Definitive Erythropoiesis Research Articles

Immunological consequences of extramedullary erythropoiesis: immunoregulatory functions of CD71+ erythroid cells.

Mammalian erythropoiesis occurs in three stages; the primitive, the fetal definitive and the adult definitive stages.[1][1] After the primitive stage, which takes place in the yolk sac, definitive erythropoiesis moves to the fetal liver and the spleen but is finally restricted to the bone marrow, as

Stat3 Is Activated By ROS to Uniquely Regulate Primitive Erythropoiesis

The mammalian embryo requires the sequential circulation of primitive and definitive erythroid cells for survival and growth. The central cytokine regulator of primitive and definitive erythropoiesis is erythropoietin (EPO). We have previously determined that primitive erythroid progenitors, unlike their definitive (CFU-E) counterparts, do not require EPO for survival. However, the mechanisms regulating the EPO-independent emergence of primitive erythropoiesis in the yolk sac remains poorly understood. Interestingly, maturing primitive erythroblasts in the murine embryo, unlike definitive erythroblasts in the fetal liver and adult bone marrow, express not only STAT5 but also STAT3, which is tyrosine phosphorylated at baseline and in response to EPO. These initial findings led is to hypothesize that STATs 5 and 3 differentially regulate terminal differentiation of primitive erythroid cells. To analyze the function of these two STATs in primary erythroid cells, we developed an imaging flow cytometry-based methodology to quantitate total and phosphorylated levels of STAT proteins in small numbers of cells isolated from staged murine embryos. We found that STAT5 plays conserved roles in primitive and definitive erythroblast survival and surface CD71 expression. In contrast, STAT3 regulates cell cycle progression and mitochondrial polarization specifically in primitive erythroblasts. In addition, STAT3, unlike STAT5, was phosphorylated in the absence of cytokine stimulation. We asked if reactive oxygen species (ROS) may be activating STAT3, since primary primitive erythroblasts, unlike definitive erythroblasts, specifically express Aquaporins 3 and 8, which can transport hydrogen peroxide, as well as water. Indeed, hydrogen peroxide exposure increased endogenous ROS, as well as phosphorylated STAT3, levels both in wild-type and EPOR-null primitive erythroblasts. Consistent with these findings, inhibition of aquaporin channel transport prevented STAT3 phosphorylation by exogenous hydrogen peroxide, but not by EPO. Taken together, our data support the concept that the primitive erythroid lineage, emerging in the hypoxic and EPO-low environment of the yolk sac in the pre-circulation murine embryo, uniquely integrates ROS-mediated STAT3 activation to regulate key aspects of terminal erythroid maturation. Disclosures Palis: Rubies Therapeutics: Consultancy.

The Condensin II Subunit, NcapH2, Is Required for Proper Embryonic Erythroid Terminal Maturation and Embryonic Viability in a Primary Mouse Model

The process of nuclear condensation is an integral part of erythroid differentiation. We have previously shown that loss of the histone methyltransferase, Setd8, results in severe embryonic (primitive) erythroid anemia due to loss of chromatin condensation and proper erythroid terminal maturation (Malik 2017). Setd8 mediated H4K20 mono-methylation is an important mediator of higher order chromatin condensation and can recruit members of the Condensin II complex (Lau 2015). ondensin I and II complexes are five protein, large complexes responsible for DNA organization and packaging during mitosis. In eukaryotes, the complexes are composed of conserved SMC components, with each Condensin having a unique kleisin bridge protein and two HEAT subunits. Subunits of Condensin II are highly expressed in erythroid cells, suggesting an erythroid specific function. Overexpression of the Condensin II HEAT subunit, Cap-G2, accelerates erythroid differentiation in MEL cells (Xu 2006). To interrogate the role of Condensin II in a primary model, we mated endogenous EpoR driven cre expressing mice with mice containing a floxed allele around Condensin II kleisin subunit, NcapH2. Embryos homozygous for EpoR specific excision of NcapH2 suffered from severe primitive erythroid anemia with visible anemia beginning at embryonic day (E11.5) and lethality occurring at approximately E13.5. Peripheral blood analysis at E11.5 was significant for erythroblasts with enlarged cell size and nuclear size, as well erythroblasts with dysplastic appearing nuclei. Knockout embryos at E12.5 exhibited peripheral blood anemia, with an increase in the number of dysplastic appearing cells, and decreased fetal liver coloration suggesting a failure of both primitive and fetal definitive erythropoiesis. Failure of the embryonic erythroid lineage to expand was confirmed in a primary cell culture system in which loss of proliferation was limited to mid to late stage precursors, suggesting that Condensin II function is critical beyond the proerythroblast stage of maturation when nuclear condensation is occurring and mass spectrometry demonstrates that H4K20 mono-methylation accumulates. Characterization of primitive erythroblasts from knockout embryos showed increased cell size, increased scatter in the size of the nucleus, and a high frequency of cells with punctate nuclear staining. In addition, the subset of erythroid cells with punctate nuclei did not appear to have a nuclear envelope, suggesting a pre-mitotic error, likely during G2 of the cell cycle. Indeed, knockout cells showed a buildup of cells in G0-G1 of the cell cycle, suggesting arrest following a defective cell division. Together, our data suggests that Condensin II is required during erythroid maturation for proper cell cycle progression and chromatin organization, and that chromatin modifications, such H4K20me1, may be a required mechanism to control the higher-level organization of chromatin. This control is required in red cell precursors, as they undergo rapid expansion, maturation, and nuclear condensation simultaneously. Understanding these mechanisms will give valuable insight into the unique sensitivity of red cells to chromatin perturbations observed in bone marrow failure and myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

