Erythroid Lineage Differentiation Research Articles

Liver X receptor activation in chronic myelogenous leukemia cells yields distinct mass fingerprints by whole cell MALDI-TOF MS, which correspond to changes in cell viability, gene expression, and differentiation markers

Liver X receptor (LXR) agonists, including synthetic ligands or naturally-occurring oxysterols, exert promising lipid-modulating, anti-inflammatory, and anti-tumor effects. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) whole cell mass spectrometry (MS) shows promise in chronic disease and food science research, clinical diagnostics of cancer cells and bacterial pathogens, and drug/nutraceutical discovery due to its relatively low-cost, rapid, reliable, and high-throughput ability to identify unique cell phenotypes and differentiation states of immune cells that play essential roles in chronic metabolic and inflammatory disease pathophysiology. However, optimization of whole cell MALDI-TOF MS-based methods to assess cellular changes in response to pharmaceutical and/or nutraceutical treatment warrants further study. Thus, we investigated whether whole cell MALDI-TOF MS could detect cellular phenotype changes of human stem-like erythroleukemia K562 cells – a model for chronic myelogenous leukemia (CML) – induced by the LXR agonist TO-901317, a synthetic ligand that mimics effects of naturally-occurring oxysterols, which are oxidized cholesterol derivatives formed under conditions of high cellular cholesterol concentrations and in food products. TO-901317 dose- and time-dependently altered mRNA expression of cholesterol flux genes, including ATP-binding cassette transporter A1 (ABCA1), LDL-receptor (LDLR), HMG-CoA reductase (HMGCR), and reduced cellular cholesterol content by 22.9% at 24 hours. TO-901317 additionally dose- and time-dependently impacted K562 cell viability, number, size, and reduced mRNA expression of the anti-apoptotic gene BCL2L1 by 43.3%. These effects corresponded to induced gene expression of erythroid markers and hemoglobin content, suggesting that LXR activation may promote erythroid lineage differentiation. Whole cell MALDI-TOF MS mass fingerprinting revealed distinct proteomic phenotypes between TO-901317 vs. untreated cells, and identified histone 2A and histone H4 as uniquely expressed proteins. These findings not only elucidate a novel role for LXR agonists in a model of CML, but demonstrate that mass fingerprints obtained by whole cell MALDI-TOF mass spectrometry effectively distinguish between treatment-induced cell phenotypes.

Abcb10 regulates murine hematopoietic stem cell potential and erythroid differentiation

Erythropoiesis in the adult bone marrow relies on mitochondrial membrane transporters to facilitate heme and hemoglobin production. Erythrocytes in the bone marrow are produced although the differentiation of erythroid progenitor cells that originate from hematopoietic stem cells (HSCs). Whether and how mitochondria transporters potentiate HSCs and affect their differentiation toward erythroid lineage remains unclear. Here, we show that the ATP-binding cassette (ABC) transporter 10 (Abcb10), located on the inner mitochondrial membrane, is essential for HSC maintenance and erythroid-lineage differentiation. Induced deletion of Abcb10 in adult mice significantly increased erythroid progenitor cell and decreased HSC number within the bone marrow (BM). Functionally, Abcb10-deficient HSCs exhibited significant decreases in stem cell potential but with a skew toward erythroid-lineage differentiation. Mechanistically, deletion of Abcb10 rendered HSCs with excess mitochondrial iron accumulation and oxidative stress yet without alteration in mitochondrial bioenergetic function. However, impaired hematopoiesis could not be rescued through the in vivo administration of a mitochondrial iron chelator or antioxidant to Abcb10-deficient mice. Abcb10-mediated mitochondrial iron transfer is thus pivotal for the regulation of physiologic HSC potential and erythroid-lineage differentiation.

Normal and dysregulated crosstalk between iron metabolism and erythropoiesis.

