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.

C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies.

In humans, specific aberrations in ß-globin results in sickle cell disease and ß-thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation. Here we briefly discuss the effects of these regulators, particularly C2H2 ZFs, in inducing HbF expression for treating ß-hemoglobin disorders, together with recent advances in developing safe and effective small-molecule therapeutics for the regulation of this well-conserved hemoglobin switch.

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating ß-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and ß-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating ß-hemoglobinopathies.

Effects of Mithramycin on BCL11A Gene Expression and on the Interaction of the BCL11A Transcriptional Complex to γ-Globin Gene Promoter Sequences.

The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from ß-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of ß-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that alterations of BCL11A gene expression and biological functions occur during MTH-mediated erythroid differentiation. Our study demonstrates that one of the mechanisms of action of MTH is a down-regulation of the transcription of the BCL11A gene, while a second mechanism of action is the inhibition of the molecular interactions between the BCL11A complex and specific sequences of the γ-globin gene promoter.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

Precision Editing as a Therapeutic Approach for ß-Hemoglobinopathies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the ß-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic ß- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with ß-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for ß-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

Resveratrol plus low-dose hydroxyurea compared to high-dose hydroxyurea alone is more effective in γ-globin gene expression and ROS reduction in K562 cells.

Hydroxyurea (HU) is an anti-cancer drug that is used for the treatment of hemoglobinopathies as a γ-globin inducer. However, its dose-dependent effects have hampered its clinical reliability. Resveratrol (RSV) is an antioxidant and γ-globin inducer. The present study aimed to assess their combined effects on the γ-globin gene expression and reactive oxygen species (ROS) level of K562 cells. The results indicated that the γ-globin gene expression was approximately two folds higher in the group treated with RSV 50 µM + HU 25 µM in comparison to HU 100 µM alone (***p < 0.001). However, there was an inverse relationship between the expression of γ-globin gene and HU concentration in the combined groups. Furthermore, the combinations of RSV and HU significantly reduced ROS levels compared to single drugs. Overall, the combination of these compounds was an appropriate strategy for increasing γ-globin expression, reducing oxidant levels, and alleviating the adverse effects of HU.

MicroRNA-92a-3p-mediated inhibition of BCL11A upregulates γ-globin expression and inhibits oxidative stress and apoptosis in erythroid precursor cells.

OBJECTIVE: This study attempted to investigate miR-92a-3p expression in peripheral blood of patients with severe ß-thalassemia, and the effect and action mechanism of miR-92a-3p on γ-globin expression and oxidative stress in erythroid precursor cells. METHODS: CD34+ hematopoietic progenitor cells (HPCs) were isolated from peripheral blood of healthy volunteers and patients with severe ß-thalassemia. The levels of miR-92a-3p, BCL11A, and γ-globin were measured in erythroid precursor cells. High-performance liquid chromatography (HPLC) was used to analyze hemoglobin F (HbF) content. HPCs were induced with erythroid differentiation and erythroid precursor cells were then obtained. The relevance between miR-92a-3p and BCL11A was studied using dual luciferase reporter gene assay, and the correlation between miR-92a-3p and HbF was assayed by Pearson correlation analysis. Reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in erythroid precursor cells were tested to evaluate oxidative stress. Cell apoptosis was examined by flow cytometry. RESULTS: Remarkably higher expression of miR-92a-3p was observed in erythroid precursor cells. Increased expression of miR-92a-3p resulted in elevated levels of γ-globin, GSH, and SOD, reduced expression of ROS and MDA, and decreased cell apoptosis. BCL11A was identified as a target of miR-92a-3p and to be downregulated by miR-92a-3p. Moreover, BCL11A knockdown alone increased the expression of γ-globin, SOD and GSH, and repressed the levels of ROS and MDA and cell apoptosis, and the following inhibition of miR-92a-3p changed these patterns. CONCLUSIONS: Our data indicated that miR-92a-3p might increase γ-globin level and reduce oxidative stress and apoptosis in erythroid precursor cells by downregulating BCL11A.

LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin.

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for ß-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF's potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the ß-type globin's expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of ß-type globin's locus, triggering the transcriptional events from γ- to ß-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an "open" chromatin conformation across the γ-δ intergenic region and accomplish ß-globin expression and hemoglobin switch.

Treatment with Hydroxyurea Leads to Fetal Hemoglobin Reactivation through CA1 and LIN28B Genes: An In Vitro Study.

