45% reduction, n=3 mice per genotype).In p21-/-th3/+, CD45- TER119+ cells also showed lesser ROS accumulation(12% less, n=3 per genotypes). However, to our surprise, the deletion of p21 on beta-thalassemic background did not have any effect on splenomegaly (n=6 mice each genotype), complete blood count, hemoglobin, RBC production or bone marrow erythroid cell maturation (n=12 mice each genotype). To further examine the underlying mechanism, we analyzed cell cycle in double mutant p21-/-th3/+ erythroblast at distinct stages of maturation identified by CD45, TER119, CD44 and cell size (n=3 mice per genotype) using ki67 staining at distinct stages of maturation. We found p21-/-th3/+erythroblasts proliferate much less than their th3/+ counterparts (basophilic erythroblasts G2 14% less, polychromatic erythroblasts 20% less, p<0.05 n=3 mice per genotype). This may partially explain lack of improvement of RBC production and anemia despite enhanced erythroblast survival. ROS levels were also reduced in double mutant p21-/-th3/+ erythroblasts as compared to controls. Next we investigated the status of p53 and Foxo3 in double mutant p21-/-th3/+ erythroblasts as compared to controls. We confirmed as we had observed earlier that nuclear p53 and Foxo3 expression were greater in th3/+ primitive erythroid (TER119-/low, c-KIT+, CD71Hi) cells than in wild type (n=3 mice per genotype) controls. Strikingly, the double mutant p21-/-th3/+ erythroblasts exhibited the greatest nuclear Foxo3 in all four groups, while nuclear p53 was dramatically reduced by over 80% (n=2 mice each genotype. Each mouse taking >=30 cells to calculate nuclear MFI) even as compared to wild type. These combined studies suggest that ameliorating apoptosis may not improve anemia in beta-thalassemia. Disclosures Liang: Hemogenyx Pharmaceuticals LLC: Current Employment.

Csnk2β Knockout during Hematopoiesis Results in Lethality at Mid/Late Gestation Mostly Due to Impaired Fetal Erythropoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4329-4329
Author(s):  
Laura Quotti Tubi ◽  
Sara Canovas Nunes ◽  
Alessandro Casellato ◽  
Elisa Mandato ◽  
Fortunato Zaffino ◽  
...  
Keyword(s):  
Transcription Factors ◽  
High Throughput ◽  
Fetal Liver ◽  
Cell Development ◽  
Conditional Knockout ◽  
Late Gestation ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Fetal Erythropoiesis

Abstract Background. CK2, a serine-threonine kinase composed of two catalytic (α) and two regulatory (β) subunits, has been clearly involved in several hematologic malignancies. This kinase regulates the PTEN/PI3K/AKT, Wnt/βcatenin, Hedgehog, JAK-STAT, cMyc and NF-κB signalling cascades, all of which are known to be of critical importance for hematopoietic stem cell (HSC) biology and normal hematopoiesis. However, the role played by CK2 during blood cell development has remained as yet unexplored. Aims and methods. CK2 function in hematopoiesis was investigated generating conditional knockout mice for CK2β by crossing Csnk2β-Flox/Flox mice with Vav1-CRE transgenic mice. Inactivation of Csnk2β started from 9.5 dpc during embryonic development. Histo-cytological methods, FACS analysis, colony-forming assays (CFA), signal transduction studies by western blotting and RT-PCR were employed to characterize the cellular and molecular phenotype. High throughput RNAseq analysis was also performed on purified Ter119-positive erythroid cells from Csnk2β knockout and Csnk2β control mice to identify differentially expressed CK2-dependent transcriptional targets. Results. Csnk2β knockout in hematopoiesis resulted lethal at mid-late gestation. Rarely some pups were found dead at birth. Macroscopic and phenotypic analysis during gestation revealed the appearance of pale and hydropic fetuses after 12.5 dpc. The majority of pups showed teleangiectasic vessels and haemorrhages. Fetal livers appeared smaller and paler. Cytological analysis and CFA studies unveiled a great depletion of hematopoietic elements belonging to both the erythroid, megakaryocytic and granulocytic-monocytic precursors. A more thorough analysis of the erythroid phenotype revealed that Csnk2β loss caused impairment/loss of red cell maturation at two developmental stages: the earlier stages of Megakaryocyte-Erythroid Precursors (MEP) and pro-erythroblasts and the later stages of terminal maturation (orthocromatic erythroblasts towards reticulocytes). Expression analysis of proteins/genes belonging to known hematopoietic and erythroid-regulating pathways showed perturbations in cell cycle regulatory molecules, cellular apoptosis, a marked reduction of total and phosphorylated Akt in Ser473 and Ser129, a decrease of GATA1 protein levels and a decrease of Hedgehog/Wnt target genes such as Gli-1 and Cyclin D1. Erythropoietin-dependent AKT activation and GATA1 phosphorylation was impaired by Csnk2β loss. Moreover, starting at 14.5 dpc, blood cells displayed a massive p53-dependent response, marked by high levels of p21 and a progressive clear apopototic phenotype. At 17.5 dpc residual hematopoietic cells in the fetal liver were represented by dying erythroid cells, immature myelo-monocytic precursors (expressing high CD11b and low Gr1 levels on the surface) and B-cells displaying an aberrant phenotype with low intensity of expression of B220 and CD19 on the surface. High throughput RNAseq analysis of Ter119-expressing fetal liver cells (erythroid lineage) obtained from 14.5 dpc pups revealed the upregulation of 145 transcripts and the downregulation of 68 transcripts. Among the most increased transcripts were the transcription factors Jun/AP1 and stress-related intermediaries and embryonal globin ε and ζ chains. Among the most decreased transcripts were sugar transporters, glycoproteins CD36 and CD59a, Duffy Blood Group Atypical Chemokine Receptor and component members. Conclusions. We found that Csnk2β plays a critical role in mouse blood development by regulating definitive hematopoiesis of all the hematopoietic cell lineages; however, Csnk2β was needed for the early and late erythropoiesis whilst its loss could be compatible with a certain extent of immature/altered myelo-monocytic and B cell development. Among the pathways found targeted by Csnk2β loss were the PI3K/Akt and the p53-p21 cascades. Our data also suggest that Csnk2β might have a role in the proper activation of the erythroid master regulator GATA1. Moreover, RNAseq analysis revealed that this kinase might have a broader impact during erythroid cell maturation by regulating the activity of critical stress related transcription factors, of molecules regulating energy-managing cellular processes and of mechanisms controlling the switch from embryonal to fetal erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Proliferation-Independent Functions of the E2F-2 Transcription Factor during Terminal Erythroid Maturation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4356-4356
Author(s):  
Kelsey Swartz ◽  
Gangjian Qin ◽  
Alexander C Minella
Keyword(s):  
Flow Cytometry ◽  
Knockout Mice ◽  
Transcriptional Activity ◽  
Fetal Liver ◽  
Cyclin E ◽  
Cell Maturation ◽  
Erythroid Cell ◽  
Wild Type ◽  
Independent Functions ◽  
Erythroid Maturation

