Human Erythroid Progenitor Cells Research Articles

Long noncoding RNA GATA2AS influences human erythropoiesis by transcription factor and chromatin landscape modulation

AbstractLong noncoding RNAs (lncRNAs) are extensively expressed in eukaryotic cells and have been revealed to be important for regulating cell differentiation. Many lncRNAs have been found to regulate erythroid differentiation in the mouse. However, given the low sequence conservation of lncRNAs between mouse and human, our understanding of lncRNAs in human erythroid differentiation remains incomplete. lncRNAs are often transcribed opposite to protein coding genes and regulate their expression. Here, we characterized a human erythrocyte-expressed lncRNA, GATA2AS, which is transcribed opposite to erythroid transcription regulator GATA2. GATA2AS is a 2080-bp long, primarily nucleus-localized noncoding RNA that is expressed in erythroid progenitor cells and decreases during differentiation. Knockout of GATA2AS in human HUDEP2 erythroid progenitor cells using CRISPR-Cas9 genome editing to remove the transcription start site accelerated erythroid differentiation and dysregulated erythroblast gene expression. We identified GATA2AS as a novel GATA2 and HBG activator. Chromatin isolation by RNA purification showed that GATA2AS binds to thousands of genomic sites and colocalizes at a subset of sites with erythroid transcription factors including LRF and KLF1. RNA pulldown and RNA immunoprecipitation confirmed interaction between GATA2AS and LRF and KLF1. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that knockout of GATA2AS reduces binding of these transcription factors genome wide. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq showed that GATA2AS is essential to maintain the chromatin regulatory landscape during erythroid differentiation. Knockdown of GATA2AS in human primary CD34+ cells mimicked results in HUDEP2 cells. Overall, our results implicate human-specific lncRNA GATA2AS as a regulator of erythroid differentiation by influencing erythroid transcription factor binding and the chromatin regulatory landscape.

Long Non-Coding RNA H19 Leads to Upregulation of γ-Globin Gene Expression during Erythroid Differentiation

Long noncoding RNAs (lncRNAs) are important because they are involved in a variety of life activities and have many downstream targets. Moreover, there is also increasing evidence that some lncRNAs play important roles in the expression and regulation of γ-globin genes. In our previous study, we analyzed genetic material from nucleated red blood cells (NRBCs) extracted from premature and full-term umbilical cord blood samples. Through RNA sequencing (RNA-Seq) analysis, lncRNA H19 emerged as a differentially expressed transcript between the two blood types. While this discovery provided insight into H19, previous studies had not investigated its effect on the γ-globin gene. Therefore, the focus of our study was to explore the impact of H19 on the γ-globin gene. In this study, we discovered that overexpressing H19 led to a decrease in HBG mRNA levels during erythroid differentiation in K562 cells. Conversely, in CD34+ hematopoietic stem cells and human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, HBG expression increased. Additionally, we observed that H19 was primarily located in the nucleus of K562 cells, while in HUDEP-2 cells, H19 was present predominantly in the cytoplasm. These findings suggest a significant upregulation of HBG due to H19 overexpression. Notably, cytoplasmic localization in HUDEP-2 cells hints at its potential role as a competing endogenous RNA (ceRNA), regulating γ-globin expression by targeting microRNA/mRNA interactions.

An Efficient Protocol for Isolation, Purification, and Characterization of Human Erythroid Progenitor Cells from Peripheral Blood Samples of Healthy Adult Volunteers

An Efficient Protocol for Isolation, Purification, and Characterization of Human Erythroid Progenitor Cells from Peripheral Blood Samples of Healthy Adult Volunteers

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.

