Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1284-1284
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
James Cormier ◽  
Junling Wang ◽  
Xuejun Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Myeloid Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Stem And Progenitor Cells ◽  
Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures: No relevant conflicts of interest to declare.

Without GABP Transcription Factor, BCR-ABL Cannot Transform HSCs to Leukemic Stem Cells Nor Induce Chronic Myelogenous Leukemia in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 965-965
Author(s):  
Zhongfa Yang ◽  
Cong Peng ◽  
Yaoyu Chen ◽  
Junling Wang ◽  
Xuejun Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Myelogenous Leukemia ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Cycle Arrest

Abstract Abstract 965 Chronic Myelogenous Leukemia (CML) is driven by the fusion oncogene, BCR-ABL, which transforms normal hematopoietic stem cells (HSCs) to leukemic stem cells (LSCs). Tyrosine kinase inhibitors, such as imatinib mesylate, control the massive expansion of leukemic cells in most patients with CML, but cannot eradicate CML LSCs. Several genetic pathways have been shown to be critical for the growth and survival of CML LSCs, including signaling molecules, tumor suppressors, and metabolic regulators. However, the role of transcription factors in functional regulation of LSCs in CML has not been widely studied. GA Binding Protein (GABP) is an ets transcription factor that is required for entry of fibroblasts into the cell cycle, and expression of Gabpa (the DNA-binding component of the complex), alone, was sufficient to induce quiescent, serum-starved cells to enter the cell cycle. Thus, Gabp is both necessary and sufficient for cell cycle entry. Conditional deletion of Gabpa in mouse bone marrow decreased hematopoietic progenitor cells more than 100-fold, but hematopoietic stem cells (HSCs) were relatively preserved. Gabpα null HSCs exhibited significant cell cycle arrest. We sought to determine if the cell cycle arrest caused by Gabpa loss could impair development of CML cells in a mouse model. We used retroviral infection of bone marrow from 5-FU-treated mice (to enrich for stem and progenitor cells) to generate a rapidly fatal CML-like syndrome in mice. Bone marrow from mice with loxP-flanked (floxed) Gabpa and wild type control mice was infected with a retrovirus that co-expresses BCR-ABL, Cre recombinase, and green fluorescent protein (GFP). As expected, transplantation into recipient mice of control mouse bone marrow infected with BCR-ABL-Cre-GFP retrovirus caused a rapidly fatal myeloproliferative neoplasm, with a median survival of approximately three weeks; mice died with massive infiltration of GFP+ myeloid cells in peripheral blood cell, spleen, bone marrow, and other organs. In floxed Gabpa bone marrow, the retrovirus deleted floxed Gabpa in cells that express the BCR-ABL fusion oncogene, and these cells were identifiable based on GFP expression. Transplantation of floxed Gabpa bone marrow infected with BCR-ABL-Cre-GFP retrovirus failed to induce CML during six months of observation. Importantly, GFP+ peripheral blood granulocytes were observed for at least 6 months after transplantation; these CD11b+, Gr1+ cells continued to express BCR-ABL and were shown to be Gabpa null. These results indicate that the lack of Gabpa severely impaired the function of LSCs. In addition, secondary transplantation of bone marrow from these mice again demonstrated the presence of BCR-ABL-expressing peripheral blood myeloid cells. We conclude that Gabp transcription factor is required for the transformation of HSCs to LSCs by BCR-ABL. Furthermore, the persistence of BCR-ABL-expressing myeloid cells without the development of leukemia provides a unique model that permits analysis of the biological properties of BCR-ABL in vivo. The continued generation of BCR-ABL-expressing cells without CML development is unprecedented, and represents a unique model of leukemia tumor suppression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The IL-33 Receptor/ST2 acts as a positive regulator of functional mouse bone marrow hematopoietic stem and progenitor cells

2020 ◽  
Vol 84 ◽  
pp. 102435 ◽  
Author(s):  
Maegan L. Capitano ◽  
Brad Griesenauer ◽  
Bin Guo ◽  
Scott Cooper ◽  
Sophie Paczesny ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Positive Regulator ◽  
Stem And Progenitor Cells

The Ets Transcription Factor, GABP, Is Required by Myeloid Progenitors for Normal Myeloid Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 256-256
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
Junling Wang ◽  
James Cormier ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Transcriptional Repressor ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Ets Domain