Abstract Lesions in the retinoblastoma (Rb) pathway induce defects in hematopoiesis that are typically thought to be due to inappropriate proliferative signaling from increased E2F transcriptional activity. Newer data demonstrate that E2F transcription factors have distinct roles in differentiating cells, but the key proliferation-independent functions of the E2Fs remain incompletely defined. We previously found that deregulated activity and expression of cyclin E induces defects in terminal erythroid cell maturation, using a mouse knock-in model (cyclin ET74A T393A). Because cyclin E-Cdk2 potentiates E2F transcriptional activity via Rb phosphorylation, we hypothesized that inhibiting E2F activity would rescue hyperactive cyclin E-associated erythroid cell defects. E2F-2 is specifically induced during terminal erythroid maturation; therefore, we crossed our cyclin E knock-in strain with E2F-2 knockout mice. Unlike cyclin E knock-in bone marrows that display obvious defects in erythroid cell maturation, cyclin E knock-in; E2F-2 knockout animals demonstrated normalized erythroid maturation by flow cytometry. However, these compound mutant mice remain anemic, suggesting red blood cell (RBC) maturation was not completely restored. We studied adult E2F-2 -/- mice further, and consistent with published data, we found that they are anemic. Interestingly, we do not detect obvious terminal erythroid maturation defects in adult E2F-2-knockout bone marrows. Using CFSE-labeled erythrocyte in vivo survival experiments, we found loss of E2F-2 results diminishes survival of adult peripheral erythroid cells within syngeneic, wild-type recipients, suggesting the anemia in the E2F-2 knockout mice is due to accelerated destruction and not solely a production defect. To study the erythroid maturation program in the absence of E2F-2 in detail, we obtained fetal liver-derived hematopoietic progenitors from E2F-2 knockout versus wild-type embryos and differentiated these towards the erythroid lineage in vitro. E2F-2 deletion results in impaired erythroid maturation as evidenced by flow cytometry-based assays of cell surface marker expression, abnormal cell morphologies, and impaired enucleation. In order to identify functionally significant E2F-2 targets during erythroid maturation, we performed microarray analyses on sorted subpopulations of fetal liver-derived erythroid cells. We found widespread gene expression changes in E2F-2 knockout cells, which included but were not limited to proliferation related pathways. Furthermore, using global histone mass spectrometry analysis, we found that loss of E2F-2 results in marked alteration of histone-H3, lysine-4 methylation during erythroid differentiation. Thus, our data demonstrate that E2F-2 has both proliferation-dependent and independent functions that include the coordination of transcriptional and epigenetic programs during terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Gene Expression Differences Between Fetal Liver-Derived and Adult Bone Marrow-Derived Murine Megakaryocyte Progenitors: Implications for DS-TMD.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1208-1208 ◽  
Author(s):  
Karen Wieland ◽  
Alan B. Cantor
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Early Stage ◽  
Fetal Liver ◽  
Expression Profiles ◽  
Normal Growth ◽  
Adult Bone Marrow ◽  
Ifn Alpha ◽  
Adult Bone ◽  
Megakaryocyte Progenitors

