Terminal Erythroid Differentiation Research Articles

DNMT1 regulates human erythropoiesis by modulating cell cycle and endoplasmic reticulum stress in a stage-specific manner.

The dynamic balance of DNA methylation and demethylation is required for erythropoiesis. Our previous transcriptomic analyses revealed that DNA methyltransferase 1 (DNMT1) is abundantly expressed in erythroid cells at all developmental stages. However, the role and molecular mechanisms of DNMT1 in human erythropoiesis remain unknown. Here we found that DNMT1 deficiency led to cell cycle arrest of erythroid progenitors which was partially rescued by treatment with a p21 inhibitor UC2288. Mechanically, this is due to decreased DNA methylation of p21 promoter, leading to upregulation of p21 expression. In contrast, DNMT1 deficiency led to increased apoptosis during terminal stage by inducing endoplasmic reticulum (ER) stress in a p21 independent manner. ER stress was attributed to the upregulation of RPL15 expression due to the decreased DNA methylation at RPL15 promoter. The upregulated RPL15 expression subsequently caused a significant upregulation of core ribosomal proteins (RPs) and thus ultimately activated all branches of unfolded protein response (UPR) leading to the excessive ER stress, suggesting a role of DNMT1 in maintaining protein homeostasis during terminal erythroid differentiation. Furthermore, the increased apoptosis was significantly rescued by the treatment of ER stress inhibitor TUDCA. Our findings demonstrate the stage-specific role of DNMT1 in regulating human erythropoiesis and provide new insights into regulation of human erythropoiesis.

Mcph1, mutated in primary microcephaly, is also crucial for erythropoiesis.

Microcephaly is a common feature in inherited bone marrow failure syndromes, prompting investigations into shared pathways between neurogenesis and hematopoiesis. To understand this association, we studied the role of the microcephaly gene Mcph1 in hematological development. Our research revealed that Mcph1-knockout mice exhibited congenital macrocytic anemia due to impaired terminal erythroid differentiation during fetal development. Anemia's cause is a failure to complete cell division, evident from tetraploid erythroid progenitors with DNA content exceeding 4n. Gene expression profiling demonstrated activation of the p53 pathway in Mcph1-deficient erythroid precursors, leading to overexpression of Cdkn1a/p21, a major mediator of p53-dependent cell cycle arrest. Surprisingly, fetal brain analysis revealed hypertrophied binucleated neuroprogenitors overexpressing p21 in Mcph1-knockout mice, indicating a shared pathophysiological mechanism underlying both erythroid and neurological defects. However, inactivating p53 in Mcph1-/- mice failed to reverse anemia and microcephaly, suggesting that p53 activation in Mcph1-deficient cells resulted from their proliferation defect rather than causing it. These findings shed new light on Mcph1's function in fetal hematopoietic development, emphasizing the impact of disrupted cell division on neurogenesis and erythropoiesis-a common limiting pathway.

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.

A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

Unveiling P. vivax invasion pathways in Duffy-negative individuals

Vivax malaria has long been thought to be absent from sub-Saharan Africa owing to the high proportion of individuals lacking the Duffy antigen receptor for chemokines (DARC) in their erythrocytes. The interaction between P.vivax Duffy-binding protein (PvDBP) and DARC is assumed to be the main pathway used by merozoites to invade reticulocytes. However, the increasing number of reports of vivax malaria cases in genotypically Duffy-negative (DN) individuals has raised questions regarding the P.vivax invasion pathway(s). Here, we show that a subset of DN erythroblasts transiently express DARC during terminal erythroid differentiation and that P.vivax merozoites, irrespective of their origin, can invade DARC+ DN erythroblasts. These findings reveal that a large number of DN individuals may represent a silent reservoir of deep P.vivax infections at the sites of active erythropoiesis with low or no parasitemia, and it may represent an underestimated biological problem with potential clinical consequences in sub-Saharan Africa.

A Mouse Model of Zhu-Tokita-Takenouchi-Kim Syndrome Reveals Indispensable Son Functions in Myeloid-Lymphoid Lineage Balance and Terminal Differentiation