Zfat Is Indispensable for the Development of Erythroid Cells in the Fetal Liver.

In mice, fetal liver is the first tissue of definitive erythropoiesis for definitive erythroid expansion and maturation. ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in primitive hematopoiesis and T cell development. The aim of this study was to examine whether or not Zfat is involved in definitive erythropoiesis in the fetal liver during mammalian development. The role of Zfat during mouse fetal erythropoiesis in the fetal liver was examined using tamoxifen-inducible CreERT2 Zfat-deficient mice. Zfat-deficient mice exhibit moderate anemia with small and pale fetal liver through a decreased number of erythroblasts by E12.5. Apoptosis sensitivity in fetal liver erythroid progenitors was enhanced by Zfat-deficiency ex vivo. Moreover, Zfat knockdown partially inhibited CD71-/lowTer119- to CD71highTer119- transition of fetal liver erythroid progenitors with impairment in the elevation of CD71 expression. Zfat plays a critical role for erythropoiesis in the fetal liver.

PS1236 COST-EFFECTIVENESS AND COST-UTILITY ANALYSIS OF MULTIPLE TREATMENT STRATEGIES USING ABVD AND/OR BEACOPP IN THE TREATMENT OF ADVANCED-STAGE HODGKIN LYMPHOMA

Background: Donor-derived red blood cells (RBCs) are the most common form of cellular therapy, coupled with donor-dependency, alloimmunization and blood borne disease risks. In vitro derivation of RBCs from an immortal source, as iPSCs provide an alternative solution to these issues. In addition, in vitro iPSC-RBCs allow genome editing for possible gene therapies, while large scale production would be beneficial in RBC drug delivery. The current limitation of iPSC-RBCs is their development immaturity, leading to low enucleation potential, which requires improvement prior clinical application. Aims: We aim to push iPSC-derived erythroid differentiation to definitive erythropoiesis yielding enucleated erythrocytes Methods: A minimum of three independent iPSC lines were used during the studies. These iPSC lines were single cell seeded and emerging colonies were differentiated towards erythroblasts using specific growth factor cocktails and media developed at Sanquin. Human mononuclear cells isolated from fetal liver, cord blood and adult blood were also differentiated to erythroblasts. Flow cytometry was used to assess culture purity, stage of erythropoiesis and enculeation. RT-PCR and HPLC was used to quantify the developmentally defined globins. RNA was isolated from the different erythroblasts and RNA-sequencing was performed and analysed. Single cell RNA-seq was performed on single cell FACS-sorted iPSC derived hematopoietic cells. Results: To further develop the process, we have established a GMP-compatible iPSC generation and differentiation protocol, that upon hematopoietic specification yielded erythroblasts (EBL). This method resulted in 2–6∗105 EBLs/iPSC with 100% CD71+/CD235+ (erythroid-specific markers). Further maturation yielded orthochromatic normoblasts containing a mix of primitive and definitive erythroid waves, leading to poor enucleation potential and/or reticulocyte stability. Total RNAseq analysis comparing iPSC to in vitro derived definitive erythroid cells from fetal liver, cord and adult revealed significant differences and identified specific genes that may be used to further augment iPSC-erythroid terminal differentiation to erythrocytes. For instance, iPSC-derived erythroblasts lack expression of specific erythroid regulators (e.g. MYB and KLF1 target genes SOX6, BCL11A and BCAM). Single-cell RNAseq of early iPSC differentiation was performed, aiming to recognize distinct waves of hematopoiesis. Five cell clusters were identified of which, one was HSPC-like, 3 lineage-committed populations and 1 of unknown origin. This data coupled with index sorting, defined the HSPCs as CD71+/CD235−, and the erythroid cells as CD71+/CD235+ expressing embryonic and fetal Hbs. In addition, we have found differentially expressed markers between the iPSC-RBCs and definitive erythroid cells that may be used to better define specific erythroid waves during iPSC differentiations and/or fetal development. We hypothesize, that the identified HSPCs potentially give rise to developmentally more mature cells, which we aim to expand. Currently we are pursuing different approaches among which forward programming, to support early iPSC-HSPCs that produce developmentally more mature definitive erythroid cells including proper enucleation potential. Summary/Conclusion: In conclusion, we have performed extensive RNA analysis of iPSC, cord, fetal and adult erythroblast and identified potential targets that may aid in proper iPSC-derived erythroid differentiation to enucleated red cells.