Abstract About ten percent of infants with Down syndrome are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. Twenty to thirty percent of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL), typically within a year or two following resolution of their DS-TMD. Recent work has shown that both DS-TMD and DS-AMKL cells harbor acquired mutations in the key megakaryocyte transcription factor GATA-1 that lead to the exclusive production of a short GATA-1 isoform (GATA-1s). The mechanism by which GATA-1s acts in DS-TMD/AMKL remains incompletely understood. Mice engineered to produce only GATA-1s exhibit hyperproliferation of fetal liver-derived megakaryocytes, but normal growth of post-natal bone marrow-derived megakaryocytes. This suggests that unique microenvironmental features of fetal liver compared to bone marrow differentially influence the effects of GATA-1s on megakaryopoiesis. In order to further understand the mechanisms involved in these stage-specific effects, we compared gene expression profiles of wild type megakaryocyte progenitors (MkPs) isolated directly from embryonic day 13.5 (e13.5) murine fetal liver (FL) and from adult bone marrow (BM). Cells were FACS sorted based on the immunophenotype lin-sca-1-c-kit+ CD41+ CD150+, which has recently been shown to mark megakaryocyte-selective progenitors. Colony forming assays of the sorted cells revealed 92–100% growth of megakaryocyte colonies in culture medium containing multilineage cytokines (SCF, IL3, IL11, GM-CSF, EPO and TPO), confirming strong enrichment for megakaryocyte lineage cells. RNA from sorted FL and BM MkPs was then used to perform Affymetrix 3′ cDNA expression microarray analysis. Expression of early-stage megakaryocyte factors, such as c-mpl, FOG1, GATA1, Runx-1 and Fli-1 were found in both sets of samples, confirming selection of megakaryocyte progenitor cells. Importantly, we observed a striking up regulation of interferon alpha (IFN alpha) inducible genes belonging to the p200 family in BM MkPs compared to FL MkPs. Gene set enrichment analyses (GSEA) confirmed broad up regulation of the IFN alpha pathway, and as well up regulation of the JAK-STAT pathway in BM MkPs compared to FL MkPs. These findings were validated by quantitative RT-PCR and in situ immunohistochemistry. Given that STAT1 is a direct GATA-1 target gene and a major downstream effector of IFN alpha signaling, we hypothesize that enhanced IFN alpha signaling in the bone marrow may compensate for potential deficiencies of STAT1 during fetal liver megakaryopoiesis in the setting of GATA-1s. Experiments are underway to test this hypothesis.

scholarly journals Unexpected Rearrangement and Expression of the Immunoglobulin λ1 Locus in Scid Mice

1999 ◽  
Vol 191 (11) ◽  
pp. 1933-1944 ◽  
Author(s):  
Norman R. Ruetsch ◽  
Gayle C. Bosma ◽  
Melvin J. Bosma
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Scid Mice ◽  
Nonhomologous End Joining ◽  
Recessive Mutation ◽  
Wild Type ◽  
End Joining ◽  
Adult Bone Marrow ◽  
B Lineage ◽  
Adult Bone

In severe combined immunodeficient (scid) mice, V(D)J recombination is severely impaired due to a recessive mutation (scid). Thus, we were surprised to find in this study that Vλ1–Jλ1 rearrangement is routinely detectable in scid fetal liver, adult bone marrow, and spleen in the apparent absence of completed VH–DJH and Vκ–Jκ rearrangements. Particularly surprising, we found the level of Vλ1–Jλ1 rearrangement in scid fetal liver to be comparable to that in fetal liver of wild-type mice. The majority of scid Vλ1–Jλ1 rearrangements contained abnormal deletions at the VJ junction, consistent with the known effect of scid. However, ∼15% of Vλ1–Jλ1 rearrangements lacked abnormal deletions. Productive λ1 transcripts resulting from in-frame rearrangements were readily detectable in scid adult bone marrow and spleen, consistent with our ability to detect λ1-expressing cells by flow cytometry in the spleens of bcl-2–transgenic scid mice. Strikingly, λ1 transcripts from individual scid mice often showed VJ junctional sequences with the same recurring palindromic (P) additions of three, four, or five nucleotides. To account for these findings, we suggest that (a) nonhomologous end joining of Vλ1 and Jλ1 coding ends in fetal B lineage cells may not be (severely) impaired by scid; (b) recurring P additions in scid λ1 transcripts may reflect certain molecular constraints imposed by scid on the resolution of Vλ1 and Jλ1 hairpin coding ends; and (c), scid lymphocytes with productively rearranged Vλ1 and Jλ1 elements may differentiate into recombinase-inactive cells and emigrate from bone marrow to spleen.

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