Publications by authors named "Mitchell J Weiss"

151 Publications

The role of globins in cardiovascular physiology.

Physiol Rev 2021 09 6. Epub 2021 Sep 6.

Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, United States.

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system. The ability of each of these globins to interact with molecular oxygen (O) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extra-erythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in non-vascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the central and peripheral nervous systems. Brain and central nervous system neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and, thus, tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme-iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scaveging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology with a focus on NO biology, and offer perspectives for future study of these functions.
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http://dx.doi.org/10.1152/physrev.00037.2020DOI Listing
September 2021

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.

Nat Genet 2021 08 2;53(8):1177-1186. Epub 2021 Aug 2.

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

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.
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http://dx.doi.org/10.1038/s41588-021-00904-0DOI Listing
August 2021

A polygenic score for acute vaso-occlusive pain in pediatric sickle cell disease.

Blood Adv 2021 07;5(14):2839-2851

Department of Hematology.

Individuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSβ0-thalassemia from Baylor College of Medicine and from the St. Jude Children's Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion -α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the β-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10-14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10-13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.
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http://dx.doi.org/10.1182/bloodadvances.2021004634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341359PMC
July 2021

RNA-Binding Proteins PCBP1 and PCBP2 Are Critical Determinants of Murine Erythropoiesis.

Mol Cell Biol 2021 08 24;41(9):e0066820. Epub 2021 Aug 24.

Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

We previously demonstrated that the two paralogous RNA-binding proteins PCBP1 and PCBP2 are individually essential for mouse development: -null embryos are peri-implantation lethal, while -null embryos lose viability at midgestation. Midgestation embryos revealed a complex phenotype that included loss of certain hematopoietic determinants. Whether PCBP2 directly contributes to erythropoietic differentiation and whether PCBP1 has a role in this process remained undetermined. Here, we selectively inactivated the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus failed to impact viability or blood formation. However, combined inactivation of the two loci resulted in midgestational repression of erythroid/hematopoietic gene expression, loss of blood formation, and fetal demise. Orthogonal analyses of primary erythroid progenitors selectively depleted of these two RNA-binding proteins revealed that they mediate a combination of overlapping and isoform-specific impacts on hematopoietic lineage transcriptome, impacting both mRNA representation and exon splicing. These data lead us to conclude that PCBP1 and PCBP2 mediate functions critical to differentiation of the erythroid lineage.
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http://dx.doi.org/10.1128/MCB.00668-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384066PMC
August 2021

Generalization of a genetic risk score for time to first albuminuria in children with sickle cell anaemia: SCCRIP cohort study results.

Br J Haematol 2021 Jul 16;194(2):469-473. Epub 2021 Jun 16.

Division of Pediatric Nephrology and Hypertension, University of Tennessee Health Science Center, Memphis, TN, USA.

Albuminuria predicts kidney disease progression in individuals with sickle cell anaemia (SCA); however, earlier prediction of kidney disease with introduction of reno-protective therapies prior to the onset of albuminuria may attenuate disease progression. A genetic risk score (GRS) for SCA-related nephropathy may provide an improved one-time test for early identification of high-risk patients. We utilized a GRS from a recent, large, trans-ethnic meta-analysis to identify three single nucleotide polymorphisms that associate individually and in a GRS with time to first albuminuria episode in children with SCA.
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http://dx.doi.org/10.1111/bjh.17647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282691PMC
July 2021

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Nature 2021 07 2;595(7866):295-302. Epub 2021 Jun 2.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB. We used a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBB) into Makassar β-globin (HBB), a non-pathogenic variant. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB to HBB. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBB base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB-to-HBB editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB, generates benign HBB, and minimizes the undesired consequences of double-strand DNA breaks.
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http://dx.doi.org/10.1038/s41586-021-03609-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266759PMC
July 2021

Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.

Nat Genet 2021 06 6;53(6):869-880. Epub 2021 May 6.

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

Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34 hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.
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http://dx.doi.org/10.1038/s41588-021-00861-8DOI Listing
June 2021

Disease severity impacts plerixafor-mobilized stem cell collection in patients with sickle cell disease.