Hydroxyurea (HU) is an effective drug to increase fetal γ-globin gene (Hb F) expression, replacing the missing adult ß-globin gene. The mechanism of Hb F induction by HU and improvement in clinical symptoms are still poorly understood. The current study aimed to improve the molecular understanding of drug-induced alterations and reveals genes related to HU treatment responsiveness in ß-thalassemia (ß-thal). We analyzed the GSE109186 dataset using system biology and weighted gene coexpression network analysis (WGCNA) to identify and quantify gene expression changes reflected in the HU-treated human erythroblastic leukemia cells. The K562 cell line was treated in 50, 100, and 150 µM concentrations of HU for 24, 48, and 72 hours with three replications. The alteration of CA1, LIN28B and Hb F gene expression in HU-treated cells was evaluated using the real-time polymerase chain (real-time PCR) technique. The results showed that LIN28B has an increase of 4.27-fold on the first day of HU-treatment in 50 µM (p < 0.01). The CA1 expression showed a decrease at all times and doses of treatment, and the most decrease happened in 48 hours and 50 µM (p < 0.04). Hb F also showed the highest increase in 100 µM after 24 hours of treatment (5.18-fold). In summary, the data suggest that alteration of LIN28B and CA1 gene expression is associated with γ-globin increasing in HU-treated cells.

Reactivation of γ-globin expression using a minicircle DNA system to treat ß-thalassemia.

Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for ß-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of ß-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of ß-thalassemia.

Cilostazol-mediated reversion of γ-globin silencing is associated with a high level of HbF production: A potential therapeutic candidate for ß-globin disorders.

Reversal of fetal hemoglobin (HbF) silencing is an attractive therapeutic intervention for ß-thalassemia and sickle cell anemia. The current study proposes the therapeutic of repurposing of cilostazol, an FDA-approved antithrombotic agent, as a promising HbF inducer. Preliminary, we report that cilostazol induced erythroid differentiation and hemoglobinization of human erythroleukemia K562 cells. The erythroid differentiation was accompanied by increased expression of γ-globin mRNA transcripts and HbF production. Cilostazol induced erythroid differentiation and HbF production, without significantly affecting proliferation and viability of hemoglobin producing cells at maximum erythroid inducing concentration. Moreover, we investigated the effect of cilostazol on human ß- and γ-globin transgenes in in vivo ß-YAC transgenic mice, harboring human ß-locus along with ß-LCR. A good in vitro correlation was found with substantial up-regulation in fetal globin mRNA; whereas, the ß-globin gene expression was not significantly changed. F-cells, analysis in the peripheral blood of cilostazol-treated mice, revealed a significant increase in the F-cells population as compared with sham control groups. Together, these findings support the potential of cilostazol as an HbF inducer, which can be evaluated further to develop a new HbF inducer.

Acyclovir induces fetal hemoglobin via downregulation of γ-globin repressors, BCL11A and SOX6 trans-acting factors.

Pharmacological reactivation of developmentally silenced fetal hemoglobin (HbF) is an attractive approach to ameliorate the clinical manifestations of ß-thalassemia and sickle cell anemia. Hydroxyurea, the only HbF inducer, has obtained regulatory approval. However, hydroxyurea non-responders and associated myelosuppression making its widespread use undesirable. A high level of HbF with safe and effective agents remains an elusive therapeutic goal for this global health burden. This study demonstrated the effect of acyclovir on γ-globin expression and erythropoiesis, associated with increased HbF production. In vitro, human erythroleukemia cells and human CD34+ erythroid progenitors, and in vivo ß-YAC transgenic mice were used as experimental models. We found that acyclovir significantly induces expression of the γ-globin gene and HbF synthesis in CD34+ erythroid progenitors, without affecting terminal erythroid differentiation and erythroid cell proliferation. In contrast to other HbF inducers, no associated cytotoxicity with acyclovir was observed. Further, we reported the effect of acyclovir on γ-globin gene transcriptional regulators including BCL11A, FOP1, KLF1 SOX6, and GATA-1. Significant downregulation of the γ-globin repressors BCL11A and SOX6 was observed at both mRNA and protein levels. Whereas, GATA-1, a master erythroid transcription factor, was upregulated in acyclovir treated human CD34+ erythroid culture. Similarly, the HbF inducing effect of acyclovir in ß-YAC transgenic mice revealed a good in vitro correlation, with a substantial increase in fetal globin mRNA, and F cells population. These findings collectively suggest acyclovir as an effective HbF inducer and pave the way to evaluate its clinical efficacy in treating ß-globin disorders.

Hypoxia-Induced Alpha-Globin Expression in Syncytiotrophoblasts Mimics the Pattern Observed in Preeclamptic Placentas.