Early Response Transcription Factors in Erythroid Regeneration

Acute anemia activates distinct mechanisms from physiological erythropoiesis to increase red blood cell production. While erythroid regeneration in this context requires some critical cytokines (e.g., BMP-4), little is known about transcription factors (TFs) connecting growth factor stimulation to anemia-specific gene expression and cellular responses. We previously discovered that GATA1 and TAL1 control a cohort of composite cis-elements which are anemia-activated in erythroid progenitors and precursors during regeneration. Intriguingly, cis-regulatory activity and gene activation at these loci are delayed by several days following initial anemia onset by the red blood cell lysing chemical phenylhydrazine. This delay suggested that these elements were utilized for erythroid precursor expansion but not activation. We hypothesize that distinct TF mechanisms are early responders to erythroid regeneration. To identify early anemia response TFs, we compared transcript levels in mouse spleen Kit + erythroid progenitors at days 0 and 1 post-anemia (anemia upregulated genes; p<0.05, fold change>1.5). Among upregulated genes, only 10 were TFs, and 3 of them ( Jun, Junb, Fos) are components of Activator protein 1 (AP-1) transcription factor. All 3 transcripts were subsequently downregulated at 3 days after anemia. In physiological hematopoiesis, AP-1 is highly expressed in most cells but low within the erythroid lineage. These findings suggested transient anemia-activated AP-1 may initiate erythroid regeneration. While AP-1 is a big family, only JUN and FOS family exhibited transient anemia-activation. Genes containing AP-1 binding motifs were cell intrinsically upregulated post-anemia in Jun and Fos-expressing cells. To determine Jun TF activity in anemia, we performed phospho-flow cytometry in Lin- spleen cells from mice after inducing anemia. We discovered that at Day 1 vs Control, the median fluorescence intensity (MFI) of Kit +CD71 +Ter119 + cells stained with total Jun or phospho-Jun (p-Jun) antibodies increased 2.0-fold (p=0.03) and 2.9-fold (p=0.04), respectively. Concurring with transcript levels, both total and p-Jun protein increased between 6- and 24-hours post-anemia and decreased at 72 hours. These changes post-anemia were absent in non-erythroid (CD71 -Ter119 -) and Kit - cells. These data demonstrated a transient increase in JUN TF activity early during the anemia response in erythroid progenitor/precursors. AP-1 activity is commonly elevated in response to a variety of stressors, including hypoxia and inflammation. Given the limited sets of transcripts upregulated at early anemia time points, direct AP-1 targets may be required for establishing an anemia-dependent transcriptome. To identify AP-1 target genes in erythroid regeneration, we selected genetic loci nearby (distance <100kb) AP-1-occupied cis-elements in human erythroleukemia (K562) or mouse erythroid progenitor cells (GEO: GSE31477, 208960, 170378, 172818). 50 potential AP-1 target genes were upregulated in stress erythroid precursors on day 3 vs day 1 post-anemia. Seven anemia-regulated transcription factors (SOX6, E2F2, TAL1, TFDP2, FOXO3, GFI1B, MAFG) reside at loci containing candidate AP-1 cis-elements. The T-cell Acute Lymphoblastic Leukemia-1 (TAL1) TF cooperates with GATA1 at E-box-GATA composite cis-elements to promote transcription of a number of erythroid-specific genes, several of which are needed for effective erythroid regeneration. Knockdown approaches will be used to test whether Jun expression and activity promotes the anemia-dependent transcriptome, stress erythroid precursor differentiation, and cell survival during erythroid regeneration. Given that Jun can be activated by BMP-4 signaling via the Jun kinase (JNK) pathway, and the requirement for BMP-4 in erythroid regeneration, we are investigating whether BMP-4 stimulation (or other cytokines) phosphorylates Jun to establish the regenerative transcriptome at early time points post-anemia. These studies will improve our understanding of transcriptional mechanisms driving erythroid regenerative responses, which can be useful for identifying new therapeutic targets for acute/chronic anemias.