SON is a DNA- and RNA-binding protein mainly localized in nuclear speckles. Compromised SON function leads to aberrant and alternative RNA splicing, particularly for transcripts bearing weak splice sites. SON also binds to DNA and suppresses H3K4me3 modifications at transcription start sites by inhibiting the MLL1/2 methyltransferase complex. More recent studies suggest that SON serves as the core of nuclear speckles, and SON is required for the association between nuclear speckles and p53 target genes to boost RNA expression. Therefore, SON governs the expression of a myriad of target genes by regulating transcription and RNA splicing as well as nuclear speckle assembly, yet exerts its effects on specific sets of genes. However, the role of SON in organ development and hematopoiesis remains poorly understood. Recently, we identified that heterozygous loss-of-function (LoF) mutations in the SON gene cause a neurodevelopmental disorder with multi-organ anomalies in human patients. This syndrome has been designated as a novel rare disease named Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome. The ZTTK SON-Shine Foundation, a family-initiated non-profit organization, was launched, and through this foundation, we work with families to characterize clinical symptoms associated with SON LoF. Among the clinical symptoms observed in individuals with ZTTK syndrome, a noticeable feature is a wide range of hematological abnormalities, including bone marrow failure, high mean corpuscular volume (MCV), polycythemia, severe anemia, and thrombosis. Furthermore, immunoglobulin deficiency, poor responses to vaccines, and frequent infections were reported, implicating SON LoF in various hematopoietic abnormalities. To study how heterozygous LoF of SON affects organ development and causes clinical features associated with ZTTK syndrome, we created a mouse model of Son haploinsufficiency ( Son +/-). While complete knockout of Son ( Son -/-) led to embryonic lethality, Son +/- mice were viable and recapitulated many clinical features of human ZTTK syndrome patients. The Son +/- mice showed severe growth retardation, signs of cognitive impairment, skeletal abnormalities, kidney agenesis/hypoplasia, and, importantly, hematopoietic abnormalities, such as high MCV, low lymphocyte counts, and immunoglobulin deficiency. To examine the hematopoietic features found in ZTTK syndrome patients are due to SON LoF, we further examined hematopoiesis of our Son +/- mice. We found that Son +/- fetal liver as well as adult mouse bone marrow contain a reduced size of the early-stage hematopoietic stem and progenitor cell (HSPC) pool (marked as Lin-Sca1+cKit+: LSK). Analysis of LSK subpopulations revealed that Son +/- mice have myeloid-lymphoid lineage imbalance at the stage of multipotent progenitor (MPP), with increased megakaryocyte/erythroid lineage-biased MPPs (MPP2) while lymphoid lineage-biased MPPs (MPP4) are decreased. In addition, erythroid terminal differentiation and B cell maturation were impaired in Son +/- mice. Single-cell RNA-sequencing of mouse bone marrow Lin-cKit+ cells and transcriptome-based clustering also revealed that Son haploinsufficiency leads to a reduction in lymphoid lineage-committed progenitor cells and decreased expression of lymphoid lineage/B cell development genes, while erythroid lineage-promoting genes were aberrantly activated in myeloid lineage progenitors. Deep bulk RNA-sequencing and alternative splicing analysis identified that that Son haploinsufficiency caused alternative splicing of chromatin modifiers genes and epigenetic regulator genes. Furthermore, from our ATAC-sequencing analysis, we found that Son haploinsufficiency causes decreased chromatin accessibility in the gene loci critical for lymphoid lineage specification. Taken together, our Son +/- mouse model revealed that the normal level of Son is required to achieve the sufficient expression levels of the genes directing lymphoid lineage specification, B cell lineage development, and erythroid terminal differentiation. Our study demonstrates the indispensable roles of SON in multiple stages of hematopoiesis and provides valuable insight into the clinical significance of abnormal SON levels in hematopoietic diseases.

A Mediator Switch Tunes Transcription Pausing to Drive Erythropoiesis

The Mediator complex regulates various aspects of hematopoietic development, but whether composition of the Mediator complex undergoes dynamic changes for diversifying transcription and functional outputs is unknown. Here, we found that MED26, a subunit in the core Mediator complex, played a distinctive role in facilitating transcription pausing essential for erythroid development. To address whether composition of the Mediator complex undergoes dynamic changes during erythropoiesis, we detected its expression in primary human erythroblasts derived from CD34 + hematopoietic stem and progenitor cells. We found that while the protein levels of most Mediator subunits decreased substantially in the terminal erythroid differentiation, MED26 remained relatively abundant. A gain-of-function assay in the primary human erythroid culture system showed that MED26 overexpression promoted erythroid differentiation. Furthermore, Med26 conditional knockout (cKO) mice exhibited severe erythroid and hematological defects. Erythroid precursors (especially the Cd71+Ter119+ stage) were absent in the bone marrow of Med26-cKO mice, which phenocopies the cKO mice of Gata1. Prussian blue staining of the spleen section showed more iron deposition in the cKO mice. Moreover, the cKO mice were unable to recover after phenylhydrazine-induced hemolytic anemia. To dissect the molecular basis underlying MED26 function, we analyzed the chromatin occupancy signals of MED26 by CUT&Tag assay and compared them to those of MED1, which is often regarded as a representative subunit of Mediator, in human CD34 + erythroid culture cells on Day 4. Intriguingly, we found that ~60% of chromatin sites with MED1 or MED26 occupancy did not colocalize, suggesting these subunits exert context-dependent gene regulation. Notably, MED26 colocalizes with GATA1 and GATA2 better than MED1. PRO-seq analysis further revealed that MED26-enriched loci were associated with RNA polymerase II (Pol II) pausing. To examine how MED26 might mediate transcription pausing, we performed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify interacting proteins of MED1 and MED26. While MED26 and MED1 similarly coimmunoprecipitated with the majority of other Mediator subunits and the elongation complex, MED26 exhibited a substantially higher interaction with transcription pausing related factors (NELF, DSIF, and PAF complexes) compared to MED1. Knocking out MED26 significantly impairs the recruitment of PAF1 to MED26 occupancy sites, indicating the presence of MED26 facilitates the recruitment of PAF1. Our results also revealed that MED26 enrichment is associated with increased transcription pausing during terminal erythroid stages, which can promote erythroid development. Moreover, we uncovered that MED26 exhibited pronounced biomolecular condensate-forming capability. The intrinsically disordered region (IDR) of MED26 was necessary for its function in promoting erythropoiesis and recruiting pausing-related factors to the MED26-enriched condensates. Taken together, our findings demonstrate that the relative enrichment of MED26 in terminal erythropoiesis leads to a shift of transcription condensates towards a MED26-enriched form. This shift, in turn, facilitates the recruitment of pausing related factors, thereby achieving genome-wide transcription repression during erythropoiesis. This study not only illustrates the indispensable function of MED26 in erythropoiesis but also elucidates the functional coordination of MED1 and MED26 during different erythroid stages. The dynamic switch of Mediator stoichiometry might serve as an important example for understanding the mechanism of the versatile Mediator function during development.

Delineating the Functions of Cohesin Stag1 during Terminal Erythroid Differentiation

Erythroid terminal differentiation is defined by a series of precise, orchestrated and dynamic changes in the genomic architecture that is consistent with the transcriptional programing towards producing mature blood cells. In recent times, these architectural shifts are under scrutiny, not only because they shed light into the mechanisms underlying erythroid differentiation, but also because corruption of the organization principle can result in compromised hematopoietic function and tumor formation. Functionally, genome organization relies upon the concerted action of proteins such as the cohesion complex and CTCF. In this study, we identify Stag1, a cohesin protein, as a factor that contributes to the erythroid transcriptional machinery during terminal differentiation. Stag1 levels increase during terminal differentiation and depletion of Stag1 either using short hairpin RNA or using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) in Hudep2 or CD34+ cells, results in a drastic reduction of both adult globins, α- and β-globin. This is not the case when Stag1's paralogue, Stag2, is knocked-down. Further, we observe faster proliferation in erythroid cells after Stag1 depletion and these cells also demonstrate an undercondensed nucleus and a larger cell-size. This could reflect the delayed differentiation, as the cells proceeded to undergo slower differentiation as compared to their WT counterparts. Metaphase spreads of the Stag1 depleted cells suggests an altered cohesion phenotype and further provides clues into a possible epigenetic defect underlying the observed phenotypes in erythroid cells, which is being further investigated.

