Erythroid-specific Transcription Factor Research Articles

Molecular and haematological characterisation of haemolytic anaemia associated with biallelic KLF1 mutations: a case series

AimsKrüppel-like factor 1 (KLF1) is an erythroid-specific transcription factor playing an important role in erythropoiesis and haemoglobin (Hb) switching. Biallelic KLF1 mutations can cause haemolytic anaemia with thalassaemia-like syndromes but...

A comparative study of two routinely used protocols for ex vivo erythroid differentiation

BackgroundErythropoiesis is a complex developmental process in which a hematopoietic stem cell undergoes serial divisions and differentiates through well-defined stages to give rise to red blood cells. Over the last decades, several protocols have been developed to perform ex vivo erythroid differentiation, allowing investigation into erythropoiesis and red cell production in health and disease. ResultsIn the current study, we compared the two commonly used protocols by assessing the differentiation kinetics, synchronisation, and cellular yield, using molecular and cellular approaches. Peripheral blood CD34+ cells were cultured in a two-phase (2P) or a four-phase (4P) liquid culture (LC) and monitored for 20 days. Both protocols could recapitulate all stages of erythropoiesis and generate reticulocytes, although to different extents. Higher proliferation and viability rates were achieved in the 4P-LC, with a higher degree of terminal differentiation and enucleation, associated with higher levels of the erythroid-specific transcription factors GATA-1, KLF-1, and TAL-1. Although the 2P-LC protocol was less efficient regarding terminal erythroid differentiation and maturation, it showed a higher yield of erythroid progenitors in the erythropoietin (EPO)-free expansion phase. ConclusionsWe provide data supporting the use of one protocol or the other to study the biological processes occurring in the early or late stages of erythroid differentiation, depending on the physiological process or pathological defect under investigation in a given study.

The role of GATA switch in benzene metabolite hydroquinone inhibiting erythroid differentiation in K562 cells.

The phenolic metabolite of benzene, hydroquinone (HQ), has potential risks for hematological disorders and hematotoxicity in humans. Previous studies have revealed that reactive oxygen species, DNA methylation, and histone acetylation participate in benzene metabolites inhibiting erythroid differentiation in hemin-induced K562 cells. GATA1 and GATA2 are crucial erythroid-specific transcription factors that exhibit dynamic expression patterns during erythroid differentiation. We investigated the role of GATA factors in HQ-inhibited erythroid differentiation in K562 cells. When K562 cells were induced with 40μM hemin for 0-120h, the mRNA and protein levels of GATA1 and GATA2 changed dynamically. After exposure to 40μM HQ for 72h, K562 cells were induced with 40μM hemin for 48h. HQ considerably reduced the percentage of hemin-induced Hb-positive cells, decreased the GATA1 mRNA, protein, and occupancy levels at α-globin and β-globin gene clusters, and increased the GATA2 mRNA and protein levels significantly. ChIP-seq analysis revealed that HQ reduced GATA1 occupancy, and increased GATA2 occupancy at most gene loci in hemin-induced K562 cells. And GATA1 and GATA2 might play essential roles in the erythroid differentiation protein interaction network. These results elucidate that HQ decreases GATA1 occupancy and increases GATA2 occupancy at the erythroid gene loci, thereby downregulating GATA1 and upregulating GATA2 expression, which in turn modulates the expression of erythroid genes and inhibits erythroid differentiation. This partially explains the mechanism of benzene hematotoxicity.

Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts

Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of β-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.

Novel variants in Krueppel like factor 1 that cause persistence of fetal hemoglobin in In(Lu) individuals

Beta-hemoglobinopathies become prominent after birth due to a switch from γ-globin to the mutated β-globin. Haploinsufficiency for the erythroid specific indispensable transcription factor Krueppel-like factor 1 (KLF1) is associated with high persistence of fetal hemoglobin (HPFH). The In(Lu) phenotype, characterized by low to undetectable Lutheran blood group expression is caused by mutations within KLF1 gene. Here we screened a blood donor cohort of 55 Lutheran weak or negative donors for KLF1 variants and evaluated their effect on KLF1 target gene expression. To discriminate between weak and negative Lutheran expression, a flow cytometry (FCM) assay was developed to detect Lu antigen expression. The Lu(a−b−) (negative) donor group, showing a significant decreased CD44 (Indian blood group) expression, also showed increased HbF and HbA2 levels, with one individual expressing HbF as high as 5%. KLF1 exons and promoter sequencing revealed variants in 80% of the Lutheran negative donors. Thirteen different variants plus one high frequency SNP (c.304 T > C) were identified of which 6 were novel. In primary erythroblasts, knockdown of endogenous KLF1 resulted in decreased CD44, Lu and increased HbF expression, while KLF1 over-expressing cells were comparable to wild type (WT). In line with the pleiotropic effects of KLF1 during erythropoiesis, distinct KLF1 mutants expressed in erythroblasts display different abilities to rescue CD44 and Lu expression and/or to affect fetal (HbF) or adult (HbA) hemoglobin expression. With this study we identified novel KLF1 variants to be include into blood group typing analysis. In addition, we provide further insights into the regulation of genes by KLF1.