Erythroid Progenitor Cells in the Murine Bone Marrow: Parallels with Human Counterparts and Response to Acute Anemia

In mammals, the erythron consists of lineage-specific progenitor cells that transition to morphologically identifiable precursors that ultimately enucleate to become mature red blood cells. Early- and late-stage erythroid progenitors, BFU-E and CFU-E respectively, were originally defined by their capacity to generate colonies of erythroid cells in semisolid media. We have refined a flow cytometry-based strategy based on Pronk, et al. (2007) and Tusi, et al. (2018) that parallels similar strategies developed for human erythroid progenitor cells (Yan et al., 2021) to facilitate identification and isolation of progressive stages of primary murine erythroid progenitors, termed EP1 to EP4, in the bone marrow. Sorted erythroid progenitor (EP1 to EP4) populations were cultured in colony-forming assays. EP1 consists almost entirely of BFU-E, while EP4 was highly enriched in CFU-E, and the intermediate EP2 and EP3 populations progress from immature to more mature erythroid colony-forming progenitors. Global RNA-Seq studies were performed on three replicates of primary bone marrow EP1-4 from 9-12 week old outbred mice. EP1/2 and EP3/4 each shared the most similarities, consistent with the enrichment of BFU-E in the former and CFU-E in the latter. We also analyzed the global transcriptome of EP1-4 derived from in vitro culture of human CD34+ hematopoietic stem and progenitor cells to compare with the murine counterparts. Approximately two-thirds of differentially expressed genes displayed similar patterns as murine and human EP1 transition progressively to EP4. The largest transition in gene expression occurred between EP2 and EP3. Analysis of genes differentially expressed between EP2 and EP3 both in human and in mouse (adj. p-value <0.05, 361 down and 242 up) revealed marked downregulation of transcription factors expected from previous studies of erythroid progenitors (Gata2, Cd34), as well as those associated with multipotential, myeloid, and megakaryocyte lineages (Sp1 and Runx1). Upregulated transcription factors included prototypical factors associated with erythropoiesis (Klf1, Gata1 and Tal1), as well as Srebf1, a central regulator of cholesterol homeostasis. In both species the transition from EP2 to EP3 was characterized by downregulation of the gene ontology terms associated with myeloid and megakaryocyte development, and the upregulation of gene ontology terms associated with erythrocyte development, cell cycle, heme metabolism, and VLDL particle assembly. Interestingly, cholesterol content increases as EP1/2 transition to EP3/4 in both primary murine and human bone marrow. These data indicate a shared progression of erythroid progenitors can be proactively delineated in human and mouse systems. To better define the response of erythroid progenitors to stress, we established an acute anemia model in mice, reproducibly lowering the hematocrit by half via phlebotomy. Induction of acute anemia leads to a marked expansion of EP3 and EP4 at 48 hours both in the bone marrow and in the spleen. Additionally, these expanded EP3/4 populations were characterized by an increase percentage of the cells in S-phase. Principal component analysis of global transcriptomic studies of EP1-4 isolated from the marrow 48 hours after the induction of anemia revealed parallel differentiation programs when compared to their steady-state murine counterparts. 70% of the shared genes upregulated in the transition from EP2 to EP3 were upregulated to a greater extent in response to anemia. Upregulated genes, particularly in EP3/4, included known EPO-responsive genes (Pim1, Socs2, Tfrc, and Cited4), as well as hypoxia-associated genes. Interestingly, key cholesterol biosynthesis genes (Hmgcr, Hmgcs1, and Sqle) were also further upregulated in anemic versus steady-state EP3/4. This latter finding suggests that cholesterol homeostasis may play a key role in the erythroid progenitor response to stress. We conclude that late-stage erythroid progenitor (EP3/4) expansion is a specific and integral component of regenerative erythropoiesis. The identification and isolation of stage-specific murine erythroid progenitor cells that are highly analogous to their human counterparts will facilitate an improved understanding of normal and disordered erythropoiesis.