Blood Adv 2021 05;5(9):2403-2411

Cellular and Molecular Therapeutics Branch, NHLBI/NIDDK, National Institutes of Health, Bethesda, MD.

Recent studies suggest that plerixafor mobilization and apheresis in patients with sickle cell disease (SCD) is safe and can allow collection of sufficient CD34+ hematopoietic stem cell (HSC) collection for clinical gene therapy applications. However, the quantities of plerixafor-mobilized CD34+ cells vary between different SCD patients for unknown reasons. Twenty-three participants with SCD underwent plerixafor mobilization followed by apheresis, processing, and HSC enrichment under a phase 1 safety and efficacy study conducted at 2 institutions. Linear regression or Spearman's correlation test was used to assess the relationships between various hematologic and clinical parameters with total CD34+ cells/kg collected. Median CD34+ cells/kg after 2 or fewer mobilization and apheresis cycles was 4.0 × 106 (range, 1.5-12.0). Similar to what is observed generally, CD34+ yield correlated negatively with age (P < .001) and positively with baseline (P = .003) and preapheresis blood CD34+ cells/µL (P < .001), and baseline white blood cell (P = .01) and platelet counts (P = .03). Uniquely for SCD, CD34+ cell yields correlated positively with the number of days hydroxyurea was held (for up to 5 weeks, P = .01) and negatively with markers of disease severity, including hospitalization frequency within the preceding year (P = .01) and the number of medications taken for chronic pain (P = .002). Unique SCD-specific technical challenges in apheresis were also associated with reduced CD34+ cell collection efficiency and purification. Here, we describe factors that impact plerixafor mobilization success in patients with SCD, confirming known factors as described in other populations in addition to reporting previously unknown disease specific factors in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT03226691.
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http://dx.doi.org/10.1182/bloodadvances.2021004232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114546PMC
May 2021

Genetic therapies for the first molecular disease.

J Clin Invest 2021 04;131(8)

Department of Hematology.

Sickle cell disease (SCD) is a monogenic disorder characterized by recurrent episodes of severe bone pain, multi-organ failure, and early mortality. Although medical progress over the past several decades has improved clinical outcomes and offered cures for many affected individuals living in high-income countries, most SCD patients still experience substantial morbidity and premature death. Emerging technologies to manipulate somatic cell genomes and insights into the mechanisms of developmental globin gene regulation are generating potentially transformative approaches to cure SCD by autologous hematopoietic stem cell (HSC) transplantation. Key components of current approaches include ethical informed consent, isolation of patient HSCs, in vitro genetic modification of HSCs to correct the SCD mutation or circumvent its damaging effects, and reinfusion of the modified HSCs following myelotoxic bone marrow conditioning. Successful integration of these components into effective therapies requires interdisciplinary collaborations between laboratory researchers, clinical caregivers, and patients. Here we summarize current knowledge and research challenges for each key component, emphasizing that the best approaches have yet to be developed.
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http://dx.doi.org/10.1172/JCI146394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262557PMC
April 2021

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.

Nat Genet 2021 06 12;53(6):895-905. Epub 2021 Apr 12.

Howard Hughes Medical Institute, Chevy Chase, MD, USA.

Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.
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http://dx.doi.org/10.1038/s41588-021-00838-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192433PMC
June 2021

Dynamic CTCF binding directly mediates interactions among cis-regulatory elements essential for hematopoiesis.

Blood 2021 03;137(10):1327-1339

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

While constitutive CCCTC-binding factor (CTCF)-binding sites are needed to maintain relatively invariant chromatin structures, such as topologically associating domains, the precise roles of CTCF to control cell-type-specific transcriptional regulation remain poorly explored. We examined CTCF occupancy in different types of primary blood cells derived from the same donor to elucidate a new role for CTCF in gene regulation during blood cell development. We identified dynamic, cell-type-specific binding sites for CTCF that colocalize with lineage-specific transcription factors. These dynamic sites are enriched for single-nucleotide polymorphisms that are associated with blood cell traits in different linages, and they coincide with the key regulatory elements governing hematopoiesis. CRISPR-Cas9-based perturbation experiments demonstrated that these dynamic CTCF-binding sites play a critical role in red blood cell development. Furthermore, precise deletion of CTCF-binding motifs in dynamic sites abolished interactions of erythroid genes, such as RBM38, with their associated enhancers and led to abnormal erythropoiesis. These results suggest a novel, cell-type-specific function for CTCF in which it may serve to facilitate interaction of distal regulatory emblements with target promoters. Our study of the dynamic, cell-type-specific binding and function of CTCF provides new insights into transcriptional regulation during hematopoiesis.
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http://dx.doi.org/10.1182/blood.2020005780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955410PMC
March 2021

St. Jude Cloud: A Pediatric Cancer Genomic Data-Sharing Ecosystem.