Identification of a Digenic Inheritance of Pklr and KLF1 Mutations Resulting in Hemolytic Anemia in a Neonate from a Study of Genotype-Phenotype Correlation of Globin, Pklr and KLF1 in Thai Patients with Pyruvate Kinase (PK) Deficiency

Introduction: Pyruvate kinase deficiency (PK def) is an inherited disorder that affects red blood cells metabolism and causes a wide range of clinical manifestation from fetal anemia, severe neonatal jaundice, and chronic transfusion dependent anemia to fully compensated hemolytic anemia. This condition is rarely reported in Southeast Asian population due to a lack of knowledge and recognition by clinicians and unavailability of laboratory for confirmation. The majority of patients with PK def result from mutations of the PKLR genes encoding this enzyme. Our group has described for the first time (Viprakasit V et al., Blood 2014) that mutations in trans of an erythroid specific transcription factor; KLF1 that regulate PKLR expression, could result in clinical presentation mimic that of classical PK def. Moreover, α and β globin mutations are highly prevalent in our region (up to 40% carrier rate) but their roles as a genetic modifier in PK def remain unknown. In addition, mutations in KLF1 seems to be highly prevalent in our population (14.6 in 1000, n= 2464 chr.), it is of interest to determine the epistatic role of α and β globin, PKLR and KLF1 mutations in patients with classical presentation of PK def.Objectives: To characterize α and β globin, PKLR and KLF 1 mutations and correlate these with clinical presentation and outcome(s) of them in patients with PK def and their family members.Methods: After obtaining informed consent, 9 index PK def patients with their available family members were evaluated for PKLR and KLF1 mutations by direct genomic sequencing as previously described. Hematological parameters and hemoglobin analyses were performed using standard techniques. DNA analysis for all common α and β globin mutations leading to thalassemia and hemoglobinopathies found in this region was performed using our standard protocol.Results: We successfully identified PKLR mutations in 7 out of 9 index patients as shown in Table 1. All patients were diagnosed with clinical PK def following several measurement of biochemical activity. We found 11 different mutations in 6 compound heterozygotes (5 of two nucleotide mutations and one with 1 missense and a large PKLR deletion) and 1 homozygote. One mutation (c.941T>C =p.I314T) seemed to be recurrent since it was found in two families; one homozygous and one compounded with N118S. We failed to identified PKLR and KLF1 in only one patient (PK-9) suggesting that this patient might have a novel molecular mechanism that remains to be found. We found only one family with globin abnormality (Hb E trait in PK-1) without any deleterious effect on patient's clinical severity. Moreover, most of PK patients (except one) have no mutation in KLF1 genes. Interestingly, the PK-3 patient (who is an offspring of the PK-2) was subsequently found to be double heterozygous of PKLR (p.V548fs) and KLF1 (G176RfsX179 (+7bp)) mutations. This patient was previously diagnosed with PK def at birth when he had serious hemolytic anemia (Hb 10 g/dL, haptoglobin < 10 IU/L) with hyperbilirubinemia requiring phototherapy, based on a very low PK enzyme activity (6.5 IU/g Hb, 20% of normal level) and the PK level from his mother was also low (11 IU/g Hb). Since then, he had an uneventful history for 6 years, except mild anemia from iron deficiency anemia during infant period. Physical examination showed no hepatosplenomegaly. Currently his baseline Hb levels were around 12-12.5 g/dL with normal reticulocyte count (1%) and hypochromic microcytosis. There is no globin abnormality in this family. The PK enzyme activity at 6-year-of age showed an improved PK activity (41% of normal activity) without further hemolysis. Our finding showed for the first time a possible interaction between PKLR and KLF1 mutations in man and this digenic inheritance seems to cause a more serious phenotype than each of these carriers alone. However, since the clinical presentation of our index case (PK-3) appeared to be apparent only at the early age, this might suggest the possible impact of KLF1 mutations that were more serious at the early age (similar to those of patients with two KLF1 mutations; KLF1 disease) and might be further compensated by other hitherto mechanisms during erythroid development.Conclusions: We found no effects of globin mutations on the clinical phenotypes of patients with classical PK def. However, a mutation in KLF1 could deteriorate clinical presentations even in a carrier of PKLR mutation at the early age. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dysregulated Transcriptional Upregulation of Cell-Cycle Regulators By KLF1 Mutation E325K Induces Cell-Cycle Arrest in Erythroid Cells Differentiated from the Congenital Dyserythropoietic Anemia (CDA)-Patient Specific Induced Pluripotent Stem Cells

The congenital dyserythropoietic anemias (CDAs) are congenital red blood cell disorders representing ineffective erythropoiesis and dyserythropoietic changes in the bone marrow. We diagnosed a female patient with undiagnosed congenital anemia as type IV CDA caused by a heterozygous missense mutation of the erythroid-specific transcription factor, KLF1; c.973G>A, p. E325K. Although the mutation has been reported in a male patient characterized as hydrops fetalis, severe neonatal jaundice and transfusion-dependent anemia (Arnaud L et al., Am J Hum Genet, 2010), the proband showed relatively mild phenotype showing moderate dyserythropoietic anemia. In order to investigate the pathological significance of mutant KLF1 during erythropoiesis, we generated induced pluripotent stem cells (iPSCs) from peripheral blood of the CDA patient (CDA-iPSCs), and utilized these cells for better understanding of its molecular basis.Hematopoietic precursors were induced from CDA-iPSCs by embryoid bodies formation. CD34(+) precursor cells were then isolated and cultured in the presence of cytokine cocktail for additional 1-3 weeks (Figure 1). Morphological analysis revealed that many erythroblastic cells derived from CDA-iPSCs were multinucleated, in accordance with that of bone marrow erythroblasts from CDA patient. Flow cytometric analysis showed that CDA-iPSC-derived cells contained significantly lower percentage of CD235a(+)/CD71(+) erythroid lineage cells than the cells derived from control iPSCs. CD71 expression decreased in time during erythroid differentiation culture in control CD235a(+) cells, whereas it maintained in CDA-iPSC-derived cells, indicating inappropriate maturation of erythroblastic cells.These data promoted us to explore the possibility of cell death in CDA-iPSC-derived cells during erythroid differentiation culture by comprehensive microarray analysis of erythroblastic cells. In CDA-iPSC-derived cells, many positive and negative regulators of cell-cycle, such as E2F2 and CDKN2C, were differentially expressed, indicating the disruption of cell-cycle control during late stage of erythropoiesis from CDA-iPSCs. In consistent with this, significantly lower percentage of BrdU-uptaking cells were detected in CD235a(+) cells derived from CDA-iPSCs after 3 hour pulse of BrdU during erythroid liquid culture.Most of the KLF1 mutations with functional alterations currently reported are classified into hypomorphic variants or truncating loss-of-function variants. KLF1 E325K has been reported to actively interfere with transcription of KLF1 target genes (Arnaud et al., 2010). Our findings suggest that KLF1 E325K variant might act to disrupt the transcriptional control of various cell cycle regulators including known KLF1 target genes E2F2 and CDKN2C, which lead to the pathological cell-cycle arrest in type IV CDA patients. Our model provides insights on understanding the mechanisms of type IV CDA, and it would be a useful tool for drug screening and identification of novel biomarkers. [Display omitted] DisclosuresKohara:neo ALA CO.,LTD.: Research Funding. Sugawara:neo ALA CO.,LTD.: Research Funding. Yoshie:neo ALA CO.,LTD.: Research Funding. Tani:neo ALA CO.,LTD.: Research Funding; SHINNIHON PHARMACEUTICAL CO., LTD: Research Funding.

A Long Noncoding RNA from the HBS1L-MYB Intergenic Region on Chr6q23 Regulates Human Fetal Hemoglobin Expression