Bif1, a new BMP signaling inhibitor, regulates embryonic hematopoiesis in the zebrafish.

Hematopoiesis maintains the entire blood system, and dysregulation of this process can lead to malignancies (leukemia), immunodeficiencies or red blood cell diseases (anemia, polycythemia vera). We took advantage of the zebrafish model that shares most of the genetic program involved in hematopoiesis with mammals to characterize a new gene of unknown function, si:ch73-299h12.2, which is expressed in the erythroid lineage during primitive, definitive and adult hematopoiesis. This gene, required during primitive and definitive erythropoiesis, encodes a C2H2 zinc-finger protein that inhibits BMP signaling. We therefore named this gene blood-inducing factor 1 and BMP inhibitory factor 1 (bif1). We identified a bif1 ortholog in Sinocyclocheilus rhinocerous, another fish, and in the mouse genome. Both genes also inhibit BMP signaling when overexpressed in zebrafish. In conclusion, we have deorphanized a new zebrafish gene of unknown function: bif1 codes for a zinc-finger protein that inhibits BMP signaling and also regulates primitive erythropoiesis and definitive hematopoiesis.

KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a+/CD71+ stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.

Definitive EMP and Pre-HSC Emerge in Myb-Null Murine Embryos and Retain Macrophage Potential

Myb-null murine embryos lack definitive erythropoiesis but can produce primitive erythroid cells, allowing their survival to embryonic day 15 (E15) (Mucenski et al., Cell, 1991). Myb expression has been used to detect emerging HSC and is required for HSC maintenance (Lieu et al., PNAS 2009; North et al., Cell 2009). These data have led to the model that myb function is required for definitive hematopoiesis. Interestingly, macrophages and megakaryocytes are still detected in myb-null embryos and, as both of these lineages are also components of primitive hematopoiesis, it is proposed that these cells are not definitive in origin. Myb-independent macrophages infiltrate fetal tissues and have been implicated as a self-renewing source for several adult tissue-resident macrophage populations (Schulz et al., Nature 2012; Gomez Perdiguero et al., Glia 2013; Hoeffel et al., Immunity 2015). We tested the hypothesis that definitive hematopoiesis is entirely myb-dependent by examining two distinct sources of definitive erythroid/myeloid potential: 1) HSCs that emerge from hemogenic endothelium, including the AGM region at E10.5 in mice and 2) HSC-independent definitive EMP that emerge after primitive hematopoiesis from yolk sac hemogenic endothelium beginning at E8.25 (Frame et al., Stem Cells 2016). By E9.5, EMP can be prospectively isolated based on immunophenotype and contain all the erythroid/myeloid progenitor activity present at this time (McGrath et al., Cell Reports 2015). Surprisingly, we found normal numbers of immunophenotypic EMP in E9.5 Myb-null yolk sacs, and immunohistochemical analysis confirmed their emergence from Runx1+ hemogenic endothelium. At E10.5, reduced numbers of Myb-null EMP were found not only in the yolk sac, but also in the bloodstream and the liver. This decrease correlated with fewer hemogenic clusters in the yolk sacs of Myb-null embryos, as well as alterations in their cell-cycle status. The presence of significant numbers of immunophenotypic EMP suggests they could serve as an alternate source of Myb-null macrophages. Clonal analysis of sorted EMP confirmed that Myb function is necessary for erythroid and granulocyte progenitors, but Myb-null EMP retain normal plating efficiencies for macrophage progenitors. Indeed, Myb-null EMP generate only macrophages in liquid culture, lacking not only erythroid and granulocyte cells, but also Ly6C+ monocytes. Consistent with these results, RNA sequencing analysis of Myb-null versus wildtype EMP demonstrated decreased expression of genes in pathways associated with cellular