PDZD2 Is Required for Normal Erythropoiesis and Its 37-KDa Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP, and erythroid progenitors, however, it is almost universally lost through hypermethylation in acute myeloid leukemia (AML). PDZD2 encodes for a 300-kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37-kDa protein known as secreted PDZD2 (sPDZD2). sPDZD2 has been reported to function as a tumor suppressor in some solid tumors, including breast, prostate, and liver cancer. Thus, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. We first sought to determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment. Using an ELISA to quantify sPDZD2 levels in BM plasma isolated from healthy donors (n=6) and primary AMLs (n=20), we confirmed its secretion by normal CD34+ HSPCs (NBM) and downregulation in AMLs (NBM: 169 ± 17.53 vs AML: 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines (n=7) secreted significantly lower levels of sPDZD2 into the culture medium than normal HSPCs (n=2) (NBM: 28.71 ± 1.76 vs AML: 4.62 ± 1.19 ng/mL; p<0.0001). Next, to study the role of PDZD2 in normal hematopoiesis we performed in vitro myeloid and erythroid differentiation and colony-forming unit assays in CRISPR-Cas9-edited human HSPCs. CD34+ cells from healthy donors were electroporated with recombinant Cas9-sgRNA ribonucleoprotein complexes targeting PDZD2 and editing confirmed by T7I endonuclease assay and Sanger sequencing. PDZD2-edited cells (PDZD2 KO) were plated either on methylcellulose or liquid differentiation cultures (n=6). PDZD2 KO resulted in decreased colony number of all types (Control: 172.5 ± 34.37 vs PDZD2 KO: 137.0 ± 18.25, p<0.05), indicating reduced colony-forming potential. In addition, loss of PDZD2 resulted in a delay in terminal erythroid differentiation with an accumulation of pro-erythroblasts (CD71+CD235a lowCD105 high, p<0.05) and late basophilic erythroblasts (CD71+CD235a highCD105 int, p<0.01) as well as decreased orthochromatic erythroblasts (CD71+CD235a highCD105 neg, p<0.01) in a tri-phasic liquid culture system. By contrast, PDZD2 KO had no impact on myeloid differentiation. To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2 Gt/Gt gene-trap (GT) mouse model and performed peripheral blood (PB) and BM analysis compared to wild-type (WT) controls. PB counts at 2 months of age (n=6-9 per genotype) showed anemia, with a significant decrease in both red blood cell counts (WT: 9.04 ± 0.26 vs GT: 7.58 ± 0.47 x10 6/mL; p=0.026) and hemoglobin levels (WT: 14.88 ± 0.46 vs GT: 12.30 ± 0.81 g/dL; p=0.036), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decrease megakaryocyte-erythroid progenitors (MEPs) and short-term HSCs in the BM of 3 months-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. Next, we sought to evaluate the tumor suppressor role of sPDZD2 in the context of AML. We treated a panel of AML cell lines (n=9) and primary human AML specimens (n=4) with recombinant sPDZD2 ( r-sPDZD2; dose: 100nM-300nM/day) for a period of 8 days. AML cells showed dose-dependent growth inhibition (CellTiter-Glo, p<0.05 for all) and increased expression of either CD11b or CD15 differentiation markers. Cell cycle analysis on day 8 revealed that AML cells treated with r-sPDZD2 were arrested at G0/G1 phase compared to control. In summary, our study is the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for r-sPDZD2 as a novel therapeutic approach for AML.

Identification of a Novel Gene Regulatory Element in Human Erythroid Progenitor Cells

Early erythropoiesis involves commitment of multi-lineage progenitors to erythroid progenitor cells and differentiation into erythroid burst-forming unit (BFU-E) cells, then erythroid colony-forming unit (CFU-E) cells. In contrast to terminal erythroid differentiation, regulation of gene expression in early erythropoiesis is poorly defined. To address this knowledge gap, we identified active enhancers in erythroid progenitor cells linked to known disease genes and genes associated with erythrocyte traits in genome wide association studies. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from mobilized peripheral blood were cultured towards the erythroid lineage. From these cultures, FACS-based methods were utilized to purify four stages of erythroid progenitor cells, termed EP1 to EP4, and five populations of terminally differentiated erythroblasts, from proerythroblasts to orthochromatic erythroblasts. ChIP-seq was performed using antibodies to H3K4me1, H3K27Ac, H3K27me3, and H3K9me2/3. Chromatin accessibility was determined by ATAC-seq. Patterns of gene expression were determined by RNA-seq. Active enhancers were identified using the ModHMM algorithm. Numerous erythroid progenitor cell-specific active enhancers were identified near genes linked to known disease genes and GWAS-associated erythrocyte traits. rs162375, a single nucleotide polymorphism (SNP) linked to mean corpuscular hemoglobin, was in a 475bp Long Terminal Repeat Retrotransposon (LTR) element upstream of the RRP1B gene. The LTR enhancer near the RRP1B gene is a member of the LTR15 family. Transposable elements are a source of novelty in primate gene regulation when they acquire binding sites for critical tissue-specific transcription factors, and then these tissue-specific regulatory regions direct tissue-specific expression of nearby genes. The SNP-containing LTR15 erythroid progenitor-specific enhancer upstream of RRP1B contains 2 conserved GATA motifs and a conserved RUNX motif. The enhancer region had a peak of open chromatin determined by ATAC-seq, and H3K27 acetylation and strong GATA1 binding determined by ChIP-seq. These findings were present only in erythroid progenitor cells, not in earlier hematopoietic cells, terminally differentiating erythroid cells, or other non-erythroid cells. CRISPR-based gene editing was utilized to delete the LTR15 enhancer in CD34+ HSPCs cultured towards the erythroid lineage into erythroid progenitor cells. mRNA expression of RRP1B was decreased to 70.2% of wild type. Luciferase-based reporter gene studies demonstrated the enhancer increased expression of a minimal promoter 3.2-fold over background. Mutation of the GATA and RUNX sites individually or together decreased reporter gene activity to background levels. Bioinformatic analyses identified 291 additional erythroid progenitor cell specific LTR15 element active enhancers, with most containing two GATA motifs and a RUNX motif. The majority of the novel enhancer regions were also marked by progenitor cell-specific ATAC peaks and strong GATA1 binding in ChIP-seq. GATA1 and RUNX, both present in early erythropoiesis, are required to activate the regulatory elements in erythroid progenitor cells. Decreasing levels of RUNX in terminal erythroid differentiation parallels loss of enhancer activity. Since LTR15 first appeared in Catarrhine, old-world monkeys, these data indicate this novel regulatory element has recently evolved to contribute to regulation of gene expression in early erythropoiesis in primates.