Fetal hemoglobin (HbF) modifies the severity of sickle cell disease (SCD) and β-thalassemia. One of the major quantitative trait loci (QTL) associated with HbF level is the HBS1L-MYB intergenic polymorphisms (HMIP) on chr6q23. MYB regulates proliferation and maturation of erythroid cells, and modulates gene expression within the HBB gene cluster. A distal enhancer located at ~84 kb upstream of MYB has been shown by GWAS (Farrell et al , Blood 2011), insertional mutation (Suzuki et al, Mol Cell Biol 2013), long-range interaction by 3C analysis (Stadhouders et al, J Clin Invest 2014), and gene editing with Cas9 nucleases (Canver et al, Nat Genet 2017). This enhancer encompasses a 3-bp deletion polymorphism (rs66650371), which is surrounded by binding sites for erythroid-specific transcription factors, and is likely the functional motif to account for most of the effect upon HbF level by this HbF QTL. Furthermore, ENCODE datasets annotated RNA polymerase 2 occupancy and a 50-bp RNA transcript adjacent to rs66650371, and led us to hypothesize that this transcript is part of a long noncoding RNA (lncRNA). LncRNAs are greater than 200 nucleotides long, are transcribed throughout the genome, and have broad functionality.We characterized a novel 1283 bp lncRNA (HMI-LNCRNA) which was transcribed from this MYB enhancer and contained binding sites for TAL1/E47, GATA, and RUNX1. Its expression in 25 human tissues and cells was examined by qPCR. The thymus and testis had by far the highest expression. Increased expression was seen in erythroblasts from cultured cord blood CD34+ cells, followed by fetal liver, bone marrow, spleen and K562 cells. Other non-hematopoietic tissues had mostly minimal expression. The expression pattern of HMI-LNCRNA displayed similar features of most lncRNAs, including being located almost entirely within nuclei in erythroid cells, suggesting that it might regulate gene expression; and being less abundant than the protein-coding gene, MYB . HMI-LNCRNA expression was significantly higher in erythroblasts from cultured adult peripheral blood CD34+ cells, which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells, which expressed much more HBG . Similarly, HMI-LNCRNA expression was higher in HUDEP-2 cells, an immortalized human adult-like erythroid cells that expressed only HBB, compared to HUDEP-1 cells that expressed predominantly HBG .Transduction of HUDEP-2 cells with HMI-lncRNA shRNA led to downregulation of HMI-LNCRNA expression to 50% of normal level, and resulted in 200-fold increase in HBG mRNA but no change in HBB or HBS1L mRNA. HBG mRNA expression in these cells amounted to 20% of the total HBB and HBG mRNA levels. The changes in HBG mRNA levels were associated with corresponding changes in HBG protein levels documented by Western blot analysis and immunofluorescence staining using anti-HBG antibody. Similar changes in mRNA and protein levels of HBG were seen when HUDEP-2 cells transduced with HMI-lncRNA shRNA were allowed to mature in culture for up to 7 days. There was no change in the expression of BCL11A and other transcription factors known to modulate HBG expression in HUDEP-2 cells transduced with HMI-lncRNA shRNA. FACS analysis of CD71 and CD235 expression showed that 55-65% of HUDEP-2 “naïve” cells or cells transduced with scrambled shRNA were double positive for both markers, while 85% of HUDEP-2 cells transduced with HMI-lncRNA shRNA were double positive, suggesting that downregulation of HMI-LNCRNA promoted maturation of HUDEP-2 cells.HMI-LNCRNA is likely an enhancer RNA (eRNA), and facilitates the interaction between the distal enhancer with the MYB promoter. Our experimental results indicate that it has an important role in regulating HBG expression. Its downregulation can result in markedly increased HBG expression. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in SCD and β-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

A Potential Role for Genes within the mTORC1 Pathway in the Regulation of Embryonic Globin Expression Revealed By ChIP-Seq, RNA-Seq and eQTL Mapping in the Nan Anemia Mouse Model

The Nan mouse model carries a mutation in the gene encoding the erythroid-specific transcription factor, Klf1 (Krüppel-like factor 1). KLF1 regulates the terminal development of erythroid cells via the transcriptional regulation of a diverse set of genes. We recently showed by ChIP-seq and RNA-seq that Nan-KLF1 fails to bind a subset of normal KLF1 gene targets and also ectopically binds a large set of genes not normally engaged by KLF1. The result is a corrupted transcriptome that produces anemia and a ~100-fold increase in βh1 expression in adult Nan spleen and bone marrow. Genetic modifiers regulating βh1 expression in mice can potentially be translated to the regulation of human fetal γ-globin expression and identify new drug targets for the treatment of Sickle Cell Disease and β-thalassemia, as increased fetal globin ameliorates disease symptoms. We identified genetic loci modifying βh1 expression by eQTL (expression quantitative trait locus/loci) mapping using two congenic Nan strains intercrossed with C57BL/6J. We identified several significant and suggestive eQTL in the two crosses, indicating that multiple genes regulate βh1 expression in adult Nan spleen. Notably, no previously described repressors of γ-globin expression mapped within the eQTL 95% confidence intervals. By integrating genes falling within eQTL confidence intervals on Chr 7, 4, 11, and 17 with ChIP-seq data obtained using an immortalized Klf1 -null fetal erythroid cell line (K1) expressing either WT or Nan Klf1, RNA-seq data from sorted WT and Nan adult spleen erythroid precursors (pro-, baso-, poly- and orthochromatic erythroblasts), and available functional annotations (DAVID and Ingenuity pathway analysis), we identified several strong candidate genes. Among them, several genes encoding components of the mTORC1 pathway are located in eQTL confidence intervals and/or have altered binding by Nan-KLF1 resulting in a dramatic decrease of their expression in Nan. The mTORC1 pathway regulates various cellular functions upon activation by nutrients and growth factors. In prior studies, decreased Mtor (mechanistic target of rapamycin) or Rptor (regulatory associated protein of MTOR complex) induced increased expression of fetal globin, providing compelling rationale that additional genes in this pathway may induce a significant increase in βh1 expression in Nan . In our eQTL dataset, Rptor, Tsc2 (tuberous sclerosis 2), and Mlst8 (MTOR associated protein, LST8 homolog), three genes that belong to the mTORC1 pathway, are located near eQTL peaks on chromosomes 11 and 17. Nan-KLF1 fails to bind Rragd (Ras-related GTP binding),a target of KLF1 required for mTOR activation, resulting in a dramatic and progressive decrease in its expression in Nan terminally differentiating erythroid precursors (6.4, 169, 1910, and 290 fold decrease during maturation from pro- to orthochromatic erythroblasts). Rptor expression is decreased 4.1 and 10.6 fold in polychromatic and orthochromatic erythroblasts, respectively. Interestingly, the top gene ontology terms annotated for the differentially expressed genes include translation, ribosome biogenesis, redox mechanisms, cell cycle control and DNA repair. These functions are regulated by the mTORC1 pathway and strongly suggest that the Nan phenotype is influenced by its downregulation. We conclude that (1) several genes from the mTORC1 pathway, including genes not previously described, are strong candidates that may contribute simultaneously to increased βh1 expression in Nan and (2) functions regulated by mTORC1 are strongly dysregulated by the Nan mutation and reinforce the hypothesis that this pathway plays a key role in the Nan phenotype. The identification of additional modifiers of βh1 in Nan that can be translated to human HbF may provide new therapeutic opportunities. Moreover, given the role of nutrition in the regulation of the mTORC1 pathway, new nutritional guidelines for patients with Sickle Cell Disease and β-thalassemia may emerge. DisclosuresNo relevant conflicts of interest to declare.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2− CD34− Lin− Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2− CD34− LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2− CD34− LSK-HSCs.