Abstract Abstract 256 GABP transcription factor regulates genes that are required for innate immunity. GABP is an obligate tetrameric protein complex that contains two molecules of GABPa, which binds to DNA through its ets domain, and two molecules of GABPb, which contains a transcription activation domain. GABP is an essential component of a multiprotein enhanceosome that is required for retinoic acid-dependent myeloid gene transcription. Disruption in mouse embryo fibroblasts of Gabpa, the mouse gene that encodes mouse Gabpa, causes profound cell cycle arrest at the G1-S boundary, due to reduced expression of DNA Polymerase a and Thymidylate Synthase, which are required for DNA synthesis, and of Skp2, a ubiquitin ligase that controls degradation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27. Thus, GABP is a key regulator of the cell cycle. In order to define the role of GABP in myeloid differentiation, we generated mice in which exons that encode the Gabpa ets domain are flanked by loxP recombination sites, and bred these floxed mice to mice that bear the Mx1-Cre transgene. Their progeny were treated with pI-C and Gabpa was efficiently deleted in hematopoietic cells of these Gabpa−/− mice. As controls for all experiments, mice that bear Mx1-Cre but which lack the floxed Gabpa allele were also injected with pI-C. Within days, the peripheral blood white blood cell count fell in the Gabpa−/− mice compared to the controls; half of the Gabpa−/− mice died within two weeks. Gabpa−/− mice exhibited a striking loss of Gr1+, CD11b+ cells in the peripheral blood, spleen, and bone marrow. Myeloid cells of Gabpa−/− mice were immature, morphologically dysplastic, and demonstrated aberrant patterns of myeloid gene expression. Bone marrow from Gabpa−/− mice formed reduced numbers of in vitro myeloid colonies in the presence of G-CSF, M-CSF, or GM-CSF; cells isolated from in vitro colonies from Gabpa−/− mice exhibited a strong bias toward macrophage-like morphology. Multicolor flow cytometry revealed a loss of granulocyte-monocyte committed progenitor cells (GMPs) in the bone marrow of Gabpa−/− mice, and these progenitors expressed aberrant patterns of key transcription factors. Especially notable in Gabpa−/− GMPs was reduced expression of Gfi-1, a transcriptional repressor that is mutated in some congenital neutropenic syndromes, and whose genetic disruption causes abnormalities in granulocyte development. We used chromatin immunoprecipitation (ChIP) to identify ets sites in the Gfi-1 promoter that are bound by GABP in vivo. We conclude that GABP is required for proliferation or survival of committed myeloid progenitor cells and for normal maturation of granulocytes. We hypothesize that defects in myeloid cell proliferation and differentiation associated with Gabpa disruption are caused, at least in part, by its regulation of the Gfi-1 transcriptional repressor. Furthermore, we propose that the regulation of Gfi-1 by GABP constitutes a key regulatory pathway in myeloid cell development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals EBF1 As a Novel Regulator of Bone Marrow Mesenchymal Stromal Progenitors

