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Inherited non-hemolytic anemia is a group of rare bone marrow disorders characterized by erythroid defects. Although concerted efforts have been made to explore the underlying pathogenetic mechanisms of these diseases, the understanding of the causative mutations are still incomplete. Here we identify in a diseased pedigree that a gain-of-function mutation in toll-like receptor 8 (TLR8) is implicated in inherited non-hemolytic anemia. TLR8 is expressed in erythroid lineage and erythropoiesis is impaired by TLR8 activation whereas enhanced by TLR8 inhibition from erythroid progenitor stage. Mechanistically, TLR8 activation blocks annexin A2 (ANXA2)-mediated plasma membrane localization of STAT5 and disrupts EPO signaling in HuDEP2 cells. TLR8 inhibition improves erythropoiesis in RPS19 +/- HuDEP2 cells and CD34 + cells from healthy donors and inherited non-hemolytic anemic patients. Collectively, we identify a gene implicated in inherited anemia and a previously undescribed role for TLR8 in erythropoiesis, which could potentially be explored for therapeutic benefit in inherited anemia., (© 2024. The Author(s).)

Abstract: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor β. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the β-globin loci, where there was loss of RNAPII and GATA1 at β-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the β-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit., (© 2023 by The American Society of Hematology.)

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B , encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD + )/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

MicroRNAs (miRNAs) are small non-coding RNAs, which play an important role in various cellular and developmental processes. The study of miRNAs in erythropoiesis is crucial to uncover the cellular pathways that are modulated during the different stages of erythroid differentiation. Using erythroid cells derived from human CD34+ hematopoietic stem and progenitor cells (HSPCs)and small RNA sequencing, our study unravels the various miRNAs involved in critical cellular pathways in erythroid maturation. We analyzed the occupancy of erythroid transcription factors and chromatin accessibility in the promoter and enhancer regions of the differentially expressed miRNAs to integrate miRNAs in the transcriptional circuitry of erythropoiesis. Analysis of the targets of the differentially expressed miRNAs revealed novel pathways in erythroid differentiation. Finally, we described the application of Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) based editing of miRNAs to study their function in human erythropoiesis.

Background: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis., Results: We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X., Conclusions: We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed., (© 2021. The Author(s).)

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.

The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in β-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in β-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Maturation of adult (α2β2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While hemoglobin alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones that may help mature the partner globins (Hb-γ or Hb-β) in erythroid cells, or may enable nonerythroid cells to express mature Hb, are unknown. We investigated a role for heat-shock protein 90 (hsp90) in Hb maturation in erythroid precursor cells that naturally express Hb-α with either Hb-γ (K562 and HiDEP-1 cells) or Hb-β (HUDEP-2) and in nonerythroid cell lines that either endogenously express Hb-αβ (RAW and A549) or that we transfected to express the globins. We found the following: ( i ) AHSP and hsp90 associate with distinct globin partners in their immature heme-free states (AHSP with apo-Hbα, and hsp90 with apo-Hbβ or Hb-γ) and that hsp90 does not associate with mature Hb. ( ii ) Hsp90 stabilizes the apo-globins and helps to drive their heme insertion reactions, as judged by pharmacologic hsp90 inhibition or by coexpression of an ATP-ase defective hsp90. ( iii ) In nonerythroid cells, heme insertion into all globins became hsp90-dependent, which may explain how mixed Hb tetramers can mature in cells that do not express AHSP. Together, our findings uncover a process in which hsp90 first binds to immature, heme-free Hb-γ or Hb-β, drives their heme insertion process, and then dissociates to allow their heterotetramer formation with Hb-α. Thus, in driving heme insertion, hsp90 works in concert with AHSP to generate functional Hb tetramers during erythropoiesis., Competing Interests: The authors declare no conflict of interest.

Iron-restricted human anemias are associated with the acquisition of marrow resistance to the hematopoietic cytokine erythropoietin (Epo). Regulation of Epo responsiveness by iron availability serves as the basis for intravenous iron therapy in anemias of chronic disease. Epo engagement of its receptor normally promotes survival, proliferation, and differentiation of erythroid progenitors. However, Epo resistance caused by iron restriction selectively impairs proliferation and differentiation while preserving viability. Our results reveal that iron restriction limits surface display of Epo receptor in primary progenitors and that mice with enforced surface retention of the receptor fail to develop anemia with iron deprivation. A mechanistic pathway is identified in which erythroid iron restriction down-regulates a receptor control element, Scribble, through the mediation of the iron-sensing transferrin receptor 2. Scribble deficiency reduces surface expression of Epo receptor but selectively retains survival signaling via Akt. This mechanism integrates nutrient sensing with receptor function to permit modulation of progenitor expansion without compromising survival., (© 2018 Khalil et al.)

The transcription factor GATA-1 plays an essential role in erythroid differentiation. To identify novel GATA-1 target genes, we analyzed a merged ChIP-seq and expression profiling dataset. We identified FAM210B as a putative novel GATA-1 target gene. Study results demonstrated that GATA-1 directly regulates FAM210B expression, presumably by binding to an intronic enhancer region. Both human and murine FAM210B are abundantly expressed in the later stages of erythroblast development. Moreover, the deduced amino acid sequence predicted that FAM210B is a membrane protein, and Western blot analysis demonstrated its mitochondrial localization. Loss-of-function analysis in erythroid cells suggested that FAM210B may be involved in erythroid differentiation. The identification and characterization of FAM210B provides new insights in the study of erythropoiesis and hereditary anemias.

Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to K562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG, HBA, and HMOX1. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOX1, and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. We have developed a robust method to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse embryonic stem (ES) cells and have established many immortalized erythroid progenitor cell lines so far. Although their precise characteristics varied among cell lines, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. Considering the number of human ES cell lines that have been established so far and the number of induced pluripotent stem cell lines that will be established in future, the intensive testing of a number of these lines for establishing immortalized erythroid progenitor cell lines may allow the establishment of such cell lines similar to the mouse erythroid progenitor cell lines.

Blood transfusion is indispensable for many clinical applications. However, the supply of transfusable material is insufficient in many countries. Human cord blood contains many hematopoietic stem and progenitor cells, providing a promising resource for the production of transfusable material in vitro. In this study, we have refined a protocol to produce abundant red blood cells (RBC) from human cord blood in an in vitro culture system. We found that erythropoietin and interleukin-3 were most effective when they were added to the culture medium sequentially rather than simultaneously. Although insulin-like growth factor-I (IGF-1) has been reported to function as a positive regulator of RBC production in some in vitro culture systems, we found that IGF-1 had a negative effect upon RBC production. However, IGF-II appeared to function as a positive regulator of RBC production. Finally, stem cell factor functioned to both expand and accelerate the differentiation of immature erythroid cells.

In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells. [ABSTRACT FROM AUTHOR]

Assesses the functions of lipocalin 2 protein in the in vitro and in vivo models of erythropoiesis. Expression and secretion of lipocalin 2 by erythroid progenitor cells; Role of lipocalin 2 in the apoptosis of erythroid progenitor cells; Impact of lipocalin 2 on the differentiation of erythroid progenitor cells.