GDF-15 negatively regulates excess erythropoiesis and its overexpression is involved in erythroid hyperplasia

Growth differentiation factor-15 (GDF-15) is a member of TGF-β superfamily. Among hematopoietic cells, this factor is mainly produced by erythroid series and is recently considered a biomarker of ineffective erythropoiesis (IE). Whether IE induces enhanced GDF-15 expression or is prompted by it, has remained elusive. In this study we investigated how high levels of GDF-15 contribute to IE-associated erythroid dysplasia. We assessed mRNA levels of GDF-15 during erythroid maturation as well as in patients with IE using qRT-PCR. Later, the erythroid colony-forming capacity of GDF-15-treated hematopoietic stem cells (HSCs) was evaluated by CFC assay. Any effect of elevated levels of GDF-15 on erythroid maturation was ultimately examined by expression analysis of erythroid-associated transcription factors and flow cytometry analysis of CD235a expression. GDF-15 mRNA expression increased during erythroid differentiation and also in β-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Treating the cells with high GDF-15 concentration (50 ng/ml) resulted in an approximate 30% decline in the capacity of erythroid colony formation of HSCs and CD235a positive cells. Additionally, erythroid-specific transcription factors showed significant down-regulation in the early stages of erythroid differentiation. According to the expression level of GDF-15 and the role it plays in the erythroid system, high-levels of this factor could be an auto-modulatory mechanism to control the excessive production of erythroid cells.

MiR-144/451 inhibits c-Myc to promote erythroid differentiation.

Ablation of miR-144/451 disrupts homeostasis of erythropoiesis. Myc, a protooncogenic protein, is essential for erythroblast proliferation but commits rapid downregulation during erythroid maturation. How erythroblasts orchestrate maturation processes through coding and non-coding genes is largely unknown. In this study, we use miR-144/451 knockout mice as in vivo model, G1E, MEL erythroblast lines and erythroblasts from fresh mouse fetal livers as in vitro systems to demonstrate that targeted depletion of miR-144/451 blocks erythroid nuclear condensation and enucleation. This is due, at least in part, to the continued high expression of Myc in erythroblasts when miR-144/451 is absent. Specifically, miR-144/451 directly inhibits Myc in erythroblasts. Loss of miR-144/451 locus derepresses, and thus, increases the expression of Myc. Sustained high levels of Myc in miR-144/451-depleted erythroblasts blocks erythroid differentiation. Moreover, Myc reversely regulates the expression of miR-144/451, forming a positive miR-144/451-Myc feedback to ensure the complete shutoff of Myc during erythropoiesis. Given that erythroid-specific transcription factor GATA1 activates miR-144/451 and inactivates Myc, our findings indicate that GATA1-miR-144/451-Myc network safeguards normal erythroid differentiation. Our findings also demonstrate that disruption of the miR-144/451-Myc crosstalk causes anemia, suggesting that miR-144/451 might be a potential therapeutic target in red cell diseases.

Small molecule inhibits T-cell acute lymphoblastic leukaemia oncogenic interaction through conformational modulation of LMO2.

Ectopic expression in T-cell precursors of LIM only protein 2 (LMO2), a key factor in hematopoietic development, has been linked to the onset of T-cell acute lymphoblastic leukaemia (T-ALL). In the T-ALL context, LMO2 drives oncogenic progression through binding to erythroid-specific transcription factor SCL/TAL1 and sequestration of E-protein transcription factors, normally required for T-cell differentiation. A key requirement for the formation of this oncogenic protein-protein interaction (PPI) is the conformational flexibility of LMO2. Here we identify a small molecule inhibitor of the SCL-LMO2 PPI, which hinders the interaction in vitro through direct binding to LMO2. Biophysical analysis demonstrates that this inhibitor acts through a mechanism of conformational modulation of LMO2. Importantly, this work has led to the identification of a small molecule inhibitor of the SCL-LMO2 PPI, which can provide a starting point for the development of new agents for the treatment of T-ALL. These results suggest that similar approaches, based on the modulation of protein conformation by small molecules, might be used for therapeutic targeting of other oncogenic PPIs.