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 96-96
Author(s):  
Marta Derecka ◽  
Senthilkumar Ramamoorthy ◽  
Pierre Cauchy ◽  
Josip Herman ◽  
Dominic Grun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSPC is still a major challenge since the cues from bone marrow microenvironment remain largely elusive. Signals coming from the bone marrow niche, and specifically mesenchymal stem and progenitor cells (MSPC), orchestrate maintenance, trafficking and stage specific differentiation of HSPCs. Although, it is generally accepted that MSPCs are essential for hematopoietic homeostasis and generating multiple types of stromal cells, the exact transcriptional networks regulating MSPCs are not well established. Early B-cell factor 1 (Ebf1) has been discovered as lineage-specific transcription factor governing B lymphopoiesis. Additionally, it has been shown to play important role in differentiation of adipocytes, which are a niche component supporting hematopoietic regeneration. Thus, in this study we seek to examine if Ebf1 has an alternative function in non-hematopoietic compartment of bone marrow, specifically in mesenchymal stromal cells that maintain proper hematopoiesis. Here, we identified Ebf1 as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblasticcolonies (CFU-F) indicating reduced self-renewal. Moreover, cells expanded from these colonies display impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. In order to test how this defective MSPCs influence maintenance of HSPCs, we performed long-term culture-initiating cell assay (LTC-IC). After 5 weeks of co-culture of Ebf1-deficient stromal cells with wild type HSPCs we could observe significantly decreased number of cobblestone and CFU colonies formed by primitive HSPCs, in comparison to co-cultures with control stromal cells. Furthermore, in vivo adoptive transfers of wild type HSPCs to Ebf1+/- recipient mice showed a decrease in the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Additionally, Prx1-Cre-mediated deletion of Ebf1 specifically in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. These results confirm that mesenchymal stromal cells lacking Ebf1 render insufficient support for HSPCs to sustain proper hematopoiesis. Interestingly, we also observed a reduced ability of HSPCs sorted from Prx1CreEbf1fl/fl mice to form colonies in methylcellulose, suggesting not only impaired maintenance but also hindered function of these cells. Moreover, HSPCs exposed to Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occupancy of AP-1, ETS, Runx and IRF motifs, which is consistent with decreased myeloid output seen in Prx1CreEbf1fl/fl mice. These results support the hypothesis that defective niche can cause epigenetic reprograming of HSPCs. Finally, single cell and bulk transcriptome analysis of MSPCs lacking Ebf1 revealed differences in the niche composition and decreased expression of lineage-instructive signals for myeloid cells. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Wtapblock Cell Differentiation of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Yuanyuan Liu ◽  
Feifei Xiao ◽  
Bijie Yang ◽  
Zhiwei Chen ◽  
Jieping Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Wilms Tumor ◽  
Conditional Knockout ◽  
Poly I:C ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Wilms' tumor 1-associating protein (WTAP) is a ubiquitously expressed nuclear protein has been associated with regulation of cell proliferation, apoptosis, embryonic development, cell cycle, RNA splicing and stabilization, N6-Methyladenosine RNA modification in various physiological processes. Recently,WTAPwas reported to promoter tumorigenicity in Glioblastoma and cholangiocarcinoma. Besides solid tumors,WTAPplays an important role in abnormal proliferation and arrested differentiation in acute myeloid leukemia (AML) cell, suggesting its oncogenic activity. For its promising novel therapeutic target in AML, a systematic investigation of the roles ofWTAPin normal hematopoiesis is warranted.To investigate the function ofWTAPin normal hematopoietic system, we firstly determined the mRNA level ofWTAPin different hematopoietic stem and progenitor cells (HSPCs) and several mature populations in C57/B6 mouse bone marrow (BM).WTAPwas ubiquitously expressed in different cell populations and especially elevated in HSPCs. For WTAP-null and heterozygous caused early embryonic lethality, we generated endothelial system conditional knockout (cKO) mice by crossing WTAP floxed mice with poly (I:C) induced Mx1-Cre transgenic mice. In poly (I:C) inducedWTAPfl/fl-Mx1-Cre, WTAP deficiency lead to approximately 2-fold increase in HSC and LSK pool size, and modest expansion of HPC, CLP and LMPP population. In competitive BM transplantation assay, lossWTAPshowed a significantly decreased repopulation capacity. WhileWTAPknockout did not significantly affect the proliferation, cell cycle and apoptosis of HSPCs tested by Brdu, Ki67 and Annexin-V straining assay. Mechanistically, deletion ofWTAPin HSC resulted in decreased transcription of myeloid cell and erythrocyte differentiation gene (including Jak3, Jun and Junb) and genes regulating pluripotency of stem cells (induding Akt2, Fzd1/9 and Mapk3).Collectively, we speculateWTAPplay important role in blocking cell differentiation of HSPCs. Currently, we are conducting a series of studies to reveal the underlying molecular mechanism(s) ofWTAPregulating normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3707-3707
Author(s):  
Adedamola Elujoba-Bridenstine ◽  
Lijian Shao ◽  
Katherine Zink ◽  
Laura Sanchez ◽  
Brian Cox ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Gene Set Enrichment Analysis ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Behavior

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior. Disclosures No relevant conflicts of interest to declare.