Dynamic Changes in Lysine Succinylation As Important Regulators of Erythropoiesis

Lysine succinylation has emerged as a recently discovered protein modification that significantly impacts the chemical environment and exhibits diverse functions in various biological processes. However, the specific role of lysine succinylation in erythropoiesis has not been fully elucidated. In this study, we investigated the levels of six common acylations (acetylation, crotonylation, succinylation, propionylation, butyrylation, and malonylation) in human erythroid cells. Interestingly, we observed a prominent accumulation of lysine succinylation during human erythroid differentiation, suggesting its potential importance in this process. To explore the functional significance of succinylation, we inhibited succinylation in human erythroid progenitor cell line by disrupting the expression of the key succinyltransferases and desuccinylases. The results revealed that succinylation inhibition led to suppressed cell proliferation, increased apoptosis, and disrupted differentiation, indicating the essential role of succinylation in erythropoiesis. Furthermore, integrative proteome and succinylome analysis identifies 939 quantifiable proteins with 2,871 Ksu sites. Notably, we observed inconsistencies between alterations in protein levels and succinylation levels, suggesting that the role of succinylation in proteins' function regulation. These succinylated proteins are widely distributed in various cellular compartments and involved in multiple cell processes, indicating that succinylation is a prevalent modification in erythropoiesis. Mechanically, we identified CYCS as a key target of succinylation during erythropoiesis, emphasizing its essential role in this process. Specially, we implicated KAT2A-mediated histone succinylation in chromatin remodeling, further highlighting the regulatory significance of lysine succinylation in erythropoiesis at the epigenetic level. Collectively, our comprehensive investigation of the succinylation landscape during erythropoiesis provides valuable insights into its regulatory role and offer potential implications for erythroid-related diseases and therapeutic strategies.

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.

Proteomics screening uncovers HMGA1 as a promising negative regulator for γ-globin expression in response to decreased β-globin levels

Reactivation of fetal hemoglobin (HbF) is a critical goal for the treatment of patients with hemoglobinopathies. β-globin disorders can trigger stress erythropoiesis in red blood cells (RBCs). Cell-intrinsic erythroid stress signals promote erythroid precursors to express high levels of fetal hemoglobin, which is also known as γ-globin. However, the molecular mechanism underlying γ-globin production during cell-intrinsic erythroid stress remains to be elucidated. Here, we utilized CRISPR-Cas9 to model a stressed state caused by reduced levels of adult β-globin in HUDEP2 human erythroid progenitor cells. We found that a decrease in β-globin expression correlates with the upregulation of γ-globin expression. We also identified transcription factor high-mobility group A1 (HMGA1; formerly HMG-I/Y) as a potential γ-globin regulator that responds to reduced β-globin levels. Upon erythroid stress, there is a downregulation of HMGA1, which normally binds −626 to −610 base pairs upstream from the STAT3 promoter, to downregulate STAT3 expression. STAT3 is a known γ-globin repressor, so the downregulation of HMGA1 ultimately upregulates γ-globin expression. SignificanceThis study demonstrated HMGA1 as a potential regulator in the poorly understood phenomenon of stress-induced globin compensation, and after further validation these results might inform new strategies to treat patients with sickle cell disease and β-thalassemia.

Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors.

Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD=103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.

Deficiency of ribosomal protein S26, which is mutated in a subset of patients with Diamond Blackfan anemia, impairs erythroid differentiation.

Introduction: Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis. Results and discussion: Here we characterized the phenotype of RPS26-deficiency in a cell line derived from human umbilical cord blood erythroid progenitors (HUDEP-1 cells). This model recapitulates cellular hallmarks of Diamond Blackfan anemia including: imbalanced production of ribosomal RNAs, upregulation of pro-apoptotic genes and reduced viability, and shows increased levels of intracellular calcium. Evaluation of the expression of erythroid markers revealed the impairment of erythroid differentiation in RPS26-silenced cells compared to control cells. Conclusions: In conclusion, for the first time we assessed the effect of RPS26 deficiency in a human erythroid progenitor cell line and demonstrated that these cells can be used as a scalable model system to study aspects of DBA pathophysiology that have been refractory to detailed investigation because of the paucity of specific cell types affected in this disorder.

Elucidation of the Role of FAM210B in Mitochondrial Metabolism and Erythropoiesis.

