ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.

CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.

BACKGROUND: Sickle cell disease is caused by a defect in the ß-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny. METHODS: We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923. RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period. CONCLUSIONS: CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).

Elevating fetal hemoglobin: recently discovered regulators and mechanisms.

ABSTRACT: It has been known for over half a century that throughout ontogeny, humans produce different forms of hemoglobin, a tetramer of α- and ß-like hemoglobin chains. The switch from fetal to adult hemoglobin occurs around the time of birth when erythropoiesis shifts from the fetal liver to the bone marrow. Naturally, diseases caused by defective adult ß-globin genes, such as sickle cell disease and ß-thalassemia, manifest themselves as the production of fetal hemoglobin fades. Reversal of this developmental switch has been a major goal to treat these diseases and has been a driving force to understand its underlying molecular biology. Several review articles have illustrated the long and at times arduous paths that led to the discovery of the first transcriptional regulators involved in this process. Here, we survey recent developments spurred by the discovery of CRISPR tools that enabled for the first time high-throughput genetic screens for new molecules that impact the fetal-to-adult hemoglobin switch. Numerous opportunities for therapeutic intervention have thus come to light, offering hope for effective pharmacologic intervention for patients for whom gene therapy is out of reach.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Evolution of nanobodies specific for BCL11A.

Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Here, we describe the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. We obtained first-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) from a synthetic yeast surface display library, and employed error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA-binding specificity. Given the ease of manipulation of nanobodies and their exquisite specificity, nanobody-mediated TPD of TFs should be suitable for dissecting regulatory relationships of TFs and gene targets and validating therapeutic potential of proteins of interest.

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

Bach1 inhibitor HPP-D mediates γ-globin gene activation in sickle erythroid progenitors.

Sickle cell disease (SCD) is the most common ß-hemoglobinopathy caused by various mutations in the adult ß-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.

Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression.

The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the ß-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δß-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult ß-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of ß-hemoglobinopathies.

Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in ß-thalassemia and sickle cell disease patients.

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both ß-thalassemia (ß-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.

Transcriptional Repressor BCL11A in Erythroid Cells.

BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2ß2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and ß-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and ß-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.

Genetic Modifiers of Stroke in Patients with Sickle Cell Disease-A Scoping Review.

Sickle cell disease (SCD) clinically manifests itself with a myriad of complications. Stroke, both ischemic and hemorrhagic, as well as silent white matter changes, occurs at a relatively high prevalence. Understanding why and in whom stroke is most likely to occur is critical to the effective prevention and treatment of individuals with SCD. Genetic studies, including genome- and exome-wide association studies (GWAS and EWAS), have found several key modifiers associated with increased stroke/stroke risk in SCD via mechanisms including Hemoglobin F (HbF) modulation, inflammation, cellular adhesion, endothelial disruption, and hemolysis. We present a review on the modifiers that have most clearly demonstrated an association to date. More studies are needed to validate other potential polymorphisms and identify new ones. Incorporating gene-focused screenings in clinical care could provide avenues for more targeted, more effective, and less toxic prevention of stroke in this population. The data from this review will be used to inform the initial GWAS performed by the International Hemoglobinopathy Research Network (INHERENT) consortium.

BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of ß-hemoglobinopathies.

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in ß-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to ß-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of ß-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in ß-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early "proof-of-concept" to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for ß-thalassemia.

Development of a double shmiR lentivirus effectively targeting both BCL11A and ZNF410 for enhanced induction of fetal hemoglobin to treat ß-hemoglobinopathies.

A promising treatment for ß-hemoglobinopathies is the de-repression of γ-globin expression leading to increased fetal hemoglobin (HbF) by targeting BCL11A. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ-globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ-globin repressor ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to a 70% reduction of both BCL11A and ZNF410 proteins. There was a consistent and significant additional 10% increase in HbF compared to targeting BCL11A alone in erythroid cells. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype compared to the SS. Erythrocytes differentiated from transduced HSCs from ß-thalassemia major patients demonstrated improved globin chain balance by increased γ-globin with reduced microcytosis. Reconstitution of DS-transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction for treating ß-hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products.

Allosteric control of hemoglobin S fiber formation by oxygen and its relation to the pathophysiology of sickle cell disease.

The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

Genome editing strategies for fetal hemoglobin induction in beta-hemoglobinopathies.