Erythroblasts possess unique characteristics as they undergo differentiation from hematopoietic stem cells. During terminal erythropoiesis, these cells incorporate large amounts of iron in order to generate hemoglobin and ultimately undergo enucleation to become mature red blood cells, ultimately delivering oxygen in the circulation. Thus, erythropoiesis is a finely tuned, multifaceted process requiring numerous properly timed physiological events to maintain efficient production of 2 million red blood cells per second in steady state. Iron is required for normal functioning in all human cells, the erythropoietic compartment consuming the majority in light of the high iron requirements for hemoglobin synthesis. Recent evidence regarding the crosstalk between erythropoiesis and iron metabolism sheds light on the regulation of iron availability by erythroblasts and the consequences of insufficient as well as excess iron on erythroid lineage proliferation and differentiation. In addition, significant progress has been made in our understanding of dysregulated iron metabolism in various congenital and acquired malignant and non-malignant diseases. Finally, we report several actual as well as theoretical opportunities for translating the recently acquired robust mechanistic understanding of iron metabolism regulation to improve management of patients with disordered erythropoiesis, such as anemia of chronic inflammation, β-thalassemia, polycythemia vera, and myelodysplastic syndromes.

Generation of Rh D-negative blood using CRISPR/Cas9.

Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD-/- cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD-/- derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.

EVALUATION OF PANCYTOPENIA WITH BONE MARROW INTERPRETATION

Background: Pancytopenia is one of the most common clinico-haematological entity observed in our day to day clinical practice. It is a disorder in which all the three major elements of blood (i.e. red blood cells, white blood cells and platelets) are decreased in number. The causes of pancytopenia may be due to decrease in hematopoietic cell production in the marrow resulting from infections, toxins, malignant cell inltration, post- chemotherapy or post-radiation. Aims and Objectives: 1) To study the etiology and clinical presentation of pancytopenia in all age groups. 2) To correlate with bone marrow interpretation Materials & Methods: This is a prospective study which was conducted among 50 patients of pancytopenia in the Clinical Pathology, Government General Hospital,Kurnool from January 2021 to October 2022 Bone marrow aspiration was done by using Salah's bone marrow puncture needle. Smears were made from bone marrow aspirate (BMA) and stained by Leishman stain and special stains like Perl`s wherever necessary. The smears were assessed for cellularity, differentiation and maturation of erythroid, myeloid and megakaryocytic lineage, M:E ratio, Plasma cells, Lymphocytes and parasites/ abnormal cells. In the present study the commonest cause of Pancytopenia was Megaloblastic anemia (70%) follo Results: wed by Dimorphic anemia (20%). The less common conditions were Multiple Myeloma (6%),Chronic Myeloid Leukemia(2%),Acute Leukemia(2%). The present study concludes that com Interpretation and Conclusion: plete primary hematological investigations along with bone marrow aspiration in pancytopenic patients are helpful for understanding disease process and to diagnose or to the rule out causes of pancytopenia. These are also helpful in planning for further investigations and management.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A

BackgroundHuman erythropoiesis is a tightly regulated, multistep process encompassing the differentiation of hematopoietic stem cells (HSCs) toward mature erythrocytes. Cellular metabolism is an important regulator of cell fate determination during the differentiation of HSCs. However, how O-GlcNAcylation, a posttranslational modification of proteins that is an ideal metabolic sensor, contributes to the commitment of HSCs to the erythroid lineage and to the terminal erythroid differentiation has not been addressed.MethodsCellular O-GlcNAcylation was manipulated using small molecule inhibition or CRISPR/Cas9 manipulation of catalyzing enzyme O-GlcNAc transferase (OGT) and removing enzyme O-GlcNAcase (OGA) in two cell models of erythroid differentiation, starting from: (i) human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) to investigate the erythroid lineage specification and differentiation; and (ii) human-derived erythroblastic leukemia K562 cells to investigate the terminal differentiation. The functional and regulatory roles of O-GlcNAcylation in erythroid differentiation, maturation, and globin production were investigated, and downstream signaling was delineated.ResultsFirst, we observed that two-step inhibition of OGT and OGA, which were established from the observed dynamics of O-GlcNAc level along the course of differentiation, promotes HSPCs toward erythroid differentiation and enucleation, in agreement with an upregulation of a multitude of erythroid-associated genes. Further studies in the efficient K562 model of erythroid differentiation confirmed that OGA inhibition and subsequent hyper-O-GlcNAcylation enhance terminal erythroid differentiation and affect globin production. Mechanistically, we found that BCL11A is a key mediator of O-GlcNAc-driven erythroid differentiation and β- and α-globin production herein. Additionally, analysis of biochemical contents using synchrotron-based Fourier transform infrared (FTIR) spectroscopy showed unique metabolic fingerprints upon OGA inhibition during erythroid differentiation, supporting that metabolic reprogramming plays a part in this process.ConclusionsThe evidence presented here demonstrated the novel regulatory role of O-GlcNAc/BCL11A axis in erythroid differentiation, maturation, and globin production that could be important in understanding erythropoiesis and hematologic disorders whose etiology is related to impaired erythroid differentiation and hemoglobinopathies. Our findings may lay the groundwork for future clinical applications toward an ex vivo production of functional human reticulocytes for transfusion from renewable cell sources, i.e., HSPCs and pluripotent stem cells.

The Macrophage Nuclear Receptor Coactivator 4 Counteracts Iron-Deficiency Anemia

The Nuclear Receptor Coactivator 4 (NCOA4) protein has been recently recognized as a novel player in iron metabolism contributing to regulation of cell and systemic iron homeostasis. NCOA4 is a cargo receptor that promotes selectively autophagy of the iron storage ferritin in conditions of iron deficiency, facilitating iron recovery from cellular stores. Inactivation of NCOA4 in cells increases ferritin aggregates [Mancias JD et al., Nature 2014; Dowdle WE et al., Nat Cell Biol 2014; Mancias JD et al., Elife 2015] and C57Bl/6 Ncoa4-ko mice show ferritin and iron accumulation in several organs, in particular spleen [Dowdle WE et al., Nat Cell Biol 2014; Bellelli R et al., Cell Rep 2016]. In these animals, iron retention in tissue ferritin reduces iron availability for erythropoiesis causing mild microcytic anaemia in iron-balanced (IB) conditions. In addition, compared to wt animals, Ncoa4-ko mice fed an iron-deficient (ID) diet develop a more severe anemia [Bellelli R et al., Cell Rep 2016].Given the important impact of mouse strain on iron metabolism, we evaluated the ability of Ncoa4-ko mice on a different background to respond to variation of dietary iron content. To this aim wt and null mice on Sv129/J background were fed an IB or an ID diet for 6 months. The ID diet induced almost no changes in hemoglobin levels of wt mice (IB=14.0±1.9 g/dL; ID=13.3±2.9 g/dL), while Ncoa4-ko developed severe microcytic anemia (Hb: IB=13.5±2.0 g/dL, ID=6.7±2.9 g/dL, p<0.05; MCV: IB=47.8±1.3 fL, ID=36.5±4.8 fL, p<0.05). These results are consistent with wt mice on Sv129/J strain being able to counteract ID by using the large reserves of iron present in their tissues. Notwithstanding such high iron deposits, Sv129/J Ncoa4 -ko fail this compensation. To better evaluate the bone marrow (BM)-derived cells (erythroid precursors, BM and spleen macrophages) contribution to the inability of Ncoa4-ko mice to respond to variation of dietary iron in comparison to other tissues (eg. liver and duodenum), we performed BM transplantation experiments in Ncoa4-ko using Sv129/J background. Lethally irradiated Ncoa4-ko mice were transplanted with wt or Ncoa4-ko BM, generating chimeric null animals with wt (Ncoa4-kowt-BM) or Ncoa4-ko BM cellsas control (Ncoa4-koko-BM). Previously published results obtained in vitro and in vivo in zebrafish suggest an essential role for NCOA4 in erythroid cells development and hemoglobinization [Mancias JD et al., Elife 2015; Ryu MS et al., JCI 2017]. However, we observed that Ncoa4-ko BM cells were able to reconstitute an almost normal erythropoiesis in lethally irradiated mice, indicating that in vivo the loss of Ncoa4 has a limited, if any, effect on the erythroid lineage differentiation. In addition, Ncoa4-ko mice with wt bone marrow have normal hematological parameters, suggesting that the mild microcytosis observed in Ncoa4-ko animals in basal conditions is due to a defect of BM-derived cells. Theoretically this effect may be due to cells belonging either to the erythroid or to the macrophage lineage; based on our findings we suggest a prevalent role for macrophages. In line with this hypothesis, Ncoa4-koko-BM mice fed an ID diet for 3 months developed a more severe anemia than Ncoa4-kowt-BM (Hb= 10.7±0.6 g/dL vs 11.7±1.0 g/dL; p=0.06), accompanied by comparable liver (LIC: Ncoa4-koko-BM=174.8±50.4 μg/g; Ncoa4-kowt-BM=157.1±49.5 μg/g) but higher spleen iron concentration (SIC: Ncoa4-koko-BM=719.0±221.3 μg/g; Ncoa4-kowt-BM=176.1±74.6 μg/g; p<0.05), consistent with decreased iron recycling by spleen macrophages of Ncoa4-koko-BM animals.Over all, our results suggest that NCOA4-mediated ferritinophagy plays a central role in macrophages in counteracting dietary ID, promoting iron release and ensuring efficient erythropoiesis. DisclosuresCamaschella:Vifor Pharma: Membership on an entity's Board of Directors or advisory committees.