growth, as well as erythroid and granulocyte fates. We further determined that Myb is not required for the emergence of immunophenotypic pre-HSC in the AGM region of E10.5 embryos. In addition, there were normal numbers of clusters in the aorta of E10.5 myb-null embryos. We were also able to detect lineage-, Kit+ (LK) cells in E14.5 livers as previously reported (Sumner et al., Oncogene 2000). While LK numbers were reduced, the Sca1+ (LSK) subset was present in normal numbers. Like EMP, sorted Myb-null E10.5 pre-HSC, as well as E14.5 liver LK and LSK, lacked erythroid or granulocyte CFC activity, but retained normal CFC-M plating efficiencies. In addition, Ly6C+ monocytes were not observed in liquid cultures of sorted Myb-null E10.5 pre-HSC, which produced only macrophages in vitro, or in Myb-null E14.5 livers. Together these data indicate that Myb is not required for hematopoietic emergence of definitive EMP or HSC, but does facilitate the expansion of these definitive stem/progenitor cells and is required for erythroid and granulocyte differentiation. Additionally, EMP and HSC contain Myb-independent macrophage potential, which does not appear to differentiate from a monocyte intermediate. DisclosuresPalis:Rubius Therapeutics: Consultancy.

Multifunctional roles of tropomodulin-3 in regulating actin dynamics.

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.

TREX1 D18N mice fail to process erythroblast DNA resulting in inflammation and dysfunctional erythropoiesis

Anaemia is commonly observed in chronic inflammatory conditions, including systemic lupus erythematosus (SLE), where ∼50% of patients display clinical signs of anaemia. Mutation at the aspartate residue 18 of the three prime repair exonuclease 1 (TREX1) gene causes a monogenic form of cutaneous lupus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. TREX1 degrades single- and double-stranded DNA (dsDNA), and the link between failed DNA degradation by nucleases, including nucleoside-diphosphate kinases (NM23H1/H2) and Deoxyribonuclease II (DNase II), and anaemia prompted our studies to investigate whether TREX1 dysfunction contributes to anaemia. Utilizing the TREX1 D18N mice we demonstrate that (1) TREX1 mutant mice develop normocytic normochromic anaemia and (2) TREX1 exonuclease participates in the degradation of DNA originating from erythroblast nuclei during definitive erythropoiesis. Gene expression, hematocrit, hemoglobin, immunohistochemistry (IHC) and flow cytometry were used to quantify dysfunctional erythropoiesis. An altered response to induced anaemia in the TREX1 D18N mice was determined through IHC, flow cytometry, and interferon-stimulated gene (ISG) expression analysis of the liver, spleen and erythroblastic islands (EBIs). IHC, flow cytometry, and ISG expression studies were performed in vitro to determine the role of TREX1 in the degradation of erythroblast DNA within EBIs. The TREX1 D18N mice exhibit altered erythropoiesis including a 20% reduction in hematocrit, 10–20 fold increased erythropoietic gene expression levels in the spleen and phenotypic signs of normocytic normochromic anaemia. Anaemia in TREX1 D18N mice is accompanied by increased erythropoietin (Epo), normal hepcidin levels and the TREX1 D18N mice display an inappropriate response to anaemic challenge. Enhanced ISG expression results from failed processing and subsequent sensing of undegraded erythroblast DNA in EBIs. TREX1 participates in the degradation of erythroblast DNA in the EBI and TREX1 D18N mice exhibit a normocytic normochromic anaemia.

Induced pluripotent stem cell–based mapping of β-globin expression throughout human erythropoietic development

AbstractRobust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish β-globin–expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with β-globin–expressing cells showing increased maturation. Compared with other cells, β-globin–expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.

Zfat expression in ZsGreen reporter gene knock‑in mice: Implications for a novel function of Zfat in definitive erythropoiesis.

Zinc finger and AT-hook domain containing (Zfat) is a transcriptional regulator harboring an AT-hook domain and 18 repeats of a C2H2 zinc-finger motif, which binds directly to the proximal region of transcription start sites in Zfat-target genes. It was previously reported that deletion of the Zfat gene in mice yields embryonic lethality by embryonic day 8.5 and impairs primitive hematopoiesis in yolk sac blood islands. In addition, Zfat has been reported to be involved in thymic T-cell development and peripheral T-cell homeostasis. In the present study, in order to obtain a precise understanding of the expression and function of Zfat, a knock-in mouse strain (ZfatZsG/+ mice), which expressed ZsGreen in the Zfat locus, was established. ZsGreen signals in tissues and cells of ZfatZsG/+ mice were examined by flow cytometric and histological analyses. Consistent with our previous studies, ZsGreen signals in ZfatZsG/+ mice were detected in the embryo and yolk sac blood islands, as well as in thymocytes, B and T cells. In the ZfatZsG/+ thymus, ZsGreen+ cells were identified not only in T-cell populations but also in thymic epithelial cells, suggesting the role of Zfat in antigen-presenting cells during thymic T-cell development. ZsGreen signals were observed in definitive erythroid progenitor cells in the fetal liver and adult bone marrow of ZfatZsG/+ mice. The proportion of ZsGreen+ cells in these tissues was highest at the early stage of erythroid differentiation, suggesting that Zfat serves particular roles in definitive erythropoiesis. Histological studies demonstrated that ZsGreen signals were detected in the pyramidal cells in the hippocampal CA1 region and the Purkinje cells in the cerebellum, suggesting novel functions of Zfat in nervous tissues. Taken together, these results indicated that the ZfatZsG/+ reporter mouse may be considered a useful tool for elucidating the expression and function of Zfat.

3084 - Distinct and Overlapping Function for Scl, Lmo2 and Ldb1 in Regulating the Divergence of Hematopoietic and Cardiac Fates

Lmo2 and Ldb1 are non-DNA binding components of a multi-factor complex containing Scl/Tal1, the master transcription factor that regulates hematopoietic fate commitment during embryogenesis. Scl has been shown to be critical for the activation of the hematopoietic transcription program in hemogenic endothelium, and repression of cardiac transcription program in endothelium and endocardium, and hence responsible for the divergence of these mutually exclusive mesodermal fates. However, the specific functions of Lmo2 and Ldb1 in this complex, especially in the repressive function, are poorly understood. Here, we employ a combination of germline and conditionally-deleted Scl, Lmo2 and Ldb1 mouse model to study the dynamic roles of each complex member. Our data suggests that similar to Scl, both Lmo2 and Ldb1 are critical for primitive and definitive erythropoiesis. However, whereas the deletion of Scl completely ablates HSPC specification, the presence of residual CD41+cKit+ HSPCs in Lmo2 and Ldb1 knockout embryos suggests that they may have partially redundant roles during HSPC specification. Lmo2, similar to Scl, is critical for the repression of cardiac fate in hemogenic endothelium and endocardium, but becomes dispensable before Scl for continued cardiac repression in the endocardium. Ldb1-/- embryos lack heart and instead show accumulation of CD31+Pdgfra+ cells in the rostral half. Moreover, hemogenic tissues of Ldb1 mutants do not show accumulation of cardiogenic progenitors but exhibit an expansion of CD31+cKit+ endothelial cells, suggesting that Ldb1 critically regulates the activation of both hematopoietic and cardiac transcriptional program in the endothelium. ChIP-seq revealed that Lmo2 predominantly acts through Scl-bound enhancers. Ldb1 also has an Scl independent function in activating cardiac genes, as well as a distinct, but Scl-dependent, function in regulating genes associated with polycomb-repression such as Ezh2 and Suz12. Our data demonstrates the dynamic and temporal interactions as well as individual functions between the members of Scl complex during the divergence of hematopoietic and cardiac lineages.

Poly(C)-Binding Protein Pcbp2 Enables Differentiation of Definitive Erythropoiesis by Directing Functional Splicing of the Runx1 Transcript.