AURKA Kinase Controls Erythroblast Enucleation Via Regulation of Centrosome Localization and ECT2 Degradation

Mature red blood cells (RBCs) in the peripheral circulation of mammals are nuclear-free. Erythroblast enucleation is proposed to be analogous to asymmetric cell division based on cellular progress. the molecular mechanisms of how spherical erythroblasts determine the direction of nuclear polarization and accomplish the final separation of the nucleus from the nascent reticulocytes remain largely unclear, until now. Aurora kinases are highly conserved serine/threonine kinases required for regulation of mitosis (AURKA and AURKB). However, up to now, the role of Aurora kinases in erythroid cells, especially in erythroblast enucleation has been fully undefined. Here, we found that AURKA and AURKB messenger RNA (mRNA) expression levels were similar early in terminal differentiation, but followed by a prominent increase in AURKA levels in polychromatic and orthochromatic erythroblasts, whereas AURKB levels were dramatically downregulated. The expression patterns of Auraro kinases were further confirmed by western blotting analysis. More notably, the expression of Aurora kinases is low or absent in most adult tissues or cells due to their lower proliferation rates. However, AURKA and AURKB are still highly expressed in orthochromatic erythroblasts compared to other precursor erythroblasts. The unconventional expression profile of Aurora kinases, especially AURKA, during terminal erythroid differentiation suggests an extraordinary role in erythroblast enucleation. To explore the role of Aurora kinases in erythroblast enucleation, we first tested whether pharmacological inhibition of these kinases blocked the enucleation of human erythroblasts. Treatment of erythroblasts with MLN8237 (Aliseritib, AURKA selective inhibitor), AZD1152(Barasertib, AURKB selective inhibitor), or AT9283 (AURKA and AURKB inhibitor) dramatically blocked enucleation in a dose-dependent manner. Furthermore, we found that knockdown of Aurora kinases by siRNA indeed resulted in impaired enucleation. We next examined whether Aurora kinases regulated nuclear polarization, which is considered the onset of erythroblast enucleation, by measuring the △centroid distribution (distance between centers of the cytoplasm and nucleus). We found that erythroblast nuclear polarization is AURKA dependent, but not AURKB dependent. Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA colocalized with the centrosome on the side of the nucleus opposite its membrane point of contact during polarization and subsequently solely translocated to the anterior end of the protrusive nucleus upon nuclear exit, whereas AURKB consistently localized to the actin contraction ring during enucleation. Colocalization of AURKA and centrosome during nuclear polarization raised the question whether AURKA controls polarization by regulating centrosome organization and maturation. Co-immunoprecipitation (co-IP) verified a direct interaction between AURKA and γ-tubulin. We found that AURKA loss did not alter the expression levels of γ-tubulin in erythroblasts. However, immunofluorescence showed that MLN8237 treatment obviously impaired the proper organization of centrosome. These phenotypes of centrinone-B, an inhibitor of the master centriole biogenesis regulator PLK4 treated erythroblasts mimic the results of AUKRA-deficient erythroblasts, suggesting a close connection between AUKRA and centrosome in polarization during erythroblast enucleation. We intriguingly found that ECT2 (Epithelial Cell Transforming 2), was only constricted at the rear of the thanslocating nucleus in enucleating erythroblasts. Inhibition of AURKA obviously impaired this transformation, exhibiting persistent cytoplasmic localization. MLN8237 treatment resulted in a significant increase of ECT2 proteins in K562 cells.In contrast to control scramble siRNA, ECT2 siRNA significantly increased the enucleation of MLN8237-treated erythroblast. The translocation of AURKA, which is originally restricted to centrosome relied on γ-tubulin interaction during nuclear polarization, was to directly degrade ECT2 at the anterior end of the protrusive nucleus for final nuclear expulsion. Our findings reveal a previously unrecognized localization and role of Aurora kinases in terminal erythroid differentiation and provide new mechanistic insights into erythroblast enucleation.