A Hypoxic Niche in the Mouse Bone Marrow Diminishes Proliferation and Differentiation of Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4777-4777
Author(s):  
Pernilla M Eliasson ◽  
Jan-Ingvar Jönsson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Active Form ◽  
Culture Conditions ◽  
Mouse Bone Marrow ◽  
Quiescent State ◽  
Hematopoietic Stem

Abstract In the bone marrow hematopoietic stem cells (HSCs) reside in specialized niches in close contact with stromal cells and endosteal osteoblasts. It is thought that this environment is hypoxic in nature, where HSCs are maintained in a quiescent state to prevent their depletion. Hypoxia stabilizes the transcription factor HIF-1α which triggers angiogenesis as well as genes slowering the cell cycle, promoting cell survival, and leading to a decrease in cellular metabolism. In this study, hypoxic effects of the maintenance of Lin−Sca1+c-kit+* (LSK) cells derived from mouse bone marrow and the involvement of the transcription factor hypoxia inducible factor 1 α (HIF-1α) were investigated. Hypoxic culture conditions led to an increase in numbers of primitive colony-forming progenitor cells and a preferential expansion of immature blast-like appearing cells. Concurrently, the immature c-kit Sca-1 phenotype was better maintained in hypoxia compared to ambient oxygen levels. Moreover, hypoxia decreased the proliferation of HSCs as measured by CFSE or PKH26 staining. This was confirmed by cell cycle analysis, and hypoxic cultivation decreased the percentage of cells in S-phase whereas cells in G0/G1 phase increased. Cells infected with a constitutively active form of HIF-1α showed the same pattern as cells cultured in hypoxia. To verify that the effect is HIF-1α mediated, we silenced HIF-1α in LSK cells with shRNA. The decrease in proliferation in hypoxic cultivation of cells infected with shRNA against HIF-1α was markedly diminished, indicating that HIF-1α play an important role in controlling proliferation of hematopoietic stem cells. These results suggest that a major function of hypoxia is to counteract proliferation and possibly differentiation, thereby sustaining maintenance. Furthermore, hypoxic culture conditions may have beneficial clinical implications for ex vivo purposes and may improve the yields of stem cells. In our ongoing-studies, we are investigating whether HIF-1α and hypoxia is an absolute prerequisite for the proper maintenance of HSCs in the bone marrow.

scholarly journals A Meta-Analytic Single-Cell Atlas of Mouse Bone Marrow Hematopoietic Development

2021 ◽  
Author(s):  
Benjamin D Harris ◽  
John Lee ◽  
Jesse Gillis
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cell Types ◽  
Marker Genes ◽  
Mouse Bone Marrow ◽  
Clear Understanding ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Stem And Progenitor Cells

The clinical importance of the hematopoietic system makes it one of the most heavily studied lineages in all of biology. A clear understanding of the cell types and functional programs during hematopoietic development is central to research in aging, cancer, and infectious diseases. Known cell types are traditionally identified by the expression of proteins on the surface of the cells. Stem and progenitor cells defined based on these markers are assigned functions based on their lineage potential. The rapid growth of single cell RNA sequencing technologies (scRNAseq) provides a new modality for evaluating the cellular and functional landscape of hematopoietic stem and progenitor cells. The popularity of this technology among hematopoiesis researchers enables us to conduct a robust meta-analysis of mouse bone marrow scRNAseq data. Using over 300,000 cells across 12 datasets, we evaluate the classification and function of cell types based on discrete clustering, in silico FACS sorting, and a continuous trajectory. We identify replicable signatures that define cell types based on genes and known cellular functions. Additionally, we evaluate the conservation of signatures associated with erythroid and monocyte lineage development across species using co-expression networks. The co-expression networks predict the effectiveness of the signature at identifying erythroid and monocyte cells in zebrafish and human scRNAseq data. Together, this analysis provides a robust reference, particularly marker genes and functional annotations, for future experiments in hematopoietic development.