Preeclampsia (PE) is a pregnancy disorder associated with placental dysfunction and elevated fetal hemoglobin (HbF). Early in pregnancy the placenta harbors hematopoietic stem and progenitor cells (HSPCs) and is an extramedullary source of erythropoiesis. However, globin expression is not unique to erythroid cells and can be triggered by hypoxia. To investigate the role of the placenta in increasing globin levels previously reported in PE, flow cytometry, histological and immunostaining and in situ analyses were used on placenta samples and ex vivo explant cultures. Our results indicated that in PE pregnancies, placental HSPC homing and erythropoiesis were not affected. Non-erythroid alpha-globin mRNA and protein, but not gamma-globin, were detected in syncytiotrophoblasts and stroma of PE placenta samples. Similarly, alpha-globin protein and mRNA were upregulated in normal placenta explants cultured in hypoxia. The upregulation was independent of HIF1 and NRF2, the two main candidates of globin transcription in non-erythroid cells. Our study is the first to demonstrate alpha-globin mRNA expression in syncytiotrophoblasts in PE, induced by hypoxia. However, gamma-globin was only expressed in erythrocytes. We conclude that alpha-globin, but not HbF, is expressed in placental syncytiotrophoblasts in PE and may contribute to the pathology of the disease.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter.

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for ß-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from ß-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in ß-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

Epigenetic activities in erythroid cell gene regulation.

Interest in the role of epigenetic mechanisms in human biology has exponentially increased over the past several decades. The multitude of opposing and context-dependent chromatin-modifying enzymes/coregulator complexes is just beginning to be understood at a molecular level. This science has benefitted tremendously from studies of erythropoiesis, in which a series of ß-globin genes are in sequence turned "on" and "off," serving as a fascinating model of coordinated gene expression. We, therefore, describe here epigenetic complexes about which we know most, using erythropoiesis as the context. The biochemical insights lay the foundation for proposing and developing novel treatments for diseases of red cells and of erythropoiesis, identifying for example epigenetic enzymes that can be drugged to manipulate ß-globin locus regulation, to favor activation of unmutated fetal hemoglobin over mutated adult ß-globin genes to treat sickle cell disease and ß-thalassemias. Other potential translational applications are in redirecting hematopoietic commitment decisions, as treatment for bone marrow failure syndromes.

CD14+ monocytes repress gamma globin expression at early stages of erythropoiesis.

In ß-hemoglobinopathies, reactivation of gamma- at the expense of beta-globin is a prominent therapeutic option. Expression of the globin genes is not strictly intrinsically regulated during erythropoiesis, supported by the observation that fetal erythroid cells switch to adult hemoglobin expression when injected in mice. We show cultured erythroblasts are a mix of HbA restrictive and HbA/HbF expressing cells and that the proportion of cells in the latter population depends on the starting material. Cultures started from CD34+ cells contain more HbA/HbF expressing cells compared to erythroblasts cultured from total peripheral blood mononuclear cells (PBMC). Depletion of CD14+ cells from PBMC resulted in higher HbF/HbA percentages. Conversely, CD34+ co-culture with CD14+ cells reduced the HbF/HbA population through cell-cell proximity, indicating that CD14+ actively repressed HbF expression in adult erythroid cultures. RNA-sequencing showed that HbA and HbA/HbF populations contain a limited number of differentially expressed genes, aside from HBG1/2. Co-culture of CD14+ cells with sorted uncommitted hematopoietic progenitors and CD34-CD36+ erythroblasts showed that hematopoietic progenitors prior to the hemoglobinized erythroid stages are more readily influenced by CD14+ cells to downregulate expression of HBG1/2, suggesting temporal regulation of these genes. This possibly provides a novel therapeutic avenue to develop ß-hemoglobinopathies treatments.

DNA methylation patterns of ß-globin cluster in ß-thalassemia patients.

BACKGROUND: Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for ß-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in ß-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. RESULTS: Here, we evaluated DNA methylation patterns of the ß-globin cluster from peripheral bloods of 105 ß0/ß0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and ß-globin promoters displayed hypomethylation in ß0/ß0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of ß0/ß0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among ß0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls. CONCLUSIONS: Our findings revealed methylation patterns in ß-globin cluster associated with ß0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in ß0 thalassemia patients and provided candidate targets for the therapies of ß-hemoglobinopathies.

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) ß chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

ATF4 Regulates MYB to Increase γ-Globin in Response to Loss of ß-Globin.

ß-Hemoglobinopathies can trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here, we use CRISPR-Cas genome editing to model the stress caused by reduced levels of adult ß-globin. We find that decreased ß-globin is sufficient to induce robust re-expression of γ-globin, and RNA sequencing (RNA-seq) of differentiating isogenic erythroid precursors implicates ATF4 as a causal regulator of this response. ATF4 binds within the HBS1L-MYB intergenic enhancer and regulates expression of MYB, a known γ-globin regulator. Overall, the reduction of ATF4 upon ß-globin knockout decreases the levels of MYB and BCL11A. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could inform strategies to treat hemoglobinopathies.