The DNA-binding protein ARID3a is Required for Human Erythrocyte Lineage Differentiation using the K562 Model Cell Line

Abstract Our previous studies demonstrated that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein associated with increased immunoglobulin heavy chain transcription and chromatin accessibility, is required for normal hematopoiesis in mice and in human cells. ARID3a knockout mice exhibited low numbers of erythrocytes and died due to failed erythropoiesis between days 9 and 12 of gestation. The role of ARID3a in human erythropoiesis has not been studied. Stimulation of the human K562 cell line with hemin induces fetal globin synthesis and erythrocyte differentiation. K562 cells constitutively express ARID3a, so we used this cell line to determine if ARID3a is important for human erythrocyte differentiation. Knockdown of ARID3a with shRNA in hemin-stimulated K562 cells resulted in &amp;gt;75% inhibition of globin. To identify genes affected by ARID3a loss, we performed RNA-seq experiments at various times after hemin stimulation with and without ARID3a. ARID3a shRNA-inhibited cells confirmed inhibition of globin gene products, including HBA1, HBA2, and HBZ. Erythroid specific transcription factors were also inhibited in samples treated with ARID3a shRNA while myeloid surface markers were induced. Several critical upstream regulators of ARID3a were also identified in these analyses. These data suggest that ARID3a is important for cell fate decisions in erythropoiesis in human cells and identifies potential important targets of ARID3a in this process.

Simultaneous regulation of miR-451 and miR-191 led to erythroid fate decision of mouse embryonic stem cell.

Objective(s):Various microRNAs (miRNAs) are expressed during development of mammalian cells, when they aid in modulating gene expression by mediating mRNA transcript cleavage and/or regulation of translation rate. miR-191 and miR-451 have been shown to be critical regulators of hematopoiesis and have important roles in the induction of erythroid fate decision. So, the aim of this study is investigation of the miR-191 and miR-451 roles in the controlling mouse embryonic stem cell (mESC) differentiation toward the erythroid lineage.Materials and Methods:mESCs were infected with either pCDH-miR-Off-191 viruses in pCDH-miR-Off-191 group or simultaneously with pCDH-miR-Off-191 and pCDH-miR-451 lentiviruses in simultaneous group. Then, the expression profiles of erythroid specific transcription factors and globin genes were analyzed using QRT-PCR on day 14 and 21 of differentiation. Flow cytometry analysis was used to evaluate of TER119 and CD235a erythroid specific surface markers. Results:Gata-1, Klf-1, Epor and globin chains were found to be expressed in pCDH-miR-Off-191 and in simultaneous groups. The majority of globin chains showed changes in their expression levels with progression of differentiation from day 14 to day 21. Flow cytometry results showed that miR-451 up- regulation and miR-191 down-regulation is associated with the expression of TER119 and CD235a. Of these two groups analyzed, simultaneous group was most significantly potent in stimulation of erythroid fate decision of mESCs. Conclusion:Together, present data demonstrate that down-regulation of miR-191 alone can enhance the differentiation of mESCs. However, the simultaneous effect of miR-451up-regulation and miR-191 down-regulation is much stronger and can have more practical use in artificial blood production.

O-GlcNAc homeostasis contributes to cell fate decisions during hematopoiesis

The addition of a single β-d-GlcNAc sugar (O-GlcNAc) by O-GlcNAc-transferase (OGT) and O-GlcNAc removal by O-GlcNAcase (OGA) maintain homeostatic O-GlcNAc levels on cellular proteins. Changes in protein O-GlcNAcylation regulate cellular differentiation and cell fate decisions, but how these changes affect erythropoiesis, an essential process in blood cell formation, remains unclear. Here, we investigated the role of O-GlcNAcylation in erythropoiesis by using G1E-ER4 cells, which carry the erythroid-specific transcription factor GATA-binding protein 1 (GATA-1) fused to the estrogen receptor (GATA-1-ER) and therefore undergo erythropoiesis after β-estradiol (E2) addition. We observed that during G1E-ER4 differentiation, overall O-GlcNAc levels decrease, and physical interactions of GATA-1 with both OGT and OGA increase. RNA-Seq-based transcriptome analysis of G1E-ER4 cells differentiated in the presence of the OGA inhibitor Thiamet-G (TMG) revealed changes in expression of 433 GATA-1 target genes. ChIP results indicated that the TMG treatment decreases the occupancy of GATA-1, OGT, and OGA at the GATA-binding site of the lysosomal protein transmembrane 5 (Laptm5) gene promoter. TMG also reduced the expression of genes involved in differentiation of NB4 and HL60 human myeloid leukemia cells, suggesting that O-GlcNAcylation is involved in the regulation of hematopoietic differentiation. Sustained treatment of G1E-ER4 cells with TMG before differentiation reduced hemoglobin-positive cells and increased stem/progenitor cell surface markers. Our results show that alterations in O-GlcNAcylation disrupt transcriptional programs controlling erythropoietic lineage commitment, suggesting a role for O-GlcNAcylation in regulating hematopoietic cell fate.