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.

Development of the Novel Cytosine Analog Aza-T-Dcyd (AB1) As a Fetal Hemoglobin Inducer to Treat Sickle Cell Disease.

Introduction: Induction of fetal hemoglobin (HbF) expression in sickle cell disease (SCD) patients using small molecules is considered an effective therapeutic strategy to ameliorate severe clinical phenotypes. With this objective in mind, several drug molecules have been tested for HbF induction. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. Studies have reported that DNA methyltransferase inhibitors such as decitabine stimulate γ-globin gene expression through DNA hypomethylation. However, decitabine has cytotoxic side effects and is most effective by intravenous administration. Thus, it is critical to develop alternative safe and orally active hypomethylating agents for inducing HbF. Our lab in collaboration with AkiraBio, conducted preclinical in vitro studies to assess a novel cytosine analog Aza-T-dCyd (AB1), for its ability to induce HbF in human K562 cells and erythroid progenitor cells derived from SCD patients. Methods: K562 cells were cultured in Iscove's Modified Dulbecco's Eagle medium, 10% fetal bovine serum and penicillin/streptomycin. The human erythroid progenitors were generated from peripheral blood mononuclear cells isolated from two SCD patients under IRB exempt protocol as previously established in our lab (Li et al., Haematologica. 2018). The cultured K562 cells were treated either with AB1 alone at gradually increasing concentrations from 3nm to 200nm, decitabine (0.2µM), hydroxyurea (HU100µM) or combined AB1 with HU for 48 hours. The cultured sickle erythroid progenitors were treated with AB1 on day 8 and then harvested on day 10. The harvested cells were analyzed for cell viability and flow cytometry to measure HbF positive cells (F-cells). The γ-globin and β-globin mRNA levels were quantified by qRT-PCR and the protein levels of HbF and DNMT-1 were detected by Western blot technique. Results: Initial studies to evaluate drug toxicity were completed. We observed higher cell viability of K562 cells treated with AB1 10nM and 30nM compared to untreated and HU 100µM (p=0.005) treated cells. Hence, subsequent experiments were conducted with the lower AB1 concentrations. By flow cytometry analysis, AB1 treatment significantly increased F-cells from 13.95% to 20.11% and 22.46% in 10nM and 30nM treated cells (p=0.0002) respectively. Moreover, mean fluorescence intensity analysis showed significant HbF expression in AB1 treated cells compared to untreated cells (p=0.00001). At the transcription level, AB1 increased γ-globin mRNA levels 1.4-fold compared to untreated cells. K562 cells treated with a combination of AB1 and HU at their maximum response concentrations (AB1 10 and 30nM; HU 100 µM) did not show additive effects on F-cells or γ-globin mRNA expression. Other combination concentrations were not assessed. In subsequent studies we investigated the HbF inducing capacity of AB1 in human erythroid progenitors generated from SCD patients (N=2). Normal cell viability was observed in sickle progenitors at 3nm, 6nM and 10nm AB1 treatment. The F-cell level increased from 16.21% to 27.68% at 10nm AB1 (p=0.02). By qRT-PCR assay, a significant 2-fold increase in γ-globin mRNA level was observed in AB1 treated erythroid cells, when normalized with total γ-globin and β-globin mRNA; β globin mRNA levels were unchanged. Finally, when we analyze the effects of AB1 on protein levels of HbF and DNMT-1 by Western blot analysis, 10nM and 30nM AB1 induced HbF and inhibited DNMT-1 expression the sickle erythroid progenitors. Conclusion: Our study findings demonstrate that AB1 induced γ-globin transcription in K562 cells and human sickle erythroid progenitors. Studies to test the effects of AB1 in the Townes SCD mouse model are in progress.