Cancer Discov 2021 05 6;11(5):1082-1099. Epub 2021 Jan 6.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Effective data sharing is key to accelerating research to improve diagnostic precision, treatment efficacy, and long-term survival in pediatric cancer and other childhood catastrophic diseases. We present St. Jude Cloud (https://www.stjude.cloud), a cloud-based data-sharing ecosystem for accessing, analyzing, and visualizing genomic data from >10,000 pediatric patients with cancer and long-term survivors, and >800 pediatric sickle cell patients. Harmonized genomic data totaling 1.25 petabytes are freely available, including 12,104 whole genomes, 7,697 whole exomes, and 2,202 transcriptomes. The resource is expanding rapidly, with regular data uploads from St. Jude's prospective clinical genomics programs. Three interconnected apps within the ecosystem-Genomics Platform, Pediatric Cancer Knowledgebase, and Visualization Community-enable simultaneously performing advanced data analysis in the cloud and enhancing the Pediatric Cancer knowledgebase. We demonstrate the value of the ecosystem through use cases that classify 135 pediatric cancer subtypes by gene expression profiling and map mutational signatures across 35 pediatric cancer subtypes. SIGNIFICANCE: To advance research and treatment of pediatric cancer, we developed St. Jude Cloud, a data-sharing ecosystem for accessing >1.2 petabytes of raw genomic data from >10,000 pediatric patients and survivors, innovative analysis workflows, integrative multiomics visualizations, and a knowledgebase of published data contributed by the global pediatric cancer community..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102307PMC
May 2021

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

Mol Cell 2021 01 9;81(2):239-254.e8. Epub 2020 Dec 9.

Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2020.11.006DOI Listing
January 2021

Integrative proteomics reveals principles of dynamic phosphosignaling networks in human erythropoiesis.

Mol Syst Biol 2020 12;16(12):e9813

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

Human erythropoiesis is an exquisitely controlled multistep developmental process, and its dysregulation leads to numerous human diseases. Transcriptome and epigenome studies provided insights into system-wide regulation, but we currently lack a global mechanistic view on the dynamics of proteome and post-translational regulation coordinating erythroid maturation. We established a mass spectrometry (MS)-based proteomics workflow to quantify and dynamically track 7,400 proteins and 27,000 phosphorylation sites of five distinct maturation stages of in vitro reconstituted erythropoiesis of CD34 HSPCs. Our data reveal developmental regulation through drastic proteome remodeling across stages of erythroid maturation encompassing most protein classes. This includes various orchestrated changes in solute carriers indicating adjustments to altered metabolic requirements. To define the distinct proteome of each maturation stage, we developed a computational deconvolution approach which revealed stage-specific marker proteins. The dynamic phosphoproteomes combined with a kinome-targeted CRISPR/Cas9 screen uncovered coordinated networks of erythropoietic kinases and pinpointed downregulation of c-Kit/MAPK signaling axis as key driver of maturation. Our system-wide view establishes the functional dynamic of complex phosphosignaling networks and regulation through proteome remodeling in erythropoiesis.
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http://dx.doi.org/10.15252/msb.20209813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706838PMC
December 2020

FBXO11-mediated proteolysis of BAHD1 relieves PRC2-dependent transcriptional repression in erythropoiesis.

Blood 2021 01;137(2):155-167

Department of Hematology.

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11-/- erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.
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http://dx.doi.org/10.1182/blood.2020007809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820877PMC
January 2021

Erythropoietin regulation of red blood cell production: from bench to bedside and back.

F1000Res 2020 18;9. Epub 2020 Sep 18.

Department of Hematology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS #355, Memphis, TN, 38105, USA.

More than 50 years of efforts to identify the major cytokine responsible for red blood cell (RBC) production (erythropoiesis) led to the identification of erythropoietin (EPO) in 1977 and its receptor (EPOR) in 1989, followed by three decades of rich scientific discovery. We now know that an elaborate oxygen-sensing mechanism regulates the production of EPO, which in turn promotes the maturation and survival of erythroid progenitors. Engagement of the EPOR by EPO activates three interconnected signaling pathways that drive RBC production via diverse downstream effectors and simultaneously trigger negative feedback loops to suppress signaling activity. Together, the finely tuned mechanisms that drive endogenous EPO production and facilitate its downstream activities have evolved to maintain RBC levels in a narrow physiological range and to respond rapidly to erythropoietic stresses such as hypoxia or blood loss. Examination of these pathways has elucidated the genetics of numerous inherited and acquired disorders associated with deficient or excessive RBC production and generated valuable drugs to treat anemia, including recombinant human EPO and more recently the prolyl hydroxylase inhibitors, which act partly by stimulating endogenous EPO synthesis. Ongoing structure-function studies of the EPOR and its essential partner, tyrosine kinase JAK2, suggest that it may be possible to generate new "designer" drugs that control selected subsets of cytokine receptor activities for therapeutic manipulation of hematopoiesis and treatment of blood cancers.
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http://dx.doi.org/10.12688/f1000research.26648.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503180PMC
February 2021

A novel algorithm comprehensively characterizes human RH genes using whole-genome sequencing data.

Blood Adv 2020 09;4(18):4347-4357

Department of Pathology, St Jude Children's Research Hospital, Memphis, TN.

RHD and RHCE genes encode Rh blood group antigens and exhibit extensive single-nucleotide polymorphisms and chromosome structural changes in patients with sickle cell disease (SCD). RH variation can drive loss of antigen epitopes or expression of new epitopes, predisposing patients with SCD to Rh alloimmunization. Serologic antigen typing is limited to common Rh antigens, necessitating a genetic approach to detect variant antigen expression. We developed a novel algorithm termed RHtyper for RH genotyping from existing whole-genome sequencing (WGS) data. RHtyper determined RH genotypes in an average of 3.4 and 3.3 minutes per sample for RHD and RHCE, respectively. In a validation cohort consisting of 57 patients with SCD, RHtyper achieved 100% accuracy for RHD and 98.2% accuracy for RHCE, when compared with genotypes obtained by RH BeadChip and targeted molecular assays and after verification by Sanger sequencing and independent next-generation sequencing assays. RHtyper was next applied to WGS data from an additional 827 patients with SCD. In the total cohort of 884 patients, RHtyper identified 38 RHD and 28 RHCE distinct alleles, including a novel RHD DAU allele, RHD* 602G, 733C, 744T 1136T. RHtyper provides comprehensive and high-throughput RH genotyping from WGS data, facilitating deconvolution of the extensive RH genetic variation among patients with SCD. We have implemented RHtyper as a cloud-based public access application in DNAnexus (https://platform.dnanexus.com/app/RHtyper), enabling clinicians and researchers to perform RH genotyping with next-generation sequencing data.
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http://dx.doi.org/10.1182/bloodadvances.2020002148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509869PMC
September 2020

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

J Clin Invest 2020 12;130(12):6677-6687

Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, Maryland, USA.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.
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http://dx.doi.org/10.1172/JCI140189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685754PMC
December 2020

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis.

Genome Res 2020 03 4;30(3):472-484. Epub 2020 Mar 4.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Thousands of epigenomic data sets have been generated in the past decade, but it is difficult for researchers to effectively use all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for aldated ystematic ntegrati of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By using IDEAS as our ntegrative and iscriminative pigenome nnotation ystem, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of more than 200,000 candidate -regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website to aid research in genomics and hematopoiesis.
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http://dx.doi.org/10.1101/gr.255760.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111515PMC
March 2020

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of hematopoietic cells.

IUBMB Life 2020 01 25;72(1):27-38. Epub 2019 Nov 25.