Whole-Genome CRISPR-Cas9 Screening Identifies Genes Required for Human and Mouse Erythroid Development

Red blood cell (RBC) development is regulated by a few external signaling molecules which act to influence the ongoing intracellular processes responsible for producing erythrocytes. While much is known about the signaling molecules important for blood production, comparatively little is known about the genes required for successfully generating erythrocytes. Several genes have already been identified as essential for RBC development through loss-of-function studies in the mouse or in human erythroid cells in vitro. However, these studies have been limited in scope to genes that are already suspected or known to influence RBC production. Since there are ~10,000 genes expressed in erythroid cells, it is unclear how many of these genes are functionally required for erythroid development. To define the repertoire of genes required for human erythroid development, we performed a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2, using the GeCKOv2 lentiviral library, which delivers Cas9 and one of 6 sgRNAs targeting virtually every gene in the genome. Following viral transduction and cell recovery for 9 days, we collected HUDEP-2 cells prior to the onset of differentiation as well as differentiated orthochromatic erythroblasts (right prior to enucleation) using flow sorting, on the basis of CD49d expression (CD49d is downregulated in the final stages of erythroid differentiation). As expected, sgRNAs targeting known erythroid essential genes (such as GATA1 and EPOR) were depleted in HUDEP-2 cells compared to the sgRNA library. Notably, we also identified genes for which sgRNAs were depleted in differentiated versus undifferentiated erythroid cells, representing genes that are likely required for terminal erythroid differentiation (such as ZFPM1, ALAS2, etc..). This supports the utility of the screen to identify novel regulators of erythropoiesis. Among highly ranked genes, NHLRC2, which was previously implicated in hemolytic anemia, was identified to be required in HUDEP-2 cells, suggesting that NHLRC2 is intrinsically required for human erythroid development. To validate the role of NHLRC2 in human erythropoiesis, we down-regulated NHLRC2 in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) undergoing erythroid differentiation, using one of 4 independent shRNAs. NHLRC2 down-regulation resulted in impaired proliferation and differentiation of human erythroid cells in vitro. To validate the results of the genome-scale screen, we generated a secondary library dedicated to examining genes that positively or negatively regulate the final stages of erythroid differentiation, while excluding common essential genes required for the survival of more than 90% of immortalized cell lines screened by the BROAD institute. The secondary screen yielded a list of over 500 genes found to influence erythroid differentiation (FDR<0.01). One of the novel genes, VAC14, was identified as a highly ranked gene in the secondary screen for erythroid differentiation. Validation experiments demonstrated that VAC14 down-regulation using one of 3 independent shRNAs result in erythroid cell proliferation defects, both in HUDEP2 cells and in erythroid cells derived from primary human HSPCs. Since germline loss of VAC14 is embryonic lethal in the mouse, to validate the role of VAC14 in erythropoiesis in vivo, we transplanted fetal livers from Vac14 null mice (or wild-type [WT] control mice) into WT recipients and examined the VAC14 deficient hematopoietic compartment. Mice transplanted with Vac14 null HSCs exhibited a profound reduction in absolute numbers of bone marrow erythroid cells with evidence of a block in erythroid maturation. Impaired bone marrow erythropoiesis was largely (but incompletely) compensated for by splenic extramedullary erythropoiesis. Vac14 null hematopoietic cells exhibited pronounced cytoplasmic vacuolation, also affecting numerous stages of erythroid cells. Altogether, we have performed an unbiased genome-scale CRISPR knock-out screen that identified (and validated) novel genes required in erythropoiesis.

Fetal Hemoglobin Decrease during Voxelotor Treatment

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to the formation of hemoglobin S (HbS). The polymerization of deoxygenated HbS is the first event in the molecular pathogenesis of SCD resulting in sickling of red blood cells (RBC), and hemolytic anemia which has been shown to be associated with organ damage and early death. Hemolytic anemia is defined by an accelerated rate of destruction RBC resulting in a lowered Hb level and a reduced gas transport capacity. Because Hb S in its oxy (R) conformation do not polymerize, drugs modifying hemoglobin (Hb) to increase the proportion of oxy-HbS have been propose to inhibit HbS polymerization. Voxelotor is such a molecule, which allosterically modifies Hb-oxygen affinity improving anemia and reducing hemolysis. However, the impact of Voxelotor on HbF levels is unknown. We describe here variations of %HbF and Mean corpuscular HbF (MCHbF) in a cohort of homozygous SCD patients treated with Voxelotor in our Sickle Cell Referral center. We conducted a prospective register study to analyze clinical and biological evolution of SCD patients treated with Voxelotor at 1500mg taken orally once daily. From December 2021 through December 2022, 25 SCD adult patients were followed during a median of 7 months [1-12]. Among them, HbF values were analyzable for 15 patients (SS gentype n=14; women in 73%; median age of 50 years [24-61]) who completed 6 months of Voxelotor with no transfusion during the same period; and with a stable dose of hydroxyurea (HU), for at least 3 months before inclusion and during the study in case of patients treated by HU (n=10). The dose of Voxelotor was stable for 13/15 patients and was decreased in 2 patients due to skin allergy (n=1) and moderate digestive intolerance. One patient died after 6 months of treatment in a context of end-stage renal failure and severe pulmonary hypertension not related to Voxelotor treatment. Mean corpuscular HbF (MCHbF) was calculated (MCHbF (pg) = MCH x %HbF). HbF percentage was measured by high performance liquid chromatography (Biorad, Variant II, beta-thal dual program). All participants provided written informed consent (CPP Erythropedie). From baseline to 6 months of follow up mean Hb (g/dL) concentration increased from 7.4 [5.4-8.5] to 8 [5-11.9] (p=0,0003; Figure A); absolute reticulocyte count (ARC ; Giga/L) decreased from 158 to 145 (p=0,005), %HbF decreased by 63% in all patients from 7,1% to 3,3% (p=0,008; Figure B) and MCHbF (pg) decreased from 3,36 to 1,03 (p=0,001; Figure D). A decrease in % HbF was observed from the first 15 days of treatment in both groups of patients treated or not with HU (Figure C) ; no changes in mean corpuscular volume (p=0.15) was observed in patients treated with HU during follow up. The HbF decrease in SCD patients treated with Voxelotor could be explained by different mechanisms: - Reduction of stress erythropoiesis which is due to an increase in erythroid output in response to anemic stress associated with a reduced expression of BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. This hypothesis is supported by the reduction in reticulocytes in patients treated with Voxelotor - The loss of an HbF-related survival advantage for SCD RBCs, while the normal RBC lifespan is approximatively 120 days, in SCD RBCs it is only a sixth of it. HbF is a major modulator of SCD and prolonge SCD RBCs survival. Its protective effect relies on its quantity and distribution among the RBC population, which is often heterogeneous in SCD patients. Protection against polymerisation of Voxelotor is probably more homogeneous among RBCs. As a result, RBCs containing low levels of HbF, which would normally disappear, have a prolonged lifespan. Similarly, it was presumed that sickling of erythroblasts could lead to ineffective terminal erythroid differentiation with an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. However we found no significant difference for index of ineffective erythropoiesis (soluble transferrin receptor / absolute reticulocyte count) before and after 6 months of treatment. The question raised is therefore that of the risk of a rebound effect upon discontinuation of Voxelotor, which has a short half-life, during the time it takes for HbF to increase. To better study this hypothesis, distribution of HbF/RBC will be studied in the HEMOPROVE trial (NCT05199766) currently in progress.