Characterization of Hemoglobin Variants Due to in-Frame Deletions in the β-Globin Gene from CRISPR/Cas9 Gene Correction for Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene (HBB). CRISPR/Cas9 based gene correction approaches rely on inducing a double-strand break (DSB) at or near the sickle mutation, which subsequently results in gene correction via the homology-directed repair (HDR) or insertions/deletions (Indels) via non-homologous end joining (NHEJ). Frameshift Indels induce a premature stop codon and disrupt HBB production. Indels that preserve the reading frame may yield structurally abnormal β-globin chain with altered amino acid sequences and protein structures that can affect the globin chain stability, leading to thalassemia of uncertain pathogenic significance. The high rate and diversity of in-frame Indels as an unintended by-product of gene correction therapy confers a challenge for predicting therapeutic outcomes when it is not possible to frame the clinical behavior of hemoglobin (Hb) variants. Here we provided the molecular and structural characterization of the most commonly identified Hb variants due to CRISPR-induced in-frame deletions and proposed a putative pathogenic mechanism for the observed phenotype using human umbilical cord-derived erythroid progenitor cells (HUDEP2). Methods: HUDEP2 clonal cell lines carrying mutated Hb variants from in-frame deletions on HBB exon1 introduced by R-66S and R-02 CRISPR sgRNAs currently in use in preclinical and clinical studies for correcting sickle mutation, respectively, were established, and clonal genotypes validated by next-generation sequencing and droplet digital PCR based copy number assay. The Hb variants derived from R-66S include p.Val7_Ser10del (-12bp), p.Val7del (-3bp), p.Glut8del (-3bp), p.Glut8_Lys9del (-6bp), pVal7Thr13del (-21bp), p.His3_Lys9delinsGln (-18bp), and p.Thr5_Lys18del (-42bp), in the order of allelic abundance observed in the bulk population of edited cells. The Hb variants derived from R-02 include p.Val12_Ala14del (-9bp), p.Val12_Thr13del (-6bp), p.Ala14del (-3bp), p.Thr13_Gly17del (-15bp), and p.Thr13_Val19delinsMet (-18bp). Selected ten clonal cell lines carrying a unique combination of genotypes and the parent HUDEP2 were cultured for expansion in serum-free media for one week. All clones were subsequently subjected to erythroid differentiation media for ten days. Samples were collected on days 4, 7, and 10 of differentiation for analysis via high-performance liquid chromatography (HPLC) using an ionic column to access the Hb profile and spectrophotometry via a Hb assay kit to assess the total Hb quantity. Results: HPLC analysis demonstrated a significant qualitative variation in Hb profile in HUDEP2 clones carrying different Hb variants. p.Val7del is Hb Leiden, and the rest of Hb variants are not listed in the HbVar database. In clones carrying Hb Leiden in homozygous or heterozygous states, Hb Leiden eluted in the position of sickle Hb (HbS). A low percentage of multiple abnormal Hb X fractions was observed in all clones carrying Hb variants compared to parent HUDEP2, which hinted at the presence of unstable Hb variants or alpha precipitates. Levels of fetal Hb (HbF) increased in mutant clones, suggesting globin chain imbalance and hematopoietic stress. Clones missing multiple amino acids showed ineffective erythropoiesis and cell death by day 4 of differentiation, most likely exacerbated by the relative excess of the α-chains. Discussion: We found that unintended gene modifications can significantly impact Hb quality and quantity, reinforcing concerns about the safety of gene-editing therapy for SCD. In patients, cells carrying in-frame deletions in the β-globin chain could produce an unstable Hb and may contribute to a β-thalassemia-like phenotype. More work is required to further assess the consequences of the diverse Hb variants in combination with other unintended modifications that disrupt HBB in a bulk population of gene-edited cells. Detecting, profiling, and reducing these unintended modifications remains a priority to increase the efficacy and safety of gene-editing therapy for treating SCD or β-thalassemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IOX1 Fails to Reduce α-Globin and Mediates γ-Globin Silencing in Adult β0-Thalassemia/Hemoglobin E Erythroid Progenitor Cells

