Molecular and cellular mechanisms that regulate human erythropoiesis

To enable effective oxygen transport, approximately 200 billion red blood cells (RBCs) need to be produced every day in the bone marrow through the fine-tuned process of erythropoiesis. Erythropoiesis is regulated at multiple levels to ensure that defective RBC maturation or overproduction can be avoided. Here, we provide an overview of different layers of this control, ranging from cytokine signaling mechanisms that enable extrinsic regulation of RBC production to intrinsic transcriptional pathways necessary for effective erythropoiesis. Recent studies have also elucidated the importance of post-transcriptional regulation and highlighted additional gatekeeping mechanisms necessary for effective erythropoiesis. We additionally discuss the insights gained by studying human genetic variation impacting erythropoiesis and highlight the discovery of BCL11A as a regulator of hemoglobin switching through genetic studies. Finally, we provide an outlook of how our ability to measure multiple facets of this process at single-cell resolution, while accounting for the impact of human variation, will continue to refine our knowledge of erythropoiesis and how this process is perturbed in disease. As we learn more about this intricate and important process, additional opportunities to modulate erythropoiesis for therapeutic purposes will undoubtedly emerge.

Down-Regulated hsa-miR-190b-5p is Correlated With BCL11A Expression in Pediatric β-Thalassemia

Background: Transcription factor BCL11A is a key regulator of hemoglobin switching in adult β-thalassemia. Several microRNAs (miRNAs) involve in the pathology of β-thalassemia by regulations of BCL11A. However, the expressions and regulators of BCL11A in pediatric β-thalassemia were unclear. Methods: 18 pediatric β- thalassemia patients and 11 healthy controls were selected in this study. We applied reverse transcript quantitative real time PCR (RT-PCR) to analyze the expression levels of hsa-miR-190b-5p and γ-globin in pediatric β-thalassemia patients and luciferase activity assay to find out the direct regulations of BCL11A . Correlation between hsa-miR-190b-5p and biochemical indicators, BCL11A was assessed by the Pearson’s correlation test. Results: The expression levels of γ-globin in pediatric β-thalassemia patients were significantly increased. Moreover, the expression levels of hsa-miR-190b-5p were significantly down-regulated in pediatric β-thalassemia patients. Furthermore, hsa-miR-190b-5p was negatively correlated with BCL11A expression in pediatric β-thalassemia patients. Through luciferase activity assay, we found that hsa-miR-190b-5p was directly interacted with BCL11A 3’UTR 499-506 regions. Conclusion: Our results suggested that hsa-miR-190b-5p played key roles in regulating of BCL11A expression, which might provide novel therapies in pediatric patients with β-thalassemia .

HIC2 Controls Developmental Hemoglobin Switching By Repressing BCL11A Transcription

One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin production in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease (SCD) and thalassemia. BCL11A is a transcriptional repressor that is thought to directly silence the fetal β-type globin (HBG1/2) genes in adult erythroid cells. Transcriptome and RNA polymerase II profiling indicate that the BCL11A gene is transcribed considerably more highly in adult erythroblasts compared to fetal cells, accounting in large part for corresponding changes in BCL11A protein levels. Yet, the mechanism governing BCL11A developmental regulation is still unclear. To identify novel regulators of the fetal-to-adult globin switch, we interrogated our recent CRISPR based genetic screens that employed single guide RNAs (sgRNAs) targeting transcription factors (Huang et al., Blood, 2020) and uncovered HIC2, a penta-dactyl zinc finger DNA binding protein bearing a BTB/POZ domain as a novel regulator of hemoglobin switching. HIC2 is expressed more highly in fetal erythroblasts compared to adult cells, a pattern inverse to that of BCL11A. Overexpression (OE) of HIC2 in the adult type erythroid HUDEP2 cell line stimulated the expression of 322 genes while impairing that of 224 genes (FDR < 0.01 and fold change ≥ 2). The most highly upregulated genes (>150-fold) were HBG1/2. Upregulation was accompanied by gains in chromatin accessibility and histone H3K27acetylation of HBG1/2, and increased chromatin contacts between the distal globin gene enhancer (LCR) and the HBG1/2 genes. Overexpression of HIC2 in primary human erythroblasts also significantly increased HBG1/2 mRNA and protein levels, sufficient to reduce cell sickling in SCD patient-derived erythroid cells. HIC2 OE lowered BCL11A mature and pre-mRNA production, indicating that HIC2 attenuates BCL11A transcription. Forced expression of BCL11A restored HBG1/2 silencing in HIC2 OE HUDEP2 cells, suggesting that BCL11A repression accounts for the effects of HIC2 on fetal globin genes. ChIP-seq revealed a strong HIC2 binding peak at the erythroid BCL11A +55 enhancer. HIC2 OE reduced chromatin accessibility and H3K27acetylation of the +55 enhancer, as well as the enhancer-promoter contacts, suggesting that HIC2 directly decommissions the enhancer to attenuate BCL11A transcription. The BCL11A +55 enhancer contains two consensus HIC2 binding motifs under the HIC2 peak adjacent to GATA:E-box and GATA motifs. CRISPR-mediated mutagenesis of both HIC2 motifs raised BCL11A basal level transcription and diminished the ability of overexpressed HIC2 to repress BCL11A transcription. Notably, HIC2 OE impaired binding of transcription factor GATA1 at the +55 enhancer, suggesting that this enhancer is under developmental control. Indeed, GATA1 binding and chromatin accessibility of +55 enhancer were virtually undetectable in HUDEP1 cells, which represent a more fetal-like state. CRISPR-mediated depletion of HIC2 in HUDEP1 cells reversed this pattern with gains in GATA1 binding, chromatin accessibility, and BCL11A transcription. In sum, HIC2 emerges as a critical regulator of hemoglobin switching that operates by imposing developmental stage-specific control onto a BCL11A transcriptional enhancer. Disclosures Blobel: Fulcrum therapeutics: Consultancy; Pfizer: Research Funding.