Identification of CNOT4 As a Positive Regulator of Erythroid Cell Growth Using a Genome Wide CRISPR Knock-out Screen

Red blood cells, also known as erythrocytes, are the principle means of oxygen delivery to the body, and inadequate or disordered erythrocyte production is a major cause of human disease. While the cell-extrinsic factors that control erythrocyte production have been well studied, our understanding of the cell-intrinsic regulation of this process is incomplete. To identify genes that regulate erythroid proliferation and/or maturation, we performed a genome-scale CRISPR knock-out screen in the immortalized human erythroid cell line, HUDEP-2. HUDEP-2 cells can be maintained at the pro-erythroblast stage and then sequentially differentiated into basophilic, polychromatic, and orthochromatic erythroblasts by changing culture conditions, which permits separate evaluation of the impact of genetic perturbations on pro-erythroblast viability and terminal erythroid differentiation capacity. To perform our screen, we used the commercially available GeCKO-v2 library, which includes 6 sgRNAs targeting virtually every protein-coding gene in the human genome. This library was packaged into lentiviral particles, each expressing Cas9, an sgRNA, and a puromycin resistance gene. HUDEP-2 cells were transduced with the pooled GeCKO-v2 library at low multiplicity of infection to ensure delivery of only one sgRNA per cell. Following puromycin selection, pro-erythroblasts were collected prior to differentiation and orthochromatic erythroblasts were sorted at day 12 of differentiation (selecting for cells that downregulated CD49d). By identifying sgRNAs that were under-represented in orthochromatic erythroblasts compared to the proerythroblasts, we identified several genes that when deleted lead to impaired erythroid differentiation. One of these genes was CNOT4, which encodes a RING E3 ligase member of the CCR4-NOT complex, a conserved transcriptional regulator. This gene has been previously reported to positively regulate JAK/STAT signaling, which is known to play a key role in erythroid growth and differentiation. Additionally, only 30 out of 1095 cell lines tested in the Depmap portal exhibit diminished growth following CNOT4 deletion, suggesting a rather specific impact on erythroid cells. To define the role of CNOT4 in erythropoiesis, we electroporated human CD34+ hematopoietic stem and progenitor cells (HSPCs) with a ribonucleoprotein (RNP) complex consisting of Cas9 protein and CNOT4-targeting sgRNA. Three unique CNOT4-targeting sgRNAs were tested. Following RNP electroporation, cells were differentiated using a four-phase erythroid differentiation culture protocol and assessed for growth, indel frequencies, and erythroid differentiation capacity. For all three sgRNAs tested, CNOT4 disruption resulted in 10-fold reduced proliferation over the course of differentiation compared to cells electroporated with non-targeting sgRNAs. Erythroid differentiation, assessed by flow cytometry using CD49d and CD233 expression, was identical in CNOT4 deleted and control cells. However, evaluation of cytospins revealed reduced cell size with diminished cytoplasm to nuclear ratio in CNOT4 deficient compared to control cells. Notably, the findings above were validated in erythroid cells differentiated from HSPCs harvested from two independent donors. Taken together, these findings suggest a novel role for CNOT4 and/or the CCR4-NOT complex in erythroid differentiation. Studies are ongoing to determine the mechanism by which CNOT4 disruption negatively impacts erythropoiesis and to examine if CNOT4 overexpression results in enhanced proliferation/differentiation of erythroid cells.

The Darc Side of Vivax Malaria in Africa: Unveiling Invasion Pathways into Duffy-Negative Erythroblasts

Background & Objectives: Plasmodium vivax malaria was long thought to be absent from sub-Saharan Africa owing to the high prevalence of people lacking the Duffy antigen receptor for chemokines (DARC) on their erythrocytes. The interaction between P. vivax Duffy binding protein (PvDBP) and DARC is assumed to be the main mechanism used by P. vivax merozoites to invade human erythrocytes. However, the increasing numbers of vivax malaria cases in Duffy-negative African individuals has raised questions about alternative P. vivax invasion pathway(s) other than PvDBP-DARC interaction. Since P. vivax has a tropism for CD71+ immature reticulocytes and the hematopoietic niches of the bone marrow may be a hidden reservoir of P. vivax parasites, we hypothesized that P. vivax merozoites may be able to invade erythroblasts derived from Duffy-positive (DP) and Duffy-negative (DN) individuals. Therefore, our objectives were to (i) investigate the expression of DARC during DP and DN erythropoiesis and (ii) examine the infection of DP and DN erythroblasts with P. vivax merozoites in vitro to study and identify host receptors used by P. vivax to invade erythroblasts. Methods: We performed in vitro erythropoiesis assays (two-phase culture) of genotypically DN and DP erythroid progenitors. Erythroid differentiation, CD71 and DARC expression profiles were monitored using flow cytometry, microscopy, and western blot over time during phase 2. In vitro invasion assays were conducted using DP and DN erythroblasts to investigate whether genotypically DN erythroblasts could be invaded by P. vivax. At D7 of phase 2, we thawed and cultured parasite isolates obtained from two Malagasy and seven Ethiopian patients infected with P. vivax. After approximately 24-30 hours of in vitro maturation (at D8 of phase 2), each P. vivax parasite culture was placed in co-culture with either DP or DN erythroblasts for 24-48 hours. We examined the infection of DP and DN erythroblasts by P. vivax parasites by light and confocal microscopy. Results: Using in vitro erythropoiesis, we show that both genotypically DP and DN erythroblasts have similar kinetics of terminal erythroid differentiation. Moreover, we found similar CD71 expression profiles in both DP and DN erythroblasts. Interestingly, we observed that a subset of DN erythroblasts transiently expressed DARC during erythropoiesis. This was confirmed using both flow cytometry and western blot analysis. In vitro invasion assays conducted on both DP and DN erythroblasts using parasite isolates, showed that P. vivax merozoites were able to invade both DP and DN erythroblasts (Figure 1). We found that all the DP and DN erythroblasts that were infected with P. vivax were CD71+ and DARC+. These results confirm that P. vivax can invade DP and DN erythroblasts expressing both DARC and CD71, irrespective of the origin of the isolates. Conclusion: This study provides the first biological evidence for the ability of P. vivax to invade genotypically DN erythroblasts and highlights that African populations may represent a silent reservoir of P. vivax infections, with major implications for the control and elimination of vivax malaria in sub-Saharan Africa.

Rps19 and Rpl5 Play Distinct Roles in hematopoietic Stem Cell maintenance and Erythroid Differentiation

Mutations in ribosomal proteins (RPs) lead to ribosomopathies. Mechanistic basis underlying the failure of erythropoiesis due to mutations in various RPs is yet to be fully defined. Lack of relevant murine models of ribosomapathies has hindered significant progress. To fill this gap, we generated Rps19 and Rpl5 floxed mice using CRISPR/Cas9 technologies and characterized their hematologic phenotype following crossing them to a Vav-iCremice. Vav-iCre;Rps19 fl/+mice are born at expected Mendelian rates with severe macrocytic anemia and reticulocytopenia. These mice die by post-natal day 10 (P10), due to bone marrow failure and absence of splenic stress erythropoiesis. Analysis of fetal erythropoiesis revealed a 40% reduction in the total number of erythroid cells in Vav-iCre;Rps19 fl/+, however erythroblasts at all stages of terminal erythroid differentiation were found. Intriguingly, studies of hematopoietic stem and progenitor cell s demonstrated a progressive exhaustion of the stem cell compartment. scRNAseq studies of Vav-iCre;Rps19 fl/+ fetal livers demonstrated increased expression of RUNX1, a transcription factor implicated in the regulation of ribosome biogenesis. Accordingly, we observed that hematopoietic progenitors of Vav-iCre,Rps19 fl/+had enhanced global protein synthesis along with increased RUNX1 protein, despite ribosomal protein haploinsufficiency demonstrated by polysome profiling. In marked contrast, Vav-iCre;Rpl5 fl/+ mice presented with a more severe phenotype and died perinatally from anemia. Further analysis revealed a failure of erythropoiesis at erythroid progenitor stages in Vav-iCre;Rpl5 fl/+. In striking contrast with the Rps19 model, Vav-iCre;Rpl5 fl/+ mice had an expansion of the hematopoietic stem and progenitor cell compartment that also had an increased protein translation rate . This increased translation occurred in the context of normal expression levels of RUNX1. Interestingly Vav-iCre;Rpl5 fl/+ micehad reduced mTORC1 activity and a dramatic reduction in the expression of other ribosomal proteins. Consistent with previous studies about the pivotal role of mTORC1 in erythropoiesis Vav-iCre,Raptor fl/fl(mTORC1 conditional knockout) had reduced expressions of RPs in fetal liver. In summary, we report different requirements for Rps19 and Rpl5 during fetal hematopoiesis in regulating erythroid progenitor biology. While Rps19 plays an essential role at the HSC level to maintain the generation of erythroid progenitors, Rpl5 is critical once progenitors are committed towards an erythroid fate. Along with the findings that translation is increased in RP haploinsufficient models, our study unravels potential compensatory mechanisms during fetal hematopoiesis.