The accumulation of unbound α-globin chains in red blood cells is a crucial pathophysiology of β-thalassemia. IOX1 (5-carboxy-8-hydroxyquinoline) is a broad-spectrum 2-oxoglutarate (2OG)-dependent oxygenase inhibitor that can reduce α-globin mRNA expression in human cord blood erythroid progenitor cells. Therefore, IOX1 has been proposed as a potential compound for β-thalassemia treatment through the decrease in α-globin chain synthesis. However, there is no empirical evidence regarding the consequences of IOX1 in β-thalassemia. In this study, the therapeutic effects of IOX1 were investigated in β0-thalassemia/hemoglobin E (HbE) erythroid progenitor cells during in vitro erythropoiesis. The results indicated that IOX1 had no impact on α-globin gene expression, but it led instead to significant decreases in γ-globin and fetal hemoglobin (HbF, α2γ2) production without affecting well-known globin regulators: KLF1, BCL11A, LRF, and GATA1. In addition, differential mRNA expression of several genes in the hypoxia response pathway revealed the induction of EGLN1, the PHD2-encoding gene, as a result of IOX1 treatment. These findings suggested that IOX1 fails to lower α-globin gene expression; on the contrary, it mediates γ-globin and HbF silencing in β0-thalassemia/HbE erythroid progenitor cells. Because of the negative correlation of EGLN1 and γ-globin gene expression after IOX1 treatment, repurposing IOX1 to study the hypoxia response pathway and γ-globin regulation may provide beneficial information for β-thalassemia.

Prime Editor 3 Mediated Beta-Thalassemia Mutations of the HBB Gene in Human Erythroid Progenitor Cells

Recently developed Prime Editor 3 (PE3) has been implemented to induce genome editing in various cell types but has not been proven in human hematopoietic stem and progenitor cells. Using PE3, we successfully installed the beta-thalassemia (beta-thal) mutations in the HBB gene in the erythroid progenitor cell line HUDEP-2. We inserted the mCherry reporter gene cassette into editing plasmids, each including the prime editing guide RNA (pegRNA) and nick sgRNA. The plasmids were electroporated into HUDEP-2 cells, and the PE3 modified cells were identified by mCherry expression and collected using fluorescence-activated cell sorting (FACS). Sanger sequencing of the positive cells confirmed that PE3 induced precise beta-thal mutations with editing ratios from 4.55 to 100%. Furthermore, an off-target analysis showed no unintentional edits occurred in the cells. The editing ratios and parameters of pegRNA and nick sgRNA were also analyzed and summarized and will contribute to enhanced PE3 design in future studies. The characterization of the HUDEP-2 beta-thal cells showed typical thalassemia phenotypes, involving ineffective erythropoiesis, abnormal erythroid differentiation, high apoptosis rate, defective alpha-globin colocalization, cell viability deterioration, and ROS resisting deficiency. These HUDEP-2 beta-thal cells could provide ideal models for future beta-thal gene therapy studies.

Multi-Omics Analysis in β-Thalassemia Using an HBB Gene-Knockout Human Erythroid Progenitor Cell Model.

β-thalassemia is a hematologic disease that may be associated with significant morbidity and mortality. Increased expression of HBG1/2 can ameliorate the severity of β-thalassemia. Compared to the unaffected population, some β-thalassemia patients display elevated HBG1/2 expression levels in their red blood cells. However, the magnitude of up-regulation does not reach the threshold of self-healing, and thus, the molecular mechanism underlying HBG1/2 expression in the context of HBB-deficiency requires further elucidation. Here, we performed a multi-omics study examining chromatin accessibility, transcriptome, proteome, and phosphorylation patterns in the HBB homozygous knockout of the HUDEP2 cell line (HBB-KO). We found that up-regulation of HBG1/2 in HBB-KO cells was not induced by the H3K4me3-mediated genetic compensation response. Deletion of HBB in human erythroid progenitor cells resulted in increased ROS levels and production of oxidative stress, which led to an increased rate of apoptosis. Furthermore, in response to oxidative stress, slower cell cycle progression and proliferation were observed. In addition, stress erythropoiesis was initiated leading to increased intracellular HBG1/2 expression. This molecular model was also validated in the single-cell transcriptome of hematopoietic stem cells from β-hemoglobinopathy patients. These findings further the understanding of HBG1/2 gene regulatory networks and provide novel clinical insights into β-thalassemia phenotypic diversity.

