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1.
J Clin Immunol ; 43(6): 1052-1066, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37407873

RESUMO

MECOM deficiency is a recently identified inborn error of immunity and inherited bone marrow failure syndrome caused by haploinsufficiency of the hematopoietic transcription factor MECOM. It is unique among inherited bone marrow failure syndromes, many of which present during later childhood or adolescence, because of the early age of onset and severity of the pancytopenia, emphasizing the importance and gene dose dependency of MECOM during hematopoiesis. B-cell lymphopenia and hypogammaglobulinemia have been described in a subset of patients with MECOM deficiency. While the mechanisms underlying the B-cell deficiency are currently unknown, recent work has provided mechanistic insights into the function of MECOM in hematopoietic stem cell (HSC) maintenance. MECOM binds to regulatory enhancers that control the expression of a network of genes essential for HSC maintenance and self-renewal. Heterozygous mutations, as seen in MECOM-deficient bone marrow failure, lead to dysregulated MECOM network expression. Extra-hematopoietic manifestations of MECOM deficiency, including renal and cardiac anomalies, radioulnar synostosis, clinodactyly, and hearing loss, have been reported. Individuals with specific genotypes have some of the systemic manifestations with isolated mild thrombocytopenia or without hematologic abnormalities, highlighting the tissue specificity of mutations in some MECOM domains. Those infants with MECOM-associated bone marrow failure require HSC transplantation for survival. Here, we review the expanding cohort of patient phenotypes and accompanying genotypes resulting in MECOM deficiency, and the proposed mechanisms underlying MECOM regulation of human HSC maintenance and B-cell development.


Assuntos
Pancitopenia , Trombocitopenia , Humanos , Criança , Pancitopenia/genética , Fatores de Transcrição/genética , Transtornos da Insuficiência da Medula Óssea , Células-Tronco Hematopoéticas , Regulação da Expressão Gênica , Síndrome Congênita de Insuficiência da Medula Óssea , Hematopoese/genética , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo
3.
Nat Immunol ; 24(1): 69-83, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36522544

RESUMO

The molecular regulation of human hematopoietic stem cell (HSC) maintenance is therapeutically important, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. Here, we have studied a rare genetic disorder due to MECOM haploinsufficiency, characterized by an early-onset absence of HSCs in vivo. By generating a faithful model of this disorder in primary human HSCs and coupling functional studies with integrative single-cell genomic analyses, we uncover a key transcriptional network involving hundreds of genes that is required for HSC maintenance. Through our analyses, we nominate cooperating transcriptional regulators and identify how MECOM prevents the CTCF-dependent genome reorganization that occurs as HSCs differentiate. We show that this transcriptional network is co-opted in high-risk leukemias, thereby enabling these cancers to acquire stem cell properties. Collectively, we illuminate a regulatory network necessary for HSC self-renewal through the study of a rare experiment of nature.


Assuntos
Leucemia , Neoplasias , Humanos , Células-Tronco Hematopoéticas , Leucemia/genética , Fatores de Transcrição/genética , Diferenciação Celular/genética
4.
Blood Adv ; 7(7): 1312-1325, 2023 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-36306384

RESUMO

Sepsis continues to be associated with high morbidity and mortality. Currently, sepsis is managed only conservatively. In sepsis, a substantial number of neutrophils is required, leading to accelerated neutrophil production. Immature neutrophils are released into the circulation to meet a demand, despite their less effective functioning in microbial eradication. Although an intervention to provide more mature neutrophils may serve as a potential sepsis treatment, the mechanism of neutrophil differentiation and maturation remains poorly understood. We discovered that CD11c, traditionally known as a dendritic cell marker, was expressed in neutrophils and regulated neutrophil maturation and effector functions. In the absence of CD11c, neutrophil maturation was impaired in the bone marrow, concomitant with a significant increase in the proliferation and apoptosis of preneutrophils, associated with less effector functions. Under lipopolysaccharide challenge, inducing an emergent neutrophil production in the bone marrow, CD11c deficiency exaggerated the release of immature neutrophils into the circulation, associated with a significant proliferation and apoptosis of preneutrophils. In contrast, constitutively active CD11c knock-in mice showed accelerated neutrophil maturation associated with enhanced effector functions, which further supports the notion that CD11c regulates neutrophil maturation. Furthermore, the constitutively active CD11c knock-in mice offered enhanced bacterial eradication. Taken together, we discovered that CD11c was critical for the regulation of neutrophil maturation, and CD11c activation could serve as a potential target for sepsis treatment.


Assuntos
Neutrófilos , Sepse , Animais , Camundongos , Medula Óssea
5.
Nat Genet ; 54(10): 1449-1450, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36138230
6.
PLoS Genet ; 17(10): e1009835, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34634037

RESUMO

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and ß-thalassemia. BCL11A has been identified as a key regulator of HbF silencing, although its precise mechanisms of action remain incompletely understood. Recent studies have identified pathogenic mutations that cause heterozygous loss-of-function of BCL11A and result in a distinct neurodevelopmental disorder that is characterized by persistent HbF expression. While the majority of cases have deletions or null mutations causing haploinsufficiency of BCL11A, several missense variants have also been identified. Here, we perform functional studies on these variants to uncover specific liabilities for BCL11A's function in HbF silencing. We find several mutations in an N-terminal C2HC zinc finger that increase proteasomal degradation of BCL11A. We also identify a distinct C-terminal missense variant in the fifth zinc finger domain that we demonstrate causes loss-of-function through disruption of DNA binding. Our analysis of missense variants causing loss-of-function in vivo illuminates mechanisms by which BCL11A silences HbF and also suggests potential therapeutic avenues for HbF induction to treat sickle cell disease and ß-thalassemia.


Assuntos
Hemoglobina Fetal/genética , Inativação Gênica/fisiologia , Mutação/genética , Proteínas Repressoras/genética , Anemia Falciforme/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Células K562 , Dedos de Zinco/genética , Talassemia beta/genética
7.
Nat Commun ; 12(1): 4991, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404810

RESUMO

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


Assuntos
Edição de Genes , Hemoglobinas/genética , Hemoglobinas/metabolismo , Sistemas CRISPR-Cas , Cromatina , Cromossomos , Proteínas de Ligação a DNA/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Expressão Gênica , Globinas , Humanos , Mutação , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Globinas beta/genética
8.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857290

RESUMO

Advances in genome sequencing have resulted in the identification of the causes for numerous rare diseases. However, many cases remain unsolved with standard molecular analyses. We describe a family presenting with a phenotype resembling inherited thrombocytopenia 2 (THC2). THC2 is generally caused by single nucleotide variants that prevent silencing of ANKRD26 expression during hematopoietic differentiation. Short-read whole-exome and genome sequencing approaches were unable to identify a causal variant in this family. Using long-read whole-genome sequencing, a large complex structural variant involving a paired-duplication inversion was identified. Through functional studies, we show that this structural variant results in a pathogenic gain-of-function WAC-ANKRD26 fusion transcript. Our findings illustrate how complex structural variants that may be missed by conventional genome sequencing approaches can cause human disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Polimorfismo de Nucleotídeo Único/genética , Trombocitopenia/genética , Adolescente , Adulto , Idoso , Linhagem Celular , Linhagem Celular Tumoral , Criança , Quebra Cromossômica , Transtornos Cromossômicos/genética , Exoma/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Trombocitopenia/congênito
9.
Blood Adv ; 4(24): 6086-6097, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33351105

RESUMO

ß2 integrins are well-known leukocyte adhesion molecules consisting of 4 members: CD11a-d. Their known biological functions range widely from leukocyte recruitment, phagocytosis, to immunological synapse formation, but the studies have been primarily focused on CD11a and CD11b. CD11c is 1 of the 4 members and is extremely homologous to CD11b. It has been well known as a dendritic cell marker, but the characterization of its function has been limited. We found that CD11c was expressed on the short-term hematopoietic stem cells and multipotent progenitor cells. The lack of CD11c did not affect the number of hematopoietic stem and progenitor cells (HSPCs) in healthy CD11c knockout mice. Different from other ß2 integrin members, however, CD11c deficiency was associated with increased apoptosis and significant loss of HSPCs in sepsis and bone marrow transplantation. Although integrins are generally known for their overlapping and redundant roles, we showed that CD11c had a distinct role of regulating the expansion of HSPCs under stress. This study shows that CD11c, a well-known dendritic cell marker, is expressed on HSPCs and serves as their functional regulator. CD11c deficiency leads to the loss of HSPCs via apoptosis in sepsis and bone marrow transplantation.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Animais , Antígenos CD11 , Antígeno CD11c , Antígenos CD18/genética , Camundongos , Camundongos Knockout
11.
Cell Stem Cell ; 27(2): 195-197, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32763180

RESUMO

As humans age, hematopoietic stem cells (HSCs) occasionally acquire mutations in genes including DNMT3A that enable them to outcompete other HSCs and increase leukemia risk. In this issue of Cell Stem Cell, Tovy et al. (2020) report a previously uncharacterized mechanism by which DNMT3A loss confers increased fitness to HSCs by analyzing a rare experiment of nature.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Hematopoese , Hematopoiese Clonal , Células Clonais , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética , Hematopoese/genética , Humanos , Mutação/genética
12.
Cell ; 180(1): 6, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31951520

RESUMO

Erythropoietin (EPO) production in the kidney is regulated by the oxygen-sensing transcription factor HIF-1α, which is degraded under normoxic conditions by HIF-prolyl hydroxylase (HIF-PHD). Inhibition of HIF-PHD by roxadustat leads to increased EPO production, better iron absorption, and amelioration of anemia in chronic kidney disease (CKD).


Assuntos
Anemia/terapia , Glicina/análogos & derivados , Fator 1 Induzível por Hipóxia/metabolismo , Isoquinolinas/uso terapêutico , Anemia/metabolismo , Glicina/uso terapêutico , Humanos , Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Prolil Hidroxilases/efeitos dos fármacos , Prolil Hidroxilases/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo
13.
Nucleic Acids Res ; 42(6): e47, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24442582

RESUMO

Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/metabolismo , Software , Algoritmos , Sequência de Bases , DNA/química , DNA/metabolismo , Células HEK293 , Humanos
14.
Nucleic Acids Res ; 42(2): 1365-78, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24157834

RESUMO

Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and ß-thalassemia are common genetic disorders caused by mutations in ß-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human ß-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous ß-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human γ-globin, a pharmacologic target in sickle cell disease therapy. Using the ß-globin and γ-globin TALENs, we generated cell lines that express GFP under the control of the endogenous ß-globin promoter and tdTomato under the control of the endogenous γ-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous ß- and γ-globin genes and identified several that preferentially upregulate γ-globin expression.


Assuntos
Desoxirribonucleases/metabolismo , Marcação de Genes , Recombinação Homóloga , Globinas beta/genética , gama-Globinas/genética , Desoxirribonucleases/química , Corantes Fluorescentes , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Engenharia de Proteínas
15.
Science ; 342(6155): 253-7, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-24115442

RESUMO

Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the ß-hemoglobinopathies.


Assuntos
Proteínas de Transporte/genética , Elementos Facilitadores Genéticos , Células Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Regulação da Expressão Gênica , Hemoglobinopatias/genética , Proteínas Nucleares/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Hemoglobina Fetal/genética , Marcação de Genes , Engenharia Genética , Variação Genética , Estudo de Associação Genômica Ampla , Hemoglobinopatias/terapia , Humanos , Camundongos , Células Precursoras de Linfócitos B/metabolismo , Proteínas Repressoras , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Mol Ