An intricate regulatory circuit between FLI1 and GATA1/GATA2/LDB1/ERG dictates erythroid vs. megakaryocytic differentiation.

During hematopoiesis, megakaryocytic erythroid progenitors (MEPs) differentiate into megakaryocytic or erythroid lineages in response to specific transcriptional factors, yet the regulatory mechanism remains to be elucidated. Using the MEP­like cell line HEL western blotting, RT­qPCR, lentivirus­mediated downregulation, flow cytometry as well as chromatin immunoprecipitation (ChIp) assay demonstrated that the E26 transformation­specific (ETS) transcription factor friend leukemia integration factor 1 (Fli­1) inhibits erythroid differentiation. The present study using these methods showed that while FLI1­mediated downregulation of GATA binding protein 1 (GATA1) suppresses erythropoiesis, its direct transcriptional induction of GATA2 promotes megakaryocytic differentiation. GATA1 is also involved in megakaryocytic differentiation through regulation of GATA2. By contrast to FLI1, the ETS member erythroblast transformation­specific­related gene (ERG) negatively controls GATA2 and its overexpression through exogenous transfection blocks megakaryocytic differentiation. In addition, FLI1 regulates expression of LIM Domain Binding 1 (LDB1) during erythroid and megakaryocytic commitment, whereas shRNA­mediated depletion of LDB1 downregulates FLI1 and GATA2 but increases GATA1 expression. In agreement, LDB1 ablation using shRNA lentivirus expression blocks megakaryocytic differentiation and modestly suppresses erythroid maturation. These results suggested that a certain threshold level of LDB1 expression enables FLI1 to block erythroid differentiation. Overall, FLI1 controlled the commitment of MEP to either erythroid or megakaryocytic lineage through an intricate regulation of GATA1/GATA2, LDB1 and ERG, exposing multiple targets for cell fate commitment and therapeutic intervention.

Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6.

The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.

miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and ß-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in ß- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in ß- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

CRKL but not CRKII contributes to hemin-induced erythroid differentiation of CML.

Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.

Developmental regulation of primitive erythropoiesis.

PURPOSE OF REVIEW: In this review, we present an overview of recent studies of primitive erythropoiesis, focusing on advances in deciphering its embryonic origin, defining species-specific differences in its developmental regulation, and better understanding the molecular and metabolic pathways involved in terminal differentiation. RECENT FINDINGS: Single-cell transcriptomics combined with state-of-the-art lineage tracing approaches in unperturbed murine embryos have yielded new insights concerning the origin of the first (primitive) erythroid cells that arise from mesoderm-derived progenitors. Moreover, studies examining primitive erythropoiesis in rare early human embryo samples reveal an overall conservation of primitive erythroid ontogeny in mammals, albeit with some interesting differences such as localization of erythropoietin (EPO) production in the early embryo. Mechanistically, the repertoire of transcription factors that critically regulate primitive erythropoiesis has been expanded to include regulators of transcription elongation, as well as epigenetic modifiers such as the histone methyltransferase DOT1L. For the latter, noncanonical roles aside from enzymatic activity are being uncovered. Lastly, detailed surveys of the metabolic and proteomic landscape of primitive erythroid precursors reveal the activation of key metabolic pathways such as pentose phosphate pathway that are paralleled by a striking loss of mRNA translation machinery. SUMMARY: The ability to interrogate single cells in vivo continues to yield new insights into the birth of the first essential organ system of the developing embryo. A comparison of the regulation of primitive and definitive erythropoiesis, as well as the interplay of the different layers of regulation - transcriptional, epigenetic, and metabolic - will be critical in achieving the goal of faithfully generating erythroid cells in vitro for therapeutic purposes.

Clinical impact and characteristics of erythroid dysplasia in adult aplastic anaemia: Results from a multicentre registry.