Formation of the mammalian hematopoietic system is under a complex set of developmental controls. Here, we report that mouse embryos lacking the KH domain poly(C) binding protein, Pcbp2, are selectively deficient in the definitive erythroid lineage. Compared to wild-type controls, transcript splicing analysis of the Pcbp2-/- embryonic liver reveals accentuated exclusion of an exon (exon 6) that encodes a highly conserved transcriptional control segment of the hematopoietic master regulator, Runx1. Embryos rendered homozygous for a Runx1 locus lacking this cassette exon (Runx1ΔE6) effectively phenocopy the loss of the definitive erythroid lineage in Pcbp2-/- embryos. These data support a model in which enhancement of Runx1 cassette exon 6 inclusion by Pcbp2 serves a critical role in development of hematopoietic progenitors and constitutes a critical step in the developmental pathway of the definitive erythropoietic lineage.

KFL1 Gene Variants in α-Thalassemia Individuals with Increased Fetal Hemoglobin in a Chinese Population

Krüppel-like factor 1 (KLF1) is a pleiotropic erythroid transcription factor that is a regulator of definitive erythropoiesis. The aim of this study was to detect KLF1 gene variants in α-thalassemia (α-thal) carriers with an increased Hb F level in a Chinese population, and determine the changes of hematological parameters as a result of interactions between KLF1 gene mutations and α-thal. Subjects with α-thal and Hb F levels of ≥1.0% were selected for further investigation. Direct sequencing was used to detect KLF1 gene mutations. Hematological parameters of subjects with α-thal and concomitant KLF1 gene mutations and those with α-thal alone were compared. The KLF1 gene variants were detected in 46 of 275 (16.7%) individuals with α-thal and Hb F levels of ≥1.0%. The detection rate of KLF1 gene mutations rose correspondingly when the Hb F level increased. For α0-thal carriers, significantly lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) values were observed in KLF1 gene mutation-positive carriers than that in KLF1 gene mutation-free carriers; conversely, significantly higher Hb A2 and Hb F levels were observed in the former condition rather than in the latter condition. The results of this study indicate that KLF1 gene variants are common in Chinese subjects with α-thal and increased Hb F levels, and KLF1 gene mutations decreased the red blood cell (RBC) indices in α-thal carriers as that in normal adults.

Mtf2-PRC2 control of canonical Wnt signaling is required for definitive erythropoiesis

Polycomb repressive complex 2 (PRC2) accessory proteins play substoichiometric, tissue-specific roles to recruit PRC2 to specific genomic loci or increase enzymatic activity, while PRC2 core proteins are required for complex stability and global levels of trimethylation of histone 3 at lysine 27 (H3K27me3). Here, we demonstrate a role for the classical PRC2 accessory protein Mtf2/Pcl2 in the hematopoietic system that is more akin to that of a core PRC2 protein. Mtf2−/− erythroid progenitors demonstrate markedly decreased core PRC2 protein levels and a global loss of H3K27me3 at promoter-proximal regions. The resulting de-repression of transcriptional and signaling networks blocks definitive erythroid development, culminating in Mtf2−/− embryos dying by e15.5 due to severe anemia. Gene regulatory network (GRN) analysis demonstrated Mtf2 directly regulates Wnt signaling in erythroblasts, leading to activated canonical Wnt signaling in Mtf2-deficient erythroblasts, while chemical inhibition of canonical Wnt signaling rescued Mtf2-deficient erythroblast differentiation in vitro. Using a combination of in vitro, in vivo and systems analyses, we demonstrate that Mtf2 is a critical epigenetic regulator of Wnt signaling during erythropoiesis and recast the role of polycomb accessory proteins in a tissue-specific context.

Deficiency in interferon type 1 receptor improves definitive erythropoiesis in Klf1 null mice

