Publications by authors named "Ryo Kurita"

72 Publications

Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines.

Commun Biol 2021 Jun 3;4(1):677. Epub 2021 Jun 3.

Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.
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http://dx.doi.org/10.1038/s42003-021-02202-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8175573PMC
June 2021

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with β-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Br J Haematol 2021 May 17. Epub 2021 May 17.

Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat β-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with β-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34 cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with β-thalassaemia.
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http://dx.doi.org/10.1111/bjh.17511DOI Listing
May 2021

Molecular analysis of the erythroid phenotype of a patient with BCL11A haploinsufficiency.

Blood Adv 2021 05;5(9):2339-2349

Academic Center for Hemoglobinopathies and Rare Anemias.

The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach.
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http://dx.doi.org/10.1182/bloodadvances.2020003753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114548PMC
May 2021

Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells.

Int J Mol Sci 2021 Mar 31;22(7). Epub 2021 Mar 31.

Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia 2371, Cyprus.

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.
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http://dx.doi.org/10.3390/ijms22073626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037168PMC
March 2021

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1.

Biochem Biophys Res Commun 2021 May 19;552:157-163. Epub 2021 Mar 19.

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200438, China. Electronic address:

β-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, αβ), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat β-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, αγ) in β-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in β-thalassemia.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.051DOI Listing
May 2021

Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia.

Am J Hum Genet 2021 04 17;108(4):709-721. Epub 2021 Mar 17.

Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515 Guangdong, China; Guangdong Engineering and Technology Research Center for Molecular Diagnostics of Human Genetic Diseases, Guangzhou, 510515 Guangdong, China; Guangdong Engineering and Technology Research Center for Genetic Testing, Guangzhou, 510515 Guangdong, China. Electronic address:

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34 erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.
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http://dx.doi.org/10.1016/j.ajhg.2021.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059375PMC
April 2021

Identification of characteristic proteins at late-stage erythroid differentiation in vitro.

Hum Cell 2021 May 22;34(3):745-749. Epub 2021 Feb 22.

Department of Research and Development, Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Tatsumi 2-1-67, Koto-ku, Tokyo , 135-8521, Japan.

The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.
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http://dx.doi.org/10.1007/s13577-021-00503-5DOI Listing
May 2021

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Mol Ther 2021 04 29;29(4):1625-1638. Epub 2021 Jan 29.

Division of Hematology, Children's Hospital of Philadelphia (CHOP), Philadelphia, PA, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Cell and Molecular Biology Affinity Group (CAMB), University of Pennsylvania, Philadelphia, PA, USA; Raymond G. Perelman Center for Cellular and Molecular Therapeutics, CHOP, Philadelphia, PA, USA; Penn Center for Musculoskeletal Disorders, CHOP, Philadelphia, PA, USA. Electronic address:

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34 cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058492PMC
April 2021

Genome-wide analysis of pseudogenes reveals HBBP1's human-specific essentiality in erythropoiesis and implication in β-thalassemia.

Dev Cell 2021 Feb 20;56(4):478-493.e11. Epub 2021 Jan 20.

State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Sciences (CAMS) & School of Basic Medicine, Peking Union Medical College (PUMC), Beijing 100005, China; Key Laboratory of RNA and Hematopoietic Regulation, Chinese Academy of Medical Sciences, Beijing 100005, China.

The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in β-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in β-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes.
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http://dx.doi.org/10.1016/j.devcel.2020.12.019DOI Listing
February 2021

PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells.

Biomaterials 2021 01 7;268:120580. Epub 2020 Dec 7.

Translational Nanobiomaterials and Imaging, Department of Radiology, Leiden University Medical Center, the Netherlands. Electronic address:

Ex vivo gene editing of CD34 hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial 'burst' of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the γ-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9-PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120580DOI Listing
January 2021

Induction of therapeutic levels of HbF in genome-edited primary β 39-thalassaemia haematopoietic stem and progenitor cells.

Br J Haematol 2021 01 20;192(2):395-404. Epub 2020 Nov 20.

Dipartimento di Scienze Mediche e Sanità Pubblica, Università degli Studi di Cagliari, Cagliari, 09124, Italy.

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of β-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δβ -thalassaemia, is able to completely rescue the β-major thalassaemia phenotype caused by the β 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from β -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous β 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of β-thalassaemia irrespective of the β-globin gene (HBB) mutations.
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http://dx.doi.org/10.1111/bjh.17167DOI Listing
January 2021

Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.

Exp Hematol 2020 11 16;91:32-38.e6. Epub 2020 Oct 16.

Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester, Rochester, NY. Electronic address:

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.
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http://dx.doi.org/10.1016/j.exphem.2020.09.201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7741183PMC
November 2020

Generation of an immortalised erythroid cell line from haematopoietic stem cells of a haemoglobin E/β-thalassemia patient.

Sci Rep 2020 10 8;10(1):16798. Epub 2020 Oct 8.

School of Biochemistry, University of Bristol, Bristol, UK.

The β-thalassemia syndromes are the most prevalent genetic disorder globally, characterised by reduced or absent β-globin chain synthesis. HbE/β-thalassemia is a subtype of β-thalassemia with extremely high frequency in Asia. Studying molecular defects behind β-thalassemia is severely impeded by paucity of material from patients and lack of suitable cell lines. Approaches to derive erythroid cells from induced pluripotent stem cells (iPSCs) created from patients are confounded by poor levels of erythroid cell expansion, aberrant or incomplete erythroid differentiation and foetal/embryonic rather than adult globin expression. In this study we generate an immortalised erythroid cell line from peripheral blood stem cells of a HbE/β-thalassemia patient. Morphological analysis shows the cells are proerythroblasts with some early basophilic erythroblasts, with no change in morphology over time in culture. The line differentiates along the erythroid pathway to orthochromatic erythroblasts and reticulocytes. Importantly, unlike iPSCs, the line maintains the haemoglobin profile of the patient's red blood cells. This is the first human cellular model for β-thalassemia providing a sustainable source of disease cells for studying underlying disease mechanisms and for use as drug screening platform, particularly for reagents designed to increase foetal haemoglobin expression as we have additionally demonstrated with hydroxyurea.
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http://dx.doi.org/10.1038/s41598-020-73991-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546635PMC
October 2020

Dual base editor catalyzes both cytosine and adenine base conversions in human cells.

Nat Biotechnol 2020 07 1;38(7):856-860. Epub 2020 Jun 1.

Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.

Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.
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http://dx.doi.org/10.1038/s41587-020-0527-yDOI Listing
July 2020

Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.

Mol Ther Methods Clin Dev 2020 Jun 18;17:999-1013. Epub 2020 Apr 18.

Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of -regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic β-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.
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http://dx.doi.org/10.1016/j.omtm.2020.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225380PMC
June 2020

A new antigen SUMI carried on glycophorin A encoded by the GYPA*M with c.91A>C (p.Thr31Pro) belongs to the MNS blood group system.

Transfusion 2020 Jun 1;60(6):1287-1293. Epub 2020 May 1.

Japanese Red Cross Central Blood Institute, Tokyo, Japan.

Background: MNS is one of the highly polymorphic blood groups comprising many antigens generated by genomic recombination among the GYPA, GYPB, and GYPE genes as well as by single-nucleotide changes. We report a patient with red blood cell (RBC) antibody against an unknown low-frequency antigen, tentatively named SUMI, and investigated its carrier molecule and causal gene.

Study Design And Methods: Standard serologic tests, including enzyme tests, were performed. Monoclonal anti-SUMI-producing cells (HIRO-305) were established by transformation and hybridization methods using lymphocytes from a donor having anti-SUMI. SUMI+ RBCs were examined by immunocomplex capture fluorescence analysis (ICFA) using HIRO-305 and murine monoclonal antibodies against RBC membrane proteins carrying blood group antigens. Genomic DNA was extracted from whole blood, and the GYPA gene was analyzed by polymerase chain reactions and Sanger sequencing.

Results: Serologic screening revealed that 23 of the 541,522 individuals (0.0042%) were SUMI+, whereas 1351 of the 10,392 individuals (13.0%) had alloanti-SUMI. SUMI antigen was sensitive to ficin, trypsin, pronase, and neuraminidase, but resistant to α-chymotrypsin and sulfydryl-reducing agents. ICFA revealed that the SUMI antigen was carried on glycophorin A (GPA). According to Sanger sequencing and cloning, the SUMI+ individuals had a GYPA*M allele with c.91A>C (p.Thr31Pro), which may abolish the O-glycan attachment site.

Conclusions: The new low-frequency antigen SUMI is carried on GPA encoded by the GYPA*M allele with c.91A>C (p.Thr31Pro). Neuraminidase sensitivity suggests that glycophorin around Pro31 are involved in the SUMI determinant.
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http://dx.doi.org/10.1111/trf.15828DOI Listing
June 2020

High level of fetal-globin reactivation by designed transcriptional activator-like effector.

Blood Adv 2020 02;4(4):687-695

Division of Experimental Hematology, Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN.

The fetal-to-adult hemoglobin switch has been a focus of a long-standing effort to potentially treat sickle cell disease and β thalassemia by induction of fetal hemoglobin. In a continuation of this effort, we designed specific transcriptional activator-like effectors (TALEs) to target both the Gγ and Aγ-globin promoters. We fused the TALEs to a LIM domain binding protein (Ldb1) dimerization domain, followed by a T2A green fluorescent protein (GFP) cassette, which were assembled into a lentiviral vector. To prevent deletions caused by the repeats of TALEs during the lentivirus packing process, we changed the TALE encoding DNA by codon optimization. Intriguingly, 5 of 14 TALEs showed forced reactivation of fetal-globin expression in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, with a significant increase in the γ-globin mRNA level by more than 70-fold. We also observed a more than 50% reduction of β-globin mRNA. High-performance liquid chromatography analysis revealed more than 30% fetal globin in TALE-induced cells compared with the control of 2%. Among several promoters studied, the β-globin gene promoter with the locus control region (LCR) enhancer showed the highest TALE expression during CD34 erythroid differentiation. At day 19 of differentiation, 2 TALEs increased fetal-globin expression more than 40-fold in the mRNA level and up to 70% of the total globin protein. These TALEs have potential for clinical translation.
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http://dx.doi.org/10.1182/bloodadvances.2019000482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042981PMC
February 2020

Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies.

Cell Res 2020 03 7;30(3):276-278. Epub 2020 Jan 7.

Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China.

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http://dx.doi.org/10.1038/s41422-019-0267-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054297PMC
March 2020

Non-transmissible MV Vector with Segmented RNA Genome Establishes Different Types of iPSCs from Hematopoietic Cells.

Mol Ther 2020 01 12;28(1):129-141. Epub 2019 Sep 12.

Project Division of ALA Advanced Medical Research, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. Electronic address:

Recent advances in gene therapy technologies have enabled the treatment of congenital disorders and cancers and facilitated the development of innovative methods, including induced pluripotent stem cell (iPSC) production and genome editing. We recently developed a novel non-transmissible and non-integrating measles virus (MV) vector capable of transferring multiple genes simultaneously into a wide range of cells through the CD46 and CD150 receptors. The MV vector expresses four genes for iPSC generation and the GFP gene for a period of time sufficient to establish iPSCs from human fibroblasts as well as peripheral blood T cells. The transgenes were expressed differentially depending on their gene order in the vector. Human hematopoietic stem/progenitor cells were directly and efficiently reprogrammed to naive-like cells that could proliferate and differentiate into primed iPSCs by the same method used to establish primed iPSCs from other cell types. The novel MV vector has several advantages for establishing iPSCs and potential future applications in gene therapy.
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http://dx.doi.org/10.1016/j.ymthe.2019.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952176PMC
January 2020

Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis.

Nat Genet 2019 07 28;51(7):1149-1159. Epub 2019 Jun 28.

Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Harvard Medical School, Boston, MA, USA.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
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http://dx.doi.org/10.1038/s41588-019-0453-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650275PMC
July 2019

levels in human hematopoietic progenitors are regulated by aging and dictate erythroid-myeloid balance.

Haematologica 2020 04 6;105(4):905-913. Epub 2019 Jun 6.

Department of Pathology, University of Virginia School of Medicine, Charlottesville, USA

Healthy bone marrow progenitors yield a co-ordinated balance of hematopoietic lineages. This balance shifts with aging toward enhanced granulopoiesis with diminished erythropoiesis and lymphopoiesis, changes which likely contribute to the development of bone marrow disorders in the elderly. In this study, RUNX3 was identified as a hematopoietic stem and progenitor cell factor whose levels decline with aging in humans and mice. This decline is exaggerated in hematopoietic stem and progenitor cells from subjects diagnosed with unexplained anemia of the elderly. Hematopoietic stem cells from elderly unexplained anemia patients had diminished erythroid but unaffected granulocytic colony forming potential. Knockdown studies revealed human hematopoietic stem and progenitor cells to be strongly influenced by RUNX3 levels, with modest deficiencies abrogating erythroid differentiation at multiple steps while retaining capacity for granulopoiesis. Transcriptome profiling indicated control by RUNX3 of key erythroid transcription factors, including and These findings thus implicate RUNX3 as a participant in hematopoietic stem and progenitor cell aging, and a key determinant of erythroid-myeloid lineage balance.
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http://dx.doi.org/10.3324/haematol.2018.208918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109730PMC
April 2020

Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors.

Methods Protoc 2018 Jul 30;1(3). Epub 2018 Jul 30.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4-7 fold higher than starting cell numbers within 9-12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.
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http://dx.doi.org/10.3390/mps1030028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481050PMC
July 2018

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Am J Hematol 2019 09 18;94(9):963-974. Epub 2019 Jun 18.

Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts.

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.
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http://dx.doi.org/10.1002/ajh.25543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6984401PMC
September 2019

Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

Exp Hematol 2019 06 18;74:19-24.e4. Epub 2019 Apr 18.

Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester, Rochester, NY. Electronic address:

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.
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http://dx.doi.org/10.1016/j.exphem.2019.04.001DOI Listing
June 2019

Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.

Haematologica 2019 12 19;104(12):2361-2371. Epub 2019 Apr 19.

Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA

As high fetal hemoglobin levels ameliorate the underlying pathophysiological defects in sickle cell anemia and beta (β)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemoglobin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex member causes a 10-20-fold increase in γ-globin gene expression in adult β-globin locus yeast artificial chromosome transgenic mice. To determine the effect of depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in γ/γ+β mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high performance liquid chromatography. In contrast, knockout had no appreciable effect on γ-globin expression. Knockdown of in primary adult erythroid cells consistently increased γ/γ+β mRNA ratios by approximately 10-fold resulting in approximately 30-40% γ/γ+β mRNA levels and a corresponding increase in γ-globin protein. exerts its repressive effects through recruitment of the chromatin remodeler CHD4 a coiled-coil domain, and the histone deacetylase core complex an intrinsically disordered region. Enforced expression of wild-type in knockout cells caused a 5-fold decrease in γ-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt complex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of as potential therapeutic targets.
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http://dx.doi.org/10.3324/haematol.2018.210963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959176PMC
December 2019

KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.

Exp Hematol 2019 05 12;73:25-37.e8. Epub 2019 Mar 12.

Project Division of ALA Advanced Medical Research, The Institute of Medical Science, University of Tokyo, Tokyo, Japan; Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Department of Advanced Molecular and Cell Therapy, Kyushu University Hospital, Fukuoka, Japan. Electronic address:

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a/CD71 stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.
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http://dx.doi.org/10.1016/j.exphem.2019.03.001DOI Listing
May 2019

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

PLoS One 2019 15;14(1):e0208237. Epub 2019 Jan 15.

Innovative Genomics Institute, University of California, Berkeley, CA, United States of America.

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208237PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333401PMC
September 2019

A natural regulatory mutation in the proximal promoter elevates fetal expression by creating a de novo GATA1 site.

Blood 2019 02 7;133(8):852-856. Epub 2019 Jan 7.

School of Biotechnology and Biomolecular Sciences, University of New South Wales (UNSW Sydney), Sydney, NSW, Australia.

β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, result from mutations in the adult gene. Reactivating the developmentally silenced fetal gene elevates fetal hemoglobin levels and ameliorates symptoms of β-hemoglobinopathies. The continued expression of fetal into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal proximal promoter can cause HPFH. The -113A>G HPFH mutation falls within the -115 cluster of HPFH mutations, a binding site for the fetal repressor BCL11A. We demonstrate that the -113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the -113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal levels. These results reveal the mechanism by which the -113A>G HPFH mutation elevates fetal and demonstrate the sensitivity of the fetal promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.
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http://dx.doi.org/10.1182/blood-2018-07-863951DOI Listing
February 2019

BAP1 regulation of the key adaptor protein NCoR1 is critical for γ-globin gene repression.

Genes Dev 2018 12 21;32(23-24):1537-1549. Epub 2018 Nov 21.

Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

Human globin gene production transcriptionally "switches" from fetal to adult synthesis shortly after birth and is controlled by macromolecular complexes that enhance or suppress transcription by elements scattered throughout the locus. The DRED (direct repeat erythroid-definitive) repressor is recruited to the ε-globin and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA-binding and epigenetic enzyme components (e.g., DNA methyltransferase 1 [DNMT1] and lysine-specific demethylase 1 [LSD1]) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: The deubiquitinase BRCA1-associated protein-1 (BAP1) is a component of the repressor complex whose activity maintains NCoR1 at sites in the β-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression.
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http://dx.doi.org/10.1101/gad.318436.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295165PMC
December 2018