Data Access and Interactive Visualization of Whole Genome Sequence of Sickle Cell Patients within the St. Jude Cloud

With the increase in availability of high depth whole genome sequencing (WGS) data of individuals with sickle cell disease (SCD), easy access to the raw sequencing data remains an issue due to technical and regulatory challenges. A compliant system that can provide facile data access would accelerate scientific discovery of genetic variants associated with clinical phenotypes. Cloud storage and computing provide an ultimate solution to this data access, which we have shown through the St. Jude Cloud (https://stjude.cloud) where over 5000 whole genome sequences for pediatric cancer patients are being shared in collaboration with DNANexus and Microsoft. Here we expand the St. Jude Cloud to sickle cell disease data through the Sickle Genome Project (SGP) Data Portal (https://pecan.stjude.org/permalink/sgp) to allow instantaneous raw data access (following data access committee approval), as well as visualization of genotype calls at individual level in a novel genome. The SGP WGS data was generated from 871 patients from St. Jude Children's Research Hospital (St. Jude) through the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Pediatr Blood Cancer. 2018 May 24: e27228) and from Baylor College of Medicine (BCM). All study participants provided informed consent for genomic study and data sharing on IRB-approved research protocols.The SGP data portal will have multi-tiered access. All users will have access to a general heat map view which shows anonymized patient clinical values (e.g., fetal hemoglobin (HbF), mean corpuscular volume (MCV), hemoglobin concentration (Hb)) and relevant SCD modifying variants (e.g., Beta-globin locus, MYB, BCL11A, HBA). The GenomePaint bowser allows for viewing coding and noncoding variants. Displayed with each variant will be a visual indication of the median fetal hemoglobin values for patients homozygous for the reference allele, heterozygous, or homozygous for the alternative allele. The browser also displays erythroid specific DNA-accessibility and epigenetic marks and indicates variant that may disrupt erythroid specific transcription factor binding sites (GATA1 and BCL11A). For anonymization purposes, within the genome browser and heat map views, clinical values and the patients age will be binned into ranges when displayed as single or low count values. Lastly, the ProteinPaint tool (Zhang and Zhou, Nature Gen, Dec 29, 2015) will enable visualization and filtering of variants with reference to protein domain and amino acid sequence.To access processed data such as BAM and VCF files for downstream analyses, a user will be required to apply for access which will be adjudicated by a data access committee. Verified researchers will be granted access to clinical data in a manner consistent with the protocol specific informed consent documentation and protocol under which the sequencing was performed. This may include coded clinical and demographic data when specified by the research protocol and informed consentThe SGP data set will be one of the first WGS datasets from primarily African American Sickle cell patients to be made available to clinicians and researchers worldwide. In addition, no SCD-centric data portal exists that contains controlled access to data and provides graphical tools for visual analysis. The combination of the visual tools and ability to download tools provides the scientific community an invaluable resource for studying sickle cell disease. DisclosuresEstepp:Daiichi Sankyo: Consultancy; NHLBI: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding. Hankins:Global Blood Therapeutics: Research Funding; bluebird bio: Consultancy; NCQA: Consultancy; Novartis: Research Funding.

Identifying ARHGEF12 As an Important Regulator of Erythropoiesis By Calculating Red Blood Cell Transfusion for Chemotherapy Induced Anemia and By Conditional Knockout Mouse Model