BCL11A promotes myeloid leukemogenesis by repressing PU.1 target genes

The transcriptional repressor, BCL11A, is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase, Trib1, in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells both in vitro and in vivo. ChIP-seq analysis showed that upon DNA-binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by both sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components, HDAC and LSD1, reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor, pracinostat, and LSD1 inhibitor, GSK2879552, resulted in growth inhibition both in vitro and in vivo. High BCL11A expression is associated with worse prognosis in human AML patients. Blocking of BCL11A expression upregulates the expression of PU.1 target genes, and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

A unified model of human hemoglobin switching through single-cell genome editing

Key mechanisms of fetal hemoglobin (HbF) regulation and switching have been elucidated through studies of human genetic variation, including mutations in the HBG1/2 promoters, deletions in the β-globin locus, and variation impacting BCL11A. While this has led to substantial insights, there has not been a unified understanding of how these distinct genetically-nominated elements, as well as other key transcription factors such as ZBTB7A, collectively interact to regulate HbF. A key limitation has been the inability to model specific genetic changes in primary isogenic human hematopoietic cells to uncover how each of these act individually and in aggregate. Here, we describe a single-cell genome editing functional assay that enables specific mutations to be recapitulated individually and in combination, providing insights into how multiple mutation-harboring functional elements collectively contribute to HbF expression. In conjunction with quantitative modeling and chromatin capture analyses, we illustrate how these genetic findings enable a comprehensive understanding of how distinct regulatory mechanisms can synergistically modulate HbF expression.

Gene Editing of KLF1 to Cure Sickle Cell Disease

Sickle cell disease (SCD) affects millions of people worldwide and represents the most common monogenic disease of mankind (1). It is due to a homozygous T to A transversion in the β-globin gene that results in an amino acid variant - G6V - and production of HbS, which polymerises in red blood cells (RBCs) under hypoxic conditions. This generates irreversibly sickled cells that fail to traverse the microcirculation, resulting in micro-infarcts, hypoxia and pain, or 'sickle cell crises'. During gestation RBCs utilise different sets of globin genes to produce embryonic and fetal hemoglobins (HbF), so it is not until after birth when adult hemoglobin (HbA) is first produced that the first signs of SCD become apparent. This process termed 'hemoglobin switching' has been the focus of research efforts for decades because it offers an opportunity to reactivate HbF in adult cells of patients with hemoglobinopathies. A number of transcription factors, including Krüppel-like factor 1 (KLF1), play critical roles in hemoglobin switching. KLF1 is an essential erythroid transcription factor that co-ordinates the expression of more than a thousand genes critical to the formation of adult RBCs. KLF1 directly binds the β-globin gene promoter to up regulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin expression (HbF). Heterozygosity for loss of function mutations in KLF1 leads to a significant increase in HbF that is beneficial to patients with β-thalassemia. We propose this can be recreated by advanced gene editing techniques to provide an effective therapy for SCD. We have employed CRISPR-based gene editing to knockout the expression of KLF1 in human cells. We designed two separate sgRNAs with corresponding HDR templates to target the second exon of KLF1 and ablate its function. We optimised transfection protocols and tested the on-target specificity of our sgRNAs achieving >90% efficacy in all cell types assayed. Using HUDEP-2 cells (2), a conditionally immortalised erythroid cell line which harbors three copies of KLF1 (3), we have demonstrated that these cells require at least one copy (>1/3) for survival; heterozygous cells (+/-/- or +/+/-) proliferate at a reduced rate, but are able to differentiate normally. Using RNA-seq, we identified some genes, including ICAM-4 and BCAM, which are down-regulated accordingly in a KLF1 gene dosage-dependent manner. ICAM-4 and BCAM are cellular adhesion molecules implicated in triggering vaso-occlusive episodes (4; 5), so it is anticipated their reduced expression may provide additional benefit in treating SCD. Gamma-globin is upregulated 10-fold, BCL11A down-regulated 3-fold, and HbF+ RBCs generated at ~20% of total RBCs in KLF1 +/-/- HUDEP-2 cell lines. We also engineered the ablation of KLF1 in CD34+ cells harvested from the peripheral blood of SCD patients undergoing exchange transfusions. Following transfection of the two guides, we performed directed differentiation using an erythroid differentiation medium and analysed the levels of HbF. We observed HbF at levels of between 40-60% of total Hb by HPLC, and HbF+ cells of ~50% by FACS. There was no measurable block in erythroid differentiation by FACS. We documented the types of gene editing using a high throughout NGS assay (6). We compared efficiencies of CRISPR repair of the HbS mutation with CRIPSR damage of the KLF1 gene. Lastly, we transplanted gene-edited CD34 cells into NSGW41 mice (where human erythropoiesis is established) to determine the efficiency and safety of editing long term HSCs from SCD patients. We will report on the results of these xenotransplantation assays. Taken together these results reveal the potential utility in targeting KLF1 to cure SCD. References: Wastnedge, E. et al..J Glob Health 8, 021103 (2018). Kurita, R. et al.PLoS One 8, e59890 (2013). Vinjamur, D. S. & Bauer, D. E. Methods Mol Biol 1698, 275-284 (2018). Bartolucci, P. et al..Blood 116, 2152-9 (2010). Zhang, J., et al. PLoS One 14, e0216467 (2019). Bell, C. C., et al. BMC Genomics 15, 1002 (2014). Perkins, A. et al..Blood 127, 1856-62 (2016). Disclosures Kaplan: Celgene: Honoraria; Novartis: Honoraria. Perkins:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Mild dyserythropoiesis and β-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.