A key regulatory gene in definitive erythropoiesis is the transcription factor Krüppel-like factor 1 (Klf1). Klf1 null mice die in utero by day 15.5 (E15.5) due to impaired definitive erythropoiesis and severe anemia. Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are formed by a central macrophage (Central Macrophage of Erythroblastic Island, CMEI) surrounded by maturating erythroblasts. Interferon-β (IFN-β) is activated in the fetal liver’s CMEI of Klf1 null mice. The inhibitory effect of IFN-β on erythropoiesis is known and, therefore, we speculated that IFN-β could have contributed to the impairment of definitive erythropoiesis in Klf1 knockout (KO) mice fetal liver. To validate this hypothesis, in this work we determined whether the inactivation of type I interferon receptor (Ifnar1) would ameliorate the phenotype of Klf1 KO mice by improving the lethal anemia. Our results show a prolonged survival of Klf1/Ifnar1 double KO embryos, with an improvement of the definitive erythropoiesis and erythroblast enucleation, together with a longer lifespan of CMEI in the fetal liver and also a restoration of the apoptotic program. Our data indicate that the cytotoxic effect of IFN-β activation in CMEI contribute to the impairment of definitive erythropoiesis associated with Klf1 deprivation.

Comparative Proteomics and Functional Analysis of Red Blood Cell Membrane Proteins in the Rare in(Lu) Blood Type

KLF1 is an essential erythroid transcription factor (EKLF) involved both in the ß-globin switch from fetal to adult and in definitive erythropoiesis. KLF1 also activates genes encoding heme biosynthetic enzymes, as well as genes involved in the cell cycle and the synthesis of membrane proteins. Heterozygosity for one or several nucleotide changes in KLF1 may be responsible for the so-called dominant Lu(a-b-) phenotype, also known as In(Lu) for “Inhibitor Lutheran” and characterized by a dramatic reduction in the expression levels of the Lutheran blood group antigens and a slight elevation in hemoglobin F (HbF). Several other blood group antigens are also under the direct control of KLF1, but their number and expression level in In(Lu) red blood cells (RBCs) remain a matter of debate. In addition, mutations in KLF1 in humans lead to anemia, acantocytosis and some of these variants, such as CDA type IV, are characterized by ineffective terminal differentiation and defects in enucleation. Given the major role of KLF1 in erythropoiesis, it would not be surprising that In(Lu) RBCs show abnormal properties. In this work, we performed proteomic studies to quantify membrane proteins in In(Lu) vs control RBCs.Results: using label-free mass spectrometry, we analyzed the expression levels of membrane proteins in 5 In(Lu) and 4 control RBCs. Hierarchical clustering allowed to identify two modulation profiles of protein expression (Figure 1). The first profile (red color) is composed of 49 proteins over-expressed in In(Lu) RBCs, with the majority of them corresponding to the “26S proteasome complex” (PSMA, PSMB, PSMD, and PSME). In addition, we confirmed the overexpression of the 26S proteasome complex in In(Lu) RBCs by western blot analyses.The second profile (green color) includes 23 membrane proteins with a lower expression level in In(Lu) RBCs; these proteins correspond to blood group antigens (e.g. Lu/BCAM, CD44), and to cytoskeletal proteins (e.g. dematin, flotillin). Five proteins carrying blood group systems show a decrease of expression in In(Lu) RBCs over 40%: Chido/Rodgers, Xg, Indian, Duffy and Lutheran: antigens. While our results indicating of decreased expression of Indian and Chido/Rodgers systems are consistent with previous reports, the Duffy expression was unexpectedly found to be decreased by 86%.Since we evidenced an important increase in the proteasome complex, and taking into account that KLF1 is involved in the regulation or erythropoiesis, we, investigated the expression profile of the 26 S proteasome expression during erythropoiesis. We observed an important decrease of all proteins of the proteasome complex during erythropoiesis.Conclusion: from our preliminary results reported in this study, we hypothesized that the increase of the 26S proteasome in the In(Lu) individuals may result from defects in organelle loss and could explain the profound dyserytropoiesis observed in the “nan” mice null for KLF1.Figure 1: Comparative proteomics of red blood cell membrane proteins in In(Lu) blood group type. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The orphan nuclear receptor TR4 regulates erythroid cell proliferation and maturation