SR-18292 Induces Fetal Hemoglobin Synthesis and Reduces Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a commonly inherited blood disorder, which affects millions around the world. Reactivation of fetal hemoglobin (HbF) synthesis in adult erythroid cells can diminish the severity of many clinical features of the disease. Up-regulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to induce HbF levels in human primary erythroid progenitor CD34 + cells. Here we reported the in vitro and in vivo effects of a small molecular compound, SR-18292, on HbF induction via modulating PGC-1α. To determine whether SR-18292 can induce HbF in human CD34 + cells, we performed RT-qPCR, flow cytometry, and western blot to measure the mRNA and protein levels of PGC-1α and γ-globin. We found that mRNA levels of PGC-1α and γ-globin were significantly increased in cells treated with SR-18292 while β-globin mRNA levels were unaffected. Protein levels of γ-globin and the ratio of F-cells were increased in treated cells. Erythroid cellular differentiation markers (CD71/CD235a) and terminal differentiation markers (CD49d/CD233) were used to examine the differentiation status. There was no significant difference in the population of CD71 +CD235a + cells among all groups. However, SR-18292 treatment increased the percentage of CD49d -CD233 + cells at 5 or 7.5 μM. These results suggest that SR-18292 at higher concentrations can promote erythroid terminal differentiation. To evaluate whether the HbF induction by SR-18292 can reduce the SCD disease pathology in SCD mice or not, we intraperitoneally administered SR-18292 to SCD mice. HbF induction was determined by flow cytometric analysis using an anti-HbF antibody. And it was shown that the HbF-high cell population was induced in SCD mice after 4 weeks of treatment. And we used transferrin receptor CD71 and erythroid-specific marker Ter119 to determine whether SR-18292 could affect erythroid differentiation. There was no significant difference in the CD71 +Ter119 + population after 4 weeks of SR-18192 treatment compared with untreated animals. The mRNA levels of γ-globin and PGC-1α were increased in treated mice. And the increase of PGC-1α and γ-globin protein levels were confirmed by western blot. Reticulocyte is a sign of premature RBC destruction (hemolysis), and the SCD mice usually have high reticulocyte counts (>50%). We measured the reticulocyte in the peripheral blood of SR-18292 treated and untreated SCD mice by flow-cytometric analyses and found that reticulocyte was significantly reduced in SR-18292 treated animals (from 55% to 20%) after 4 weeks of treatment. Complete blood cell counts (CBC) showed that SR-18292 treated mice had slightly elevated numbers of red blood cells (RBCs) and more hemoglobin, but lower counts of white blood cells, lymphocytes, and neutrophils. In addition, the RBC distribution width (RDW) was significantly reduced after SR-18292 treatment, indicating that the size of circulating RBCs was more uniform in treated mice. In addition, erythroid cell morphology was evaluated by blood smears with Wright-Giemsa stain. The number of sickled RBCs were reduced in SCD mice treated with SR-18292 for 4 weeks. This is consistent with our observation that RDW was reduced in SCD mice treated with SR-18292. Furthermore, SR-18292 treated mice had a statistically significant reduction in spleen size and weight in contrast to the spleens of untreated SCD mice. The indices of RBCs were improved in accompany with reduced hemolysis and a higher fraction of circulating mature erythrocytes in treated SCD mice. There were no observable adverse effects on other hematopoietic cell lineages. We also performed single-cell RNA-seqs in human CD34 + cells treated with SR-18292 and found that some known HbF repressors (such as BCL11A and ZBTB7A) were decreased in treated groups. Besides that, there were many differentially expressed genes (DEGs), which might be novel HbF regulators. We are verifying those DEGs genes by qPCR and using CRISPR/cas9 to examine their functions in globin regulation. In summary, our findings suggest that SR-18292 might be a promising therapeutic agent to treat SCD and modulating PGC-1α activity or the signaling pathways that it regulates might benefit therapeutically patients with SCD.

Activating transcription factor 4 in erythroid development and [Formula: see text]-thalassemia: a powerful regulator with therapeutic potential.

Activating transcription factor 4 (ATF4) is a fundamental basic region/leucine zipper transcription factor, responds to various stress signals, and plays crucial roles in normal metabolic and stress response processes. Although its functions in human health and disease are not completely understood, compelling evidence underscores ATF4 is indispensable for multiple stages and lineages of erythroid development, including the regulation of fetal liver hematopoietic stem cells, induction of terminal erythroid differentiation, and maintenance of erythroid homeostasis. [Formula: see text]-Thalassemia is a blood disorder arising from mutations in the [Formula: see text]-globin gene. Reactivating the expression of the [Formula: see text]-globin gene in adult patients has emerged as a promising therapeutic strategy for ameliorating clinical symptoms associated with [Formula: see text]-thalassemia. Recent research has suggested that ATF4 contributes to decreased fetal hemoglobin (HbF) level through its binding to potent negative regulators of HbF, such as BCL11A and MYB. Notably, evidence also suggests a contrasting outcome where increased ATF4 protein levels are associated with enhanced HbF at the transcriptional level. Consequently, the identification of mechanisms that modulate ATF4-mediated [Formula: see text]-globin transcription and its effects on erythroid development may unveil novel targets for [Formula: see text]-thalassemia treatment.

Metabolic regulation in erythroid differentiation by systemic ketogenesis in fasted mice

Erythroid terminal differentiation and maturation depend on an enormous energy supply. During periods of fasting, ketone bodies from the liver are transported into circulation and utilized as crucial fuel for peripheral tissues. However, the effects of fasting or ketogenesis on erythroid behavior remain unknown. Here, we generated a mouse model with insufficient ketogenesis by conditionally knocking out the gene encoding the hepatocyte-specific ketogenic enzyme hydroxymethylglutary-CoA synthase 2 (Hmgcs2 KO). Intriguingly, erythroid maturation was enhanced with boosted fatty acid synthesis in the bone marrow of a hepatic Hmgcs2 KO mouse under fasting conditions, suggesting that systemic ketogenesis has a profound effect on erythropoiesis. Moreover, we observed significantly activated fatty acid synthesis and mevalonate pathways along with reduced histone acetylation in immature erythrocytes under a less systemic ketogenesis condition. Our findings revealed a new insight into erythroid differentiation, in which metabolic homeostasis and histone acetylation mediated by ketone bodies are essential factors in adaptation toward nutrient deprivation and stressed erythropoiesis.

Vitamin C derivative/AA2P promotes erythroid differentiation by upregulating CA1

Abstract Vitamin C is used to treat anaemia; however, the mechanism through which vitamin C promotes erythroid differentiation is not comprehensively understood. The in vitro erythroid differentiation induction system can reveal the differentiation mechanism and provide erythrocytes for clinical transfusion and anaemia treatment. This process can be promoted by adding small-molecule compounds. In this study, we added l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P), a derivative of vitamin C, to an erythroid differentiation system induced from umbilical cord blood haematopoietic stem and progenitor cells in vitro and detected its effect on erythroid differentiation using single-cell transcription sequencing technology combined with non-targeted metabolism detection. AA2P increased the proportion of late basophilic erythroblasts, upregulating the expression of erythroid-related regulatory molecules GATA1, KLF1, ALAS2, and the globins HBG and HBB. CA1 is a target gene of AA2P, and CA1 knockdown affected the expression of globin-related genes. AA2P also increased glycolysis and decreased oxidative phosphorylation to facilitate terminal erythroid differentiation and enhanced the proliferation of early erythroid progenitors by altering the cell cycle. These results provide a reliable basis for using vitamin C to improve the efficiency of erythropoiesis in vitro and for the clinical treatment of anaemia.