C/EBPβ Isoforms Distinctively and Collaboratively Regulate the Behavior of Hematopoietic Stem and Progenitor Cells in Regenerative Conditions

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3580-3580
Author(s):  
Atsushi Sato ◽  
Hideyo Hirai ◽  
Asumi Yokota ◽  
Akihiro Tamura ◽  
Tsukimi Shoji ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Cell Cycle Regulation ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Stem And Progenitor Cells ◽  
Cycle Regulation ◽  
Cell Cycle Status

Abstract CCAAT Enhancer Binding Protein b (C/EBPb) is a leucine zipper type transcription factor. While C/EBPa plays a critical role in maintaining steady-state granulopoiesis, C/EBPb is required for stress-induced granulopoiesis (Hirai et al., 2006). We have been focusing on the functions of C/EBPb in the regulation of hematopoietic stem and progenitor cells (HSPCs) especially under stressed conditions. Last year in this meeting, we have shown that 1) C/EBPb was upregulated at protein level in HSPCs after hematopoietic stresses, 2) C/EBPb was required for initial expansion of HSPCs after transplantation, and 3) C/EBPb promoted exhaustion of HSPCs under repetitive hematopoietic stresses (56th ASH, abstract #67850). Here, we further investigated the significance of C/EBPb in cell cycle regulation of HSPCs and the distinct roles of C/EBPb isoforms in HSPCs during regenerative conditions. To clarify the involvement of C/EBPb in cell cycle regulation of HSPCs, we compared the cell cycle status of wild-type (WT) and Cebpb knockout (KO) HSPCs by intracellular Ki67 staining and short-term BrdU incorporation assay in combination with multi-color flow cytometric analysis. In order to exclude the difference in the bone marrow microenvironment, CD45.2+ WT or Cebpb KO bone marrow (BM) cells were transplanted into lethally irradiated CD45.1+ WT mice. At steady state (12 weeks after the BM transplantation), the cell cycle status of Cebpb KO HSPCs was identical to that of WT HSPCs. Then cell cycle status of HSPCs was assessed at various time points during regeneration after intraperitoneal administration of 5-fluorouracil (5-FU, 150mg/kg). We found that significantly more Cebpb KO HSPCs remained in the G0 phase than WT HSPCs (in LT-HSCs on days 3-10; in MPPs on days 6-12). Significantly less Cebpb KO HSPCs were BrdU+ and were in the S/G2/M phase on day 7. These findings suggest that C/EBPb, in a cell-intrinsic manner, facilitates cell cycle entry, progression and consequent earlier expansion of HSPCs in response to hematopoietic stresses. Next, we investigated the distinct roles of C/EBPb isoforms in regulation of HSPCs. C/EBPb is a unique single exon gene and utilization of three different initiating codons result in three distinct isoforms. Liver-enriched activating protein* (LAP*) and LAP are the longer isoforms containing transactivating domains, DNA binding and dimerization domains, and liver-enriched inhibitory protein (LIP) is the shortest isoform which lacks the transactivating domains. In order to examine the expression pattern of C/EBPb isoforms in vivo in scarce populations of regenerating HSPCs, we developed a novel flow cytometric method to distinguish the cells predominantly expressing shorter isoform (LIP) from the cells expressing both LIP and the longer isoforms (LAP* and LAP) by intracellular double staining. Using this method, we found that predominantly LIP-expressing cells transiently emerged within MPP fraction in the regenerating bone marrow (on days 5-6 after administration of 5-FU, Figure below), while overall C/EBPb expression levels were significantly upregulated in most cells. To examine the roles of respective C/EBPb isoforms in regulation of HSPCs, EML cells, a murine hematopoietic stem cell line, were retrovirally transduced with one of the C/EBPb isoforms and the transduced cells were subjected to further analysis (vectors are kind gifts from Dr Watanabe-Okouchi N and Dr Kurokawa M, Tokyo Univ). LIP-expressing EML cells were more proliferative and actively cycling than EML cells transduced with a control vector, whereas the proliferation of LAP*- or LAP-expressing cells were markedly suppressed. LIP-expressing cells remained undifferentiated status (c-kithigh CD11b-) for more than 2 weeks, while LAP*- or LAP-expressing cells rapidly differentiated into c-kitlow CD11b+ myeloid cells and eventually exhausted within a week. These results indicate LIP plays quite distinct roles from LAP* and LAP in regulation of HSPCs. Collectively, our data suggest that C/EBPb isoforms distinctively and collaboratively regulate HSPCs in regenerative conditions: early transient elevation of LIP contributes to cell cycle activation and rapid expansion of HSPC population, which is in turn converted into supply of mature myeloid cells by more abundant upregulation of LAP* and LAP. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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