Interrogating Post-Transcriptional Mechanisms of Fetal Hemoglobin Regulation

Elevated levels of fetal hemoglobin (HbF) significantly ameliorate clinical outcomes for patients with beta-hemoglobinopathies, such as sickle cell disease (SCD). The only FDA-approved drug for treating SCD through inducing HbF is hydroxyurea, however the mechanism of action is unknown with variable effectiveness among patients. Thus, there remains a strong interest to identify more robust means of upregulating HbF, such as specific inhibition of HbF repressors. BCL11A and LRF are well-characterized transcription factors that independently repress the fetal type b-globin like genes HBG1 and HBG2 but their therapeutic potential is limited by challenging druggability and critical developmental function. However, upstream regulation of these factors, such as post-transcriptional mechanisms, are not well studied and may house novel therapeutic targets. To this end, we employed a CRISPR/Cas9 based screening approach to interrogate a library of RNA binding proteins (RBP) in the context of HbF regulation. Using HUDEP2 cells, a human adult-type erythroid progenitor cell line, we screened 341 human RBPs and identified four candidate RBPs, none of which have previously been implicated in HbF regulation. Of these candidates, RNA Binding Motif 12 (RBM12) showed the greatest level of HbF induction following in vitro depletion.Depletion of RBM12 protein in HUDEP2 cells and human CD34 + hematopoietic stem and progenitor cells (HSPC) via CRISPR/Cas9 editing raised HbF production 2-4 fold as assessed by HbF flow cytometry, HBG1/2 mRNA, and protein (γ-globin). Cell viability and maturation of RBM12 perturbed cells were largely intact. Additionally, RBM12 depletion in CD34 + HSPCs derived from SCD patients resulted in reduced percentage of sickled cells under hypoxic conditions. Unexpectedly, reduction of RBM12 had minimal effect on BCL11A and LRF expression suggesting that RBM12 may regulate HbF through a pathway that is indirectly related or independent of these transcription factors.RBM12 is an RBP that is widely expressed across diverse cell types and contains multiple RNA recognition motifs (RRM). While it has been implicated in various cancers and neurological disorders, its functions are not well studied. As an RBP, RBM12 can carry out several roles of post-transcriptional regulation, such as pre-mRNA splicing, mRNA transport, stabilization, and translation. As these activities are executed in different cellular compartments, we set out to narrow down RBM12 function by assessing its subcellular localization. Immunofluorescence staining revealed strong nuclear presence of RBM12, suggesting that it functions via mRNA biogenesis and/or processing. RNASeq and LC-MS/MS analysis of RBM12 KO CD34 + HSPCs revealed modest changes in the transcriptome and proteome. In order to gain mechanistic insight into RBM12 in the context of HbF regulation, we performed cDNA rescue experiments in RBM12-deficient HUDEP2 clones. Overexpression of full length RBM12 restored HbF repression. Notably, four out of the five RRMs were dispensable for HbF silencing, but RRM1 was essential for this activity. Interestingly, an extended form of RRM1 was also sufficient for HbF silencing. Mechanistic studies of this RRM1 module are underway and will be discussed.In sum, the identification of RBM12 as a regulator of HbF production represents a previously undescribed post-transcriptional layer of hemoglobin gene regulation. In pursuing this path, we hope to gain a deeper understanding of this understudied RBP in the context of HbF regulation which might in turn lead to the identification of potential therapeutic targets for the treatment of SCD and other hemoglobinopathies. DisclosuresBlobel: Pfizer: Consultancy; Fulcrum Therapeutics, Inc.: Consultancy.

High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 μM) without prominent cytotoxicity (CC50 = 71.8 μM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.