Department of Hematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for 20 cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions-along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for the regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.
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http://dx.doi.org/10.1002/iub.2195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972633PMC
January 2020

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Blood Adv 2019 11;3(21):3379-3392

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

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and β-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.
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http://dx.doi.org/10.1182/bloodadvances.2019000820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855127PMC
November 2019

The autophagy-activating kinase ULK1 mediates clearance of free α-globin in β-thalassemia.

Sci Transl Med 2019 08;11(506)

Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

In β-thalassemia, accumulated free α-globin forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin, although the associated mechanisms are poorly understood. We show that loss of the autophagy-activating Unc-51-like kinase 1 () gene in β-thalassemic mice reduces autophagic clearance of α-globin in red blood cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy-related 5 () gene has relatively minor effects. Systemic treatment with the mTORC1 inhibitor rapamycin reduces α-globin precipitates and lessens pathologies in β-thalassemic mice via an ULK1-dependent pathway. Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from CD34 cells of β-thalassemic individuals. Our findings define a drug-regulatable pathway for ameliorating β-thalassemia.
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http://dx.doi.org/10.1126/scitranslmed.aav4881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441525PMC
August 2019

Mechanisms of Progression of Myeloid Preleukemia to Transformed Myeloid Leukemia in Children with Down Syndrome.

Cancer Cell 2019 08 11;36(2):123-138.e10. Epub 2019 Jul 11.

MRC MHU, BRC Hematology Theme, Oxford Biomedical Research Centre, Oxford Centre for Haematology, WIMM, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DU, UK; Department of Paediatrics, University of Oxford, Oxford OX3 9DS, UK.

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.
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http://dx.doi.org/10.1016/j.ccell.2019.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863161PMC
August 2019

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

N Engl J Med 2019 04;380(16):1525-1534

From the Departments of Bone Marrow Transplantation and Cellular Therapy (E.M., B.T., W.J., S.G.), Hematology (S.Z., Z.M., J.C., J.D., X.T., B.Y.R., M.J.W., B.P.S.), Therapeutics Production and Quality (T.L., M.M.M.), Immunology (H.A., B.Y.), Pharmaceutical Sciences (S.J.C.), Biostatistics (G.K., C.L.), and Infectious Diseases (G.M.), St. Jude Children's Research Hospital, Memphis, TN; the Allergy and Clinical Immunology Division, Hospital Nacional Edgardo Rebagliati Martins, Lima, Peru (J.C.A.B.); the Department of Pediatrics, Allergy-Immunology Division, Children's Hospital Los Angeles, Los Angeles (J.A.C.), and the Department of Pediatrics, Division of Pediatric Allergy-Immunology-Bone Marrow Transplantation, University of California, San Francisco (UCSF) Benioff Children's Hospital, San Francisco (J.R.L.-B., J.M.P., M.J.C.) - both in California; the Department of Pediatrics, Pediatric Allergy and Immunology, University of New Mexico, Albuquerque (E.D.); University of Oklahoma Health Sciences Center, Tulsa (J.T.L.); Departamento de Pediatria da Universidade de Taubaté, Conselho Nacional de Medicina, São Paulo (A.C.M.A.); Copperfield Childcare, Claremont, South Africa (H.W.); and the Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (S.S.D.R., H.L.M.).

Background: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia.

Methods: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1.

Results: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven.

Conclusions: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).
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http://dx.doi.org/10.1056/NEJMoa1815408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636624PMC
April 2019

Regulation of gene expression by miR-144/451 during mouse erythropoiesis.

Blood 2019 06 10;133(23):2518-2528. Epub 2019 Apr 10.

Division of Hematology/Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

The microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound ∼1400 erythroblast mRNAs in a miR-144/451-dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only ∼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, , facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.
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http://dx.doi.org/10.1182/blood.2018854604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557621PMC
June 2019

Children with sickle cell anemia and APOL1 genetic variants develop albuminuria early in life.

Haematologica 2019 09 19;104(9):e385-e387. Epub 2019 Mar 19.

Division Pediatric Hematology and Oncology, University of Alabama at Birmingham, Birmingham, AL, USA

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http://dx.doi.org/10.3324/haematol.2018.212779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717565PMC
September 2019

Complexities of genetic diagnosis illustrated by an atypical case of congenital hypoplastic anemia.

Cold Spring Harb Mol Case Stud 2018 12 17;4(6). Epub 2018 Dec 17.

Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Diamond-Blackfan Anemia (DBA) is a rare polygenic disorder defined by congenital hypoplastic anemia with marked decrease or absence of bone marrow erythroid precursors. Identifying the specific genetic etiology is important for counseling and clinical management. A 6-yr-old boy with a clinical diagnosis of DBA has been followed by our pediatric hematology team since birth. His clinical course includes transfusion-dependent hypoplastic anemia and progressive autoimmune cytopenias. Genetic testing failed to identify a causative mutation in any of the classical DBA-associated genes. He and his parents underwent trio whole-exome sequencing (WES) with no genetic etiology identified initially. Clinical persistence and suspicion led to testing for adenosine deaminase 2 (ADA2) activity and whole-genome sequencing (WGS) that identified compound heterozygous pathogenic mutations in the ADA2-encoding gene, a recently appreciated etiology for congenital hypoplastic anemia. This case illustrates current challenges in genetic testing and how they can be overcome by multidisciplinary expertise in clinical medicine and genomics.
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http://dx.doi.org/10.1101/mcs.a003384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318771PMC
December 2018

Testicular endothelial cells are a critical population in the germline stem cell niche.

Nat Commun 2018 10 22;9(1):4379. Epub 2018 Oct 22.

Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA, 19104, USA.

Maintenance of adult tissues depends on stem cell self-renewal in local niches. Spermatogonial stem cells (SSC) are germline adult stem cells necessary for spermatogenesis and fertility. We show that testicular endothelial cells (TECs) are part of the SSC niche producing glial cell line-derived neurotrophic factor (GDNF) and other factors to support human and mouse SSCs in long-term culture. We demonstrate that FGF-2 binding to FGFR1 on TECs activates the calcineurin pathway to produce GDNF. Comparison of the TEC secretome to lung and liver endothelial cells identified 5 factors sufficient for long-term maintenance of human and mouse SSC colonies in feeder-free cultures. Male cancer survivors after chemotherapy are often infertile since SSCs are highly susceptible to cytotoxic injury. Transplantation of TECs alone restores spermatogenesis in mice after chemotherapy-induced depletion of SSCs. Identifying TECs as a niche population necessary for SSC self-renewal may facilitate fertility preservation for prepubertal boys diagnosed with cancer.
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http://dx.doi.org/10.1038/s41467-018-06881-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197186PMC
October 2018

Endothelial cell α-globin and its molecular chaperone α-hemoglobin-stabilizing protein regulate arteriolar contractility.

J Clin Invest 2018 11 8;128(11):5073-5082. Epub 2018 Oct 8.

Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Arteriolar endothelial cell-expressed (EC-expressed) α-globin binds endothelial NOS (eNOS) and degrades its enzymatic product, NO, via dioxygenation, thereby lessening the vasodilatory effects of NO on nearby vascular smooth muscle. Although this reaction potentially affects vascular physiology, the mechanisms that regulate α-globin expression and dioxygenase activity in ECs are unknown. Without β-globin, α-globin is unstable and cytotoxic, particularly in its oxidized form, which is generated by dioxygenation and recycled via endogenous reductases. We show that the molecular chaperone α-hemoglobin-stabilizing protein (AHSP) promotes arteriolar α-globin expression in vivo and facilitates its reduction by eNOS. In Ahsp-/- mice, EC α-globin was decreased by 70%. Ahsp-/- and Hba1-/- mice exhibited similar evidence of increased vascular NO signaling, including arteriolar dilation, blunted α1-adrenergic vasoconstriction, and reduced blood pressure. Purified α-globin bound eNOS or AHSP, but not both together. In ECs in culture, eNOS or AHSP enhanced α-globin expression posttranscriptionally. However, only AHSP prevented oxidized α-globin precipitation in solution. Finally, eNOS reduced AHSP-bound α-globin approximately 6-fold faster than did the major erythrocyte hemoglobin reductases (cytochrome B5 reductase plus cytochrome B5). Our data support a model whereby redox-sensitive shuttling of EC α-globin between AHSP and eNOS regulates EC NO degradation and vascular tone.
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http://dx.doi.org/10.1172/JCI99933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205378PMC
November 2018
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