Morphological dysplasia in haematopoietic cells, defined by a 10% threshold in each lineage, is one of the diagnostic criteria for myelodysplastic neoplasms. Dysplasia limited to the erythroid lineage has also been reported in some cases of aplastic anaemia (AA); however, its significance remains unclear. We herein examined the impact of erythroid dysplasia on immunosuppressive therapy responses and survival in AA patients. The present study included 100 eligible AA patients without ring sideroblasts. Among them, 32 had dysplasia in the erythroid lineage (AA with minimal dysplasia [mini-D]). No significant sex or age differences were observed between AA groups with and without erythroid dysplasia. In severe/very severe AA and non-severe AA patients, a response to anti-thymocyte globulin + ciclosporin within 12 months was observed in 80.0% and 60.0% of AA with mini-D and 42.9% and 90.0% of those without dysplasia, with no significant difference (p = 0.29 and p = 0.24 respectively). Overall survival and leukaemia-free survival did not significantly differ between the groups. Collectively, the present results indicate that the presence of erythroid dysplasia did not significantly affect clinical characteristics or outcomes in AA patients, suggesting that its presence in AA is acceptable. Therefore, erythroid dysplasia should not exclude an AA diagnosis.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Nuclear Factor Erythroid 2-Related Factor (NRF2), Heme Oxygenase 1 (HO-1) and Total Oxidant-Antioxidant Status in Patients with COVID-19 / Factor nuclear relacionado con el eritroide 2 (NRF2), hemo oxigenasa 1 (HO-1) y estado oxidante-antoxidante total en pacientes con COVID-19

Introduction: Studies on nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) levels in COVID-19 patients are limited. This study aimed to investigate the relationship between some biomarkers of oxidant-antioxidant status with COVID-19 disease. Material and methods: The patients older than 18 years of age who tested positive for SARS CoV-2 PCR (polymerase chain reaction) with clinical symptoms and signs were included in this study. Total antioxidant status (TAS), total antioxidant status (TOS), oxidative stress index (OSI) and HO-1 and Nrf2 levels were analyzed from serum samples taken before and after treatment. Results: In this study, 16 patients followed up with the diagnosis of COVID-19 were included. 9 (56.3%) of the patients were female and 7 (43.8%) were male. The mean age was 33.75 ± 17.03 years. All patients were symptomatic and were hospitalized to be followed up. It was determined that Nrf2 and HO-1 values increased significantly after treatment. Moreover, there was a significant positive correlation between Nrf2 and TAS values and TAS increases significantly in parallel to an increase in Nrf2, and there was a significant but negative correlation between Nrf2 and TOS and OSI values, and thus an increase in Nrf2 led to a decrease in TOS and OSI values. There was a significant positive correlation between HO-1 and TAS, and TAS increased significantly, as HO-1 increased. Conclusions: The decrease in TOS and OSI and the increase in Nrf2 and HO-1 during the follow-up period in COVID-19 patients suggest that the body tries to prevent ROS-related oxidative stress via Nrf2 and HO-1 and that oxidative stress may have a key role in the pathophysiology of COVID-19. (AU)