Various degree of anemia post chemotherapy was frequently observed among patients even with the same treatment protocol and similar intensity. In this study, we performed whole exon sequencing in 31 individuals having multiple RBC transfusions (MRTs) and 31 patients with no RBC transfusion (NRT), all from the SCMC-ALL 2005 cohort. For children with different age and mass, the RBC transfusion amount was normalized by body surface area, and only post-remission transfusions were calculated to minimize the effect of the disease itself. Patients who received more than 8 RBC units/m2 were defined as MRT. By a genome-wide association study (GWAS), 1708 single nucleotide polymorphisms (SNPs) of 281 genes passed the criterion of the primary sort: a call rate of >95% and P<0.01. Of interest, most of the SNPs were located in introns adjacent to exons, suggesting that these polymorphisms are relevant to chemotherapy-induced anemia by regulating gene expression. Thus, these genes should be highly expressed in erythroid. Sorted according to the Differentiation Map Portal (DMAP) database, 10 genes were at the top of the list TFDP2, CHPT1, LPIN2, TNS1, BSG, ANXA7, EPB42, RAP1GAP, ARHGEF12, and ABCC4 which expressed in erythroid 2 fold above the average. Indeed, some of these genes have well-known associations with RBC function, such as with cytotoxicity susceptibility to chemotherapy agents and with erythropoiesis. Of note, 3 of them are related to small GTPases, including ARHGEF12, a RhoA specific guanine exchange factor.Among SNPs in ARHGEF12, the most significant association was with rs76693355 (P=3.469E-03, odds ratio 5.864). Because whole exome sequencing can miss most regulatory SNPs located in introns, we screened all SNPs with linkage disequilibrium 0.2<r2<1 related to rs76693355 in the 5-kb flanking regions of ARHGEF12 and found rs10892563 at a binding site of the erythroid-specific transcription factor GATA1. The minor allele of this variant disrupted this binding site, which was confirmed by dual luciferase assay in vitro and ARHGEF12 expression quantitation of sorted the CD71-positive erythroid cells from banked bone marrow samples of ALL patients (A). Targeted SNP genotyping of 452 ALL patients enrolled in the SCMC-2005 protocols showed that the genotype frequencies of rs10892563 were CC 7.52%, CT 41.37%, and TT 51.11%. The average normalized RBC transfusion was 4.533 units/m2 in patients with CC, and 2.353 and 2.335 in patients with CT and TT genotypes, respectively. All patients who were homozygous needed RBC transfusion to maintain hemoglobin above 65 g/L, whereas among those who were heterozygous or wild-type, the RBC transfusion frequencies were 61.497% and 70.996%. Considering MRTs frequency, both those who were homozygous and heterozygous had significantly higher than patients carrying the wild type (B). The above results suggested that the ARHGEF12 polymorphism rs10892563 affects chemotherapy-induced anemia susceptibility.To study whether ARHGEF12 related chemotherapy-induced anemia susceptibility is due to ARHGEF12 regulated erythroid regeneration, conditional knockout mouse model was explored. At steady state, arhgef12 deletion selectively perturbed erythroid development in the stage where erythroid precursors and erythroblasts differentiate to late erythroblast (C,D). When challenged by hemolysis induced by Phenylhydrazine at dose of 40mg/kg, arhgef12null mice exhibited worsened anemia and delayed recovery (E). Whle increasing phenylhydrazine dose to 80mg/kg, arhgef12-null mice failed to generate enough mature RBCs to support life (F).In conclusion, ARHGEF12 plays an important role in erythroid hematopoiesis, and its genetic polymorphism may also influence anemia susceptibility of individuals experience chemotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PML-RARα interferes with erythropoiesis by repressing LMO2 in acute promyelocytic leukaemia.

The PML‐RARα fusion gene, generated by the t(15;17) chromosome translocation, is regarded as the initiating factor of acute promyelocytic leukaemia (APL). In addition to the well‐known effects on blocking myeloid differentiation at the promyelocytic stage, promyelocytic leukaemia‐retinoic acid receptor α (PML‐RARα) has also been reported to interfere with multiple differentiation processes, including erythroid differentiation. However, the detailed molecular mechanism by which PML‐RARα impairs erythropoiesis has not yet been fully addressed. By chromatin immunoprecipitation‐PCR assay, we found that PML‐RARα bound to the distal promoter region of LMO2 (LIM‐only protein 2), a critical erythroid‐specific transcription factor. Luciferase reporter assays and qRT‐PCR results demonstrated that PML‐RARα down‐regulated the expression of the LMO2 distal transcript through transrepressing its promoter activity. Analysis of gene expression profiling data from large cohorts of acute myeloid leukaemia (AML) patients confirmed that LMO2 expressed at a markedly lower level in APL patients in comparison to non‐APL AML patients. Further flow cytometry analysis demonstrated that PML‐RARα inhibited erythropoietin‐induced erythroid differentiation by down‐regulating LMO2 expression. Our findings reveal a previously unidentified mechanism, by which PML‐RARα interferes with erythropoiesis through directly targeting and transrepressing LMO2 expression in the development of APL.

Role of tissue-specific promoter DNA methylation in regulating the human EKLF gene

Erythroid Krüppel-like factor (EKLF/KLF1) is an erythroid-specific transcription factor whose activity is essential for erythropoiesis. The underlying mechanisms for EKLF specifically restricted to erythroid cells are of great interest but remain incompletely understood. To explore the epigenetic regulation of EKLF expression by promoter DNA methylation, we investigated the methylation status of the EKLF promoter and EKLF gene expression from a panel of human tissues. We observed that erythroid-specific hypomethylation of the EKLF promoter in adult erythroid cells was positively associated with EKLF expression. Demethylation of the EKLF promoter by 5-aza-2′-deoxycytidine led to elevated EKLF expression in non-erythroid cells. We further uncovered that EKLF promoter DNA methylation reduced the binding affinity for the transcription factors GATA1 and c-myb (MYB), which in turn silenced EKLF expression. These results suggest that hypomethylation of the EKLF promoter has functional significance in the establishment and maintenance of erythroid-specific gene expression.