AbstractThe orphan nuclear receptors TR4 (NR2C2) and TR2 (NR2C1) are the DNA-binding subunits of the macromolecular complex, direct repeat erythroid-definitive, which has been shown to repress ε- and γ-globin transcription during adult definitive erythropoiesis. Previous studies implied that TR2 and TR4 act largely in a redundant manner during erythroid differentiation; however, during the course of routine genetic studies, we observed multiple variably penetrant phenotypes in the Tr4 mutants, suggesting that indirect effects of the mutation might be masked by multiple modifying genes. To test this hypothesis, Tr4+/− mutant mice were bred into a congenic C57BL/6 background and their phenotypes were reexamined. Surprisingly, we found that homozygous Tr4 null mutant mice expired early during embryogenesis, around embryonic day 7.0, and well before erythropoiesis commences. We further found that Tr4+/− erythroid cells failed to fully differentiate and exhibited diminished proliferative capacity. Analysis of Tr4+/− mutant erythroid cells revealed that reduced TR4 abundance resulted in decreased expression of genes required for heme biosynthesis and erythroid differentiation (Alad and Alas2), but led to significantly increased expression of the proliferation inhibitory factor, cyclin dependent kinase inhibitor (Cdkn1c). These studies support a vital role for TR4 in promoting erythroid maturation and proliferation, and demonstrate that TR4 and TR2 execute distinct, individual functions during embryogenesis and erythroid differentiation.

Hematopoietic-Specific CSNK2B Loss in Mice Causes Impaired Erythropoiesis

CK2 (Csnk2, casein kinase 2) is a Ser-Thr kinase composed by two catalytic (α) and two regulatory (β) subunits and involved in the regulation of various signaling cascades, which are critical for stem cell biology and hematopoietic development. However, a direct role for CK2 during blood cell differentiation is still undefined. Here, we examined the function of CK2 in erythropoiesis by using a hematopoietic-specific conditional knockout mouse model of the β regulatory subunit (Vav1-CRE x Csnk2β f/f mice). Since CK2β knockout mice died in utero, the study was carried out during gestation collecting fetuses from 12.5 to 17.5 days post conception (dpc) and performing the analysis on fetal liver. CK2β knockout fetuses were pale and hydropic, displayed a smaller liver, disarrayed vascularization and haemorrhages. Lack of CK2β caused depletion of hematopoietic/precursor cells, in particular of common lymphoid progenitors and megakaryocyte-erythrocyte progenitors. CK2β loss resulted to affect both early and late erythroid maturation and red cell viability. CK2β knockout contained lower numbers of TER119 positive cells, which displayed a down modulation of the surface expression of transferrin receptor (CD71) and an increased spontaneous apoptosis. Erythroid cells showed alterations in morphology compatible with myelodysplastic changes. Loss of CK2β caused alterations of erythroid cell proliferation, which was different depending on the stage of erythroid maturation: indeed, BrdU and 7AAD staining showed that less mature erythroid cells (CD71+Ter119-) had a lower rate of proliferation but a normal viability; on the contrary, more mature (CD71-Ter119+) erythroid cells suffered in part of apoptosis and in part accumulated in the S phase. RNA seq analysis performed on purified Ter119+ cells revealed upregulation of TP53 -associated genes as well as of Cdkn1a (p21); on the contrary, there was a down-modulation of Stat5 (an erythropoietin receptor down-stream effector) and genes involved in red cell survival and differentiation in particular c-kit and genes associated to the PI3/Akt pathway. The expression of adhesion molecules and surface carriers for inorganic cations/anionsimportant for the osmotic equilibrium and cell membrane integrity was also found markedly dysregulated. Real time quantitative PCR and Western Blot (WB) analyses confirmed the expression data of Cdkn1a, c-Kit, Bcl-xL, Jak-Stat5 as well as of Akt-Gata-1 axis. Gata-1, the key transcription factor for definitive erythropoiesis, was reduced in CK2β knockout mice as were its downstream target genes such as Alas-2, Lrf, Eklf, Epo-R, β-globin. Immature fetal globins accumulated. In order to find a molecular mechanism, we used an in vitro model of erythroid differentiation based on G1ER cells, an estrogen inducible GATA-1 null murine erythroblast cell line; the combined treatment of β-estradiol and inhibition of CK2 through the chemical inhibitor CX-4945 or RNA interference against CK2β confirmed the negative effect on differentiation. Western blot analysis indicated a potential role of the kinase in the regulation of Akt, Gata-1 and Stat5 protein stability. Moreover, the blockade or down modulation of CK2 caused changes in Gata-1 nuclear distribution with loss of the speckled pattern induced by β-estradiol. Thus, CK2 is a likely essential controller of GATA-1 transcriptional function. Altogether, our work demonstrates that CK2 is a master regulator of erythroid development, by impinging on Stat5, Akt and Gata-1 signaling and influencing red cell viability, bioenergetics, proliferation and maturation. DisclosuresNo relevant conflicts of interest to declare.