Genome editing to correct a defective ß-globin gene or induce fetal globin (HbF) for patients with beta-hemoglobinopathies has the potential to be a curative strategy available to all. HbF reactivation has long been an area of intense interest given the HbF inhibition of sickle hemoglobin (HbS) polymerization. Patients with HbS who also have high HbF tend to have less severe or even minimal clinical manifestations. Approaches to genetically engineer high HbF include de novo generation of naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations, editing of transcriptional HbF repressors or their binding sites and/or regulating epigenetic intermediates controlling HbF expression. Recent preclinical and early clinical trial data show encouraging results; however, long-term follow-up is lacking, and the safety and efficacy concerns of genome editing remain.

Pharmacologic induction of PGC-1α stimulates fetal haemoglobin gene expression.

Sickle cell disease (SCD) is a genetic disorder that affects millions around the world. Enhancement of fetal γ-globin levels and fetal haemoglobin (HbF) production in SCD patients leads to diminished severity of many clinical features of the disease. We recently identified the transcriptional co-activator PGC-1α as a new protein involved in the regulation of the globin genes. Here, we report that upregulation of PGC-1α by infection with a lentivirus expressing PGC-1α or by the small-molecule PGC-1α agonist ZLN005 in human primary erythroid progenitor CD34+ cells induces both fetal γ-globin mRNA and protein expression as well as the percentage of HbF-positive cell (F cells) without significantly affecting cell proliferation and differentiation. We further found that the combination of ZLN005 and hydroxyurea (hydroxycarbamide) exhibited an additive effect on the expression of γ-globin and the generation of F cells from cultured CD34+ cells. In addition, ZLN005 induced robust expression of the murine embryonic ßh1-globin gene and to a lesser extent, human γ-globin gene expression in sickle mice. These findings suggest that activation of PGC-1α by ZLN005 might provide a new path for modulating HbF levels with potential therapeutic benefit in ß-hemoglobinopathies.

Novel histone deacetylase inhibitor CT-101 induces γ-globin gene expression in sickle erythroid progenitors with targeted epigenetic effects.

Induction of fetal hemoglobin (HbF) expression ameliorates the clinical severity and prolong survival in persons with sickle cell disease (SCD). Hydroxyurea (HU) is the only FDA-approved HbF inducer however, additional therapeutics that produce an additive effect in SCD are needed. To this end, development of potent Class I histone deacetylase inhibitors (HDACi) for HbF induction represents a rational molecularly targeted approach. In studies here, we evaluated CT-101, a novel Class I-restricted HDACi, a Largazole derivative, for pharmacodynamics, cytotoxicity, and targeted epigenetic effects. In SCD-derived erythroid progenitors, CT-101 induced HbF expression with additive activity in combination with HU. CT-101 preferentially activated γ-globin gene transcription, increased acetylated histone H3 levels, and conferred an open chromatin conformation in the γ-globin promoter. These data indicate CT-101 represents a strong potential candidate as a molecularly targeted inducer of HbF.

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.

miRNA Expression Associated with HbF in Saudi Sickle Cell Anemia.

Background and Objectives: Sickle cell anemia (SCA) is a hereditary monogenic disease due to a single ß-globin gene mutation that codes for the production of sickle hemoglobin. Its phenotype is modulated by fetal hemoglobin (HbF), a product of γ-globin genes. Exploring the molecules that regulate γ-globin genes at both transcriptional and translational levels, including microRNA (miRNA), might help identify alternative therapeutic targets. Materials and Methods: Using next-generation sequencing we identified pre-miRNAs and mature miRNA expression signatures associated with different HbF levels in patients homozygous for the sickle hemoglobin gene. The involvement of identified miRNAs in potential SCD-related pathways was investigated with the DIANA TOOL and miRWalk 2.0 database. Results: miR-184 were most highly upregulated in reticulocytes. miR-3609 and miR-483-5p were most highly downregulated in sickle cell anemia with high HbF. miR-370-3p that regulates LIN28A, and miR-451a which is effective in modulating α- and ß- globin levels were also significantly upregulated. miRNA targeted gene pathway interaction identified BCL7A, BCL2L1, LIN28A, KLF6, GATA6, solute carrier family genes and ZNF genes associated with erythropoiesis, cell cycle regulation, glycosphingolipid biosynthesis, cAMP, cGMP-PKG, mTOR, MAPK and PI3K-AKT signaling pathways and cancer pathways. Conclusions: miRNA signatures and their target genes identified novel miRNAs that could regulate fetal hemoglobin production and might be exploited therapeutically.