Introducción: Los estudios sobre los niveles del factor 2 relacionado con el factor nuclear eritroide 2 (Nrf2) y la hemo oxigenasa-1 (HO-1) en pacientes con COVID-19 son limitados. Este estudio tuvo como objetivo investigar la relación entre algunos biomarcadores del estado oxidante-antioxidante con la enfermedad COVID-19. Material y métodos: Se incluyeron en este estudio los pacientes mayores de 18 años que dieron positivo a PCR (reacción en cadena de la polimerasa) de SARS CoV-2 con síntomas y signos clínicos. Se analizaron el estado antioxidante total (TAS), el estado antioxidante total (TOS), el índice de estrés oxidativo (OSI) y los niveles de HO-1 y Nrf2 a partir de muestras de suero tomadas antes y después del tratamiento. Resultados: En este estudio se incluyeron 16 pacientes seguidos con diagnóstico de COVID-19. 9 (56,3%) de los pacientes eran mujeres y 7 (43,8%) eran hombres. La edad media fue 33,75 ± 17,03 años. Todos los pacientes presentaban síntomas y fueron hospitalizados para seguimiento. Se determinó que los valores de Nrf2 y HO-1 aumentaron significativamente después del tratamiento. Además, hubo una correlación positiva significativa entre los valores de Nrf2 y TAS y TAS aumenta significativamente en paralelo a un aumento en Nrf2, y también hubo una correlación significativa pero negativa entre Nrf2 y los valores de TOS y OSI y, por lo tanto, un aumento en Nrf2 condujo a una disminución en los valores TOS y OSI. Hubo una correlación positiva significativa entre HO-1 y TAS, y TAS aumentó significativamente a medida que aumentaba HO-1. Conclusiones: La disminución de TOS y OSI y el aumento de Nrf2 y HO-1 durante el período de seguimiento en pacientes con COVID-19 sugieren que el cuerpo intenta prevenir el estrés oxidativo relacionado con ROS a través de Nrf2 y HO-1 y que el estrés oxidativo puede tener un papel clave en la fisiopatología de COVID-19. (AU)

Bach1 inhibitor HPP-D mediates γ-globin gene activation in sickle erythroid progenitors.

Sickle cell disease (SCD) is the most common ß-hemoglobinopathy caused by various mutations in the adult ß-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.

Phenotypic Alterations in Erythroid Nucleated Cells of Spleen and Bone Marrow in Acute Hypoxia.

Hypoxia leads to metabolic changes at the cellular, tissue, and organismal levels. The molecular mechanisms for controlling physiological changes during hypoxia have not yet been fully studied. Erythroid cells are essential for adjusting the rate of erythropoiesis and can influence the development and differentiation of immune cells under normal and pathological conditions. We simulated high-altitude hypoxia conditions for mice and assessed the content of erythroid nucleated cells in the spleen and bone marrow under the existing microenvironment. For a pure population of CD71+ erythroid cells, we assessed the production of cytokines and the expression of genes that regulate the immune response. Our findings show changes in the cellular composition of the bone marrow and spleen during hypoxia, as well as changes in the composition of the erythroid cell subpopulations during acute hypoxic exposure in the form of a decrease in orthochromatophilic erythroid cells that are ready for rapid enucleation and the accumulation of their precursors. Cytokine production normally differs only between organs; this effect persists during hypoxia. In the bone marrow, during hypoxia, genes of the C-lectin pathway are activated. Thus, hypoxia triggers the activation of various adaptive and compensatory mechanisms in order to limit inflammatory processes and modify metabolism.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Human cellular model systems of ß-thalassemia enable in-depth analysis of disease phenotype.

ß-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for ß-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in ß-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.

Single-cell RNA seq analysis of erythroid cells reveals a specific sub-population of stress erythroid progenitors.

BACKGROUND: : Erythroid cells play important roles in hemostasis and disease. However, there is still significant knowledge gap regarding stress erythropoiesis. METHODS: : Two single-cell RNAseq datasets of erythroid cells on GEO with accession numbers GSE149938 and GSE184916 were obtained. The datasets from two sources, bone marrow and peripheral blood were analyzed using Seurat v4.1.1, and other tools in R. QC metrics were performed, data were normalized and scaled. Principal components that capture the variation of the data were determined. In clustering the cells, KNN graph was constructed and Louvain algorithm was applied to optimize the standard modularity function. Clusters were defined via differential expression of features. RESULTS: We identified 9 different cell types, with a particular cluster representing the stress erythroids. The clusters showed differentially expressed genes as observed from the gene signature plot. The stress erythroid cluster differentially expressed some genes including ALAS2, HEMGN, and GUK1. CONCLUSION: The erythroid population was found to be heterogeneous, with a distinct sub-cell type constituting the stress erythroids; this may have important implications for our knowledge of steady-state and stress erythropoiesis, and the markers found in this cluster may prove useful for future research into the dynamics of stress erythroid progenitor cell differentiation.

Erythroid Cells as Full Participants in the Tumor Microenvironment.

The tumor microenvironment is an important factor that can determine the success or failure of antitumor therapy. Cells of hematopoietic origin are one of the most important mediators of the tumor-host interaction and, depending on the cell type and functional state, exert pro- or antitumor effects in the tumor microenvironment or in adjacent tissues. Erythroid cells can be full members of the tumor microenvironment and exhibit immunoregulatory properties. Tumor growth is accompanied by the need to obtain growth factors and oxygen, which stimulates the appearance of the foci of extramedullary erythropoiesis. Tumor cells create conditions to maintain the long-term proliferation and viability of erythroid cells. In turn, tumor erythroid cells have a number of mechanisms to suppress the antitumor immune response. This review considers current data on the existence of erythroid cells in the tumor microenvironment, formation of angiogenic clusters, and creation of optimal conditions for tumor growth. Despite being the most important life-support function of the body, erythroid cells support tumor growth and do not work against it. The study of various signaling mechanisms linking tumor growth with the mobilization of erythroid cells and the phenotypic and functional differences between erythroid cells of different origin allows us to identify potential targets for immunotherapy.

Elucidation of the low-expressing erythroid CR1 phenotype by bioinformatic mining of the GATA1-driven blood-group regulome.

Genetic determinants underlying most human blood groups are now clarified but variation in expression levels remains largely unexplored. By developing a bioinformatics pipeline analyzing GATA1/Chromatin immunoprecipitation followed by sequencing (ChIP-seq) datasets, we identify 193 potential regulatory sites in 33 blood-group genes. As proof-of-concept, we aimed to delineate the low-expressing complement receptor 1 (CR1) Helgeson phenotype on erythrocytes, which is correlated with several diseases and protects against severe malaria. We demonstrate that two candidate CR1 enhancer motifs in intron 4 bind GATA1 and drive transcription. Both are functionally abolished by naturally-occurring SNVs. Erythrocyte CR1-mRNA and CR1 levels correlate dose-dependently with genotype of one SNV (rs11117991) in two healthy donor cohorts. Haplotype analysis of rs11117991 with previously proposed markers for Helgeson shows high linkage disequilibrium in Europeans but explains the poor prediction reported for Africans. These data resolve the longstanding debate on the genetic basis of inherited low CR1 and form a systematic starting point to investigate the blood group regulome.

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

CD36 relative mean fluorescence intensity of CD105+ nucleated erythroid cells can be used to differentiate myelodysplastic syndrome from megaloblastic anemia.

This study aims to evaluate the differences in CD105+ nucleated erythroid cell (NEC) immunophenotypes between myelodysplastic syndrome (MDS) and megaloblastic anemia (MA) using multiparameter flow cytometry and to screen potential markers. We analyzed bone marrow sample data from 37 patients with MDS, 35 with MA, 53 with iron-deficiency anemia (anemic controls), and 35 without anemia (normal controls). Compared with normal controls, the MDS and MA groups showed a decrease in the proportion of CD117+CD105+NEC and the relative mean fluorescence intensity (RMFI) of CD71 in CD105+NEC, accompanied by an increase in the coefficient of variation (CV) of CD71 and CD36. Additionally, CD36 RMFI of CD105+NEC increased in the MA group. Compared with anemia controls, the MDS and MA groups showed a significant increase in CD36 CV of CD105+NEC, and the CD36 RMFI in the MA group increased while that in the MDS group decreased. The proportions of CD117+CD105+NEC, CD36 CV, and CD36 RMFI in CD105+NEC differed significantly between MDS and MA groups. Among them, CD36 RMFI had good diagnostic performance (area under the curve: 0.844, 95% confidence interval: 0.753-0.935). CD36 RMFI of CD105+NEC may be a helpful marker in differentiating MDS and MA using multiparameter flow cytometry.

VPS37C facilitates erythroid differentiation by promoting EKLF stability.

The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.

A mitochondrial iron-responsive pathway regulated by DELE1.

The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.