Cabozantinib promotes erythroid differentiation in K562 erythroleukemia cells through global changes in gene expression and JNK activation.

Cabozantinib is a potent tyrosine kinase inhibitor with multiple targets including MET, VEGFR2, RET, KIT, and FLT3. Cabozantinib is widely used for the treatment of medullary thyroid cancer and renal cell carcinoma. We recently suggested cabozantinib as a potential therapeutic alternative for acute myeloid leukemia (AML) patients with FLT3-internal tandem duplication (FLT3-ITD). Here, we report that cabozantinib can promote differentiation in erythroid leukemia cells. We found that K562 erythroid leukemia cells treated with 1 μM cabozantinib for 72 h underwent erythroid lineage differentiation. Transcriptomic analysis revealed that various pathways associated with heme biosynthesis, hemoglobin production, and GATA1 targets were upregulated, whereas cell survival pathways were downregulated. Further examination revealed that cabozantinib-induced erythroid differentiation is at least in part regulated by JNK activation and phosphorylation. Levels of phosphorylated BCR-ABL, AKT, STAT5, ERK, and p38 also decreased following cabozantinib treatment. Therefore, we indicate that cabozantinib has dual functions. First, it induces K562 cell differentiation toward the erythroid lineage by upregulating heme biosynthesis, globin synthesis, and erythroid-associated reactions. Second, cabozantinib inhibits K562 cell proliferation by inhibiting the phosphorylation of BCR-ABL and the downstream MAPK, PI3K-AKT, and JAK-STAT signaling pathways.

Role of c-Myc haploinsufficiency in the maintenance of HSCs in mice

AbstractThis study was conducted to determine the dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/β-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence and by promoting apoptosis. We have identified Nr4a1, Nr4a2, and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2, and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediates the function of c-Myc in regulating HSC quiescence, whereas all 3 genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/β-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induces a blockage of erythroid lineage differentiation through promoting secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc–heterozygous mice and to significantly prolong the survival of those mice. Furthermore, we showed that c-Myc–mediated Apc loss induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc-deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.

Epigenetic modifiers in normal and aberrent erythropoeisis

Erythroid differentiation program is comprised of lineage commitment, erythroid progenitor proliferation, and termination differentiation. Each stage of the differentiation program is heavily influenced by epigenetic modifiers that alter the epigenome in a dynamic fashion influenced by cytokines/humeral factors and are amicable to target by drugs. The epigenetic modifiers can be classified as DNA modifiers (DNMT, TET), mRNA modifiers (RNA methylases and demethylases) and histone protein modifiers (methyltransferases, acetyltransferases, demethylases, and deacetylases). Here we describe mechanisms by which these epigenetic modifiers influence and guide erythroid-lineage differentiation during normal and malignant erythropoiesis and also benign diseases that arise from their altered structure or function.

Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors

SUMMARYThe mammalian SWitch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling BAF (BRG1/BRM-associated factor) complex plays an essential role in developmental and pathological processes. We show that the deletion of Baf155, which encodes a subunit of the BAF complex, in the Tie2(+) lineage (Baf155 (CKO) leads to defects in yolk sac myeloid and definitive erythroid (EryD) lineage differentiation from erythromyeloid progenitors (EMPs). The chromatin of myeloid gene loci in Baf155 CKO EMPs is mostly inaccessible and enriched mainly by the ETS binding motif. BAF155 interacts with PU.1 and is recruited to PU.1 target gene loci together with p300 and KDM6a. Treatment of Baf155 CKO embryos with GSK126, an H3K27me2/3 methyltransferase EZH2 inhibitor, rescues myeloid lineage gene expression. This study uncovers indispensable BAF-mediated chromatin remodeling of myeloid gene loci at the EMP stage. Future studies exploiting epigenetics in the generation and application of EMP derivatives for tissue repair, regeneration, and disease are warranted.

Molecular Signature of Megakaryocyte-Erythroid Progenitors Reveals Role of Cell Cycle in Fate Specification

Megakaryocytic-Erythroid Progenitors (MEP) produce megakaryocytes (Mk) and erythroid (E) cells. The detailed molecular mechanisms underlying the MEP fate decision have not been determined. One of the challenges in studying the fate decisions in MEP has been the lack of high purity populations of the specific cell type. We established an improved method for enriching primary adult human MEP, in which CFU-Mk/E (single cells that give rise to colonies containing exclusively Mk and E) are enriched to ~50% with the remaining cells being CFU-Mk and BFU-E.. We applied single cell RNA sequencing to identify the molecular signature of this enriched MEP population, and compared this to that of CMP, and enriched populations of Mk or E committed progenitors (MKP or ERP), which produce >90% Mk or E colonies in CFU assays). Single cell sequencing results indicate that MEP have a unique gene expression signature consistent with a transition state from CMP to MKP and ERP. MEP have random co-expression of a fraction of 60 genes that are otherwise expressed exclusively in CMP, MKP or ERP.Amongst the most differentially expressed groups of genes between MEP, MKP, and ERP are those related to cell cycle. Bioinformatic analysis suggested that MYC and E2F may accelerate MEP cell cycling as cells commit toward the E or Mk lineage.To determine whether the change in cell cycle is the consequence of cell fate determinant or itself can also regulate the cell fate decision, we used chemical and molecular approaches to modify cell cycling of MEP. Our data show that ATRA and mTOR can each reduce the MEP proliferation rate, and bias MEP toward Mk lineage differentiation (see Figure, > 1.65-fold increased in Mk colony number). We tested whether effect is mediated by downstream MYC pathways, and found that suppression of MYC or MAX (heterodimeric partner of MYC) similarly slowed proliferation and induced an Mk bias in primary human MEP (1.5 and 1.8 fold increased in Mk colony number). If slowing the cell cycle promotes Mk fate commitment, then acceleration of the cell cycle may promote erythroid fate commitment. Indeed, MEP cycling was enhanced by lentiviral-mediated overexpression of Cyclins-CDKs or shRNA mediated p53, and these MEP were significantly (p < 0.05) biased toward erythroid lineage differentiation (> 1.7-fold increased in E colony number). These results support that the speed or frequency of the cell cycle regulates cell fate decisions.In summary, we apply single cell sequencing on pure human CMP, MEP, MKP and ERP and identify the unique MEP gene signature. Thus, by enriching primary human subpopulations, functionally confirming their fate commitment potential, performing single cell RNA sequencing, analyzing the data for gene expression patterns, and testing by both genetic and pharmacological approaches, we have confirmed that the fate commitment of primary human bipotent MEP can be toggled by cell cycle speed. Now that we have proven that cell cycle activity mechanistically controls MEP fate decisions, specific genetic and epigenetic mechanisms by which Mk vs erythroid specification is determined are being explored. The data obtained from healthy cells can now be applied to the mechanisms underlying benign and malignant disease states of Mk and E production. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aid is a key regulator of myeloid/erythroid differentiation and DNA methylation in hematopoietic stem/progenitor cells

AbstractRecent studies have reported that activation-induced cytidine deaminase (AID) and ten-eleven-translocation (TET) family members regulate active DNA demethylation. Genetic alterations of TET2 occur in myeloid malignancies, and hematopoietic-specific loss of Tet2 induces aberrant hematopoietic stem cell (HSC) self-renewal/differentiation, implicating TET2 as a master regulator of normal and malignant hematopoiesis. Despite the functional link between AID and TET in epigenetic gene regulation, the role of AID loss in hematopoiesis and myeloid transformation remains to be investigated. Here, we show that Aid loss in mice leads to expansion of myeloid cells and reduced erythroid progenitors resulting in anemia, with dysregulated expression of Cebpa and Gata1, myeloid/erythroid lineage-specific transcription factors. Consistent with data in the murine context, silencing of AID in human bone marrow cells skews differentiation toward myelomonocytic lineage. However, in contrast to Tet2 loss, Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD to induce myeloid transformation. Genome-wide transcription and differential methylation analysis uncover the critical role of Aid as a key epigenetic regulator. These results indicate that AID and TET2 share common effects on myeloid and erythroid lineage differentiation, however, their role is nonredundant in regulating HSC self-renewal and in myeloid transformation.

CBFβ-SMMHC creates aberrant megakaryocyte-erythroid progenitors prone to leukemia initiation in mice

AbstractAcute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFβ-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.

The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells.

MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, β, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14thand 21thafter transduction. Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, β, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines.

Setd1a and NURF mediate chromatin dynamics and gene regulation during erythroid lineage commitment and differentiation

The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult β-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation.

Essential role of Ufm1 conjugation in the hematopoietic system.

Protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins plays a pivotal role in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. Despite its discovery more than a decade ago, its biological functions and working mechanism remains poorly understood. Recent genetic studies using knockout mouse models provide unambiguous evidence for the indispensable role of the Ufm1 system in animal development and hematopoiesis, especially erythroid development. In this short review, we summarize the recent progress on this important protein modification system and highlight potential challenges ahead. Further elucidation of the function and working mechanism of the ufmylation pathway would provide insight into disease pathogenesis and novel therapeutic targets for blood-related diseases such as anemia.

Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

The Tet Dioxygenase Co-Factor Ascorbic Acid Reduces DNA Methylation and Increases Expression of the γ-Globin Gene and Acts in a Combinatorial Manner with HbF-Inducing Drugs Targeting Repressive Epigenetic Modifications

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). Differences in DNA methylation of the γ-globin gene promoter between adult and fetal liver erythroid cells are highly associated with developmental differences in γ-globin expression. Mechanisms that establish and/or modulate DNA methylation of the γ-globin promoter during adult and fetal erythroid differentiation are important in the regulation of γ-globin expression. Pharmacological manipulation of DNA methylation increases HbF in nonhuman primates and SCD patients. Decitabine, a DNA methyltransferase inhibitor that demethylates DNA and increases HbF, is currently in clinical trials. Recent studies have shown that 5-hydroxymethylcytosine (5-hmC), an oxidative product of 5-methylcytosine (5-mC) catalyzed by activity of the TET dioxygenase family, is an intermediate in developmental processes that demethylate DNA. Previously we showed that the γ-globin gene promoter was demethylated during fetal liver erythroid differentiation and to a lesser extent during adult bone marrow (BM) erythroid differentiation. We have investigated the role of 5-hmC in the mechanism of γ-globin gene demethylation by analyzing 5-hmC levels at the HpaII site located at position -51 5’ to the γ-globin transcription start site using a T4-MspI assay in DNA isolated from FACS-purified subpopulations of baboon BM cells enriched for different stages of erythroid lineage differentiation. Levels of 5-hmC were >3 fold higher (p<0.001) in the CD117+CD36+ subpopulation enriched in CFUe (7.15+1.34%) compared to the terminal erythroid precursors (2.33+0.84%) showing that 5-hmC levels are dynamically regulated during erythroid differentiation. Although baboon BM erythroid subpopulations express both TET2 and TET3, higher levels of TET3 were observed in terminal erythroid precursors than in the more primitive CD117+CD36+ subpopulation. High levels of TET3 were also observed in FACS-purified erythroid cells derived from cultured CD34+ baboon BM, human peripheral blood, and human cord blood cells suggesting a role for TET3 in erythroid differentiation. To investigate the relationship between 5-hmC, 5-mC, and γ-globin expression, levels of γ-globin promoter 5-hmC and 5-mC were determined in purified erythroid cells derived from baboon BM CD34+ erythroid progenitors grown in culture conditions resulting in either high (liquid culture) or low (AFT024 murine fetal liver stromal cell line co-culture) levels of γ-globin expression. Levels of γ-globin promoter 5-hmC (mean difference 4.93% total cytosine; p<0.005) and γ-globin chain expression (mean difference γ/γ+β=0.44; p<0.001) were higher and γ-globin promoter 5-mC levels lower (mean difference -25.2% total cytosine; p<0.01) in erythroid progenitors grown in liquid cultures compared to stromal cell line co-cultures. Supplementation of culture media with ascorbic acid, a co-factor of the TET dioxygenases, increased γ-globin expression (mean difference γ/γ+β=0.12; p<0.005) and reduced the level of γ-globin promoter DNA methylation (mean difference -29.0% total cytosine; p<0.001) in baboon BM erythroid progenitors grown in both liquid and co-cultures compared to untreated controls. Ascorbic acid also increased γ-globin expression in cultures derived from human peripheral blood CD34+ progenitors (mean difference γ/γ+β=0.08; p<0.05). In addition, in baboon BM erythroid progenitor cultures ascorbic acid increased γ-globin expression in an additive manner in combination with either the DNA methyltransferase inhibitor decitabine (p<0.001) or the LSD1 inhibitor tranylcypromine (p<0.001) compared to either drug alone, while no combinatorial effects on γ-globin expression were observed with hydroxyurea. These results demonstrate that ascorbic acid is a DNA hypomethylating agent that increases γ-globin gene expression and are consistent with a role for the TET-mediated 5-hmC pathway in the regulation of DNA methylation and expression of the γ-globin gene. Furthermore, these results suggest that vitamin C deficiency, observed in approximately 50% of patients with sickle cell disease, may limit HbF induction by drugs that target epigenetic silencing mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Regulation of the human ABCB10 gene by E2F transcription factors

Here, we report for the first time a functional study of transcriptional regulation of the human ABCB10 gene. We cloned a functional promoter sequence, and then showed that E2F2, E2F3 and E2F4 can activate transcription from the ABCB10 promoter. We identified sites responsible for this activation and confirmed direct binding of E2F4 to these sites in EMSA and ChIP assays. Finally, by silencing the expression of E2F factors we demonstrated their importance in maintenance of the basal ABCB10 expression. This study provides important atypical examples of E2F4 being a transcriptional activator rather than repressor as well as directly binding to a promoter and regulating it through an alternative and classical DNA consensus response element sequences. It also provides a mechanistic link between E2F4 and ABCB10, both of which are involved in the same physiological phenomena: erythroid lineage differentiation and maturation as well as protection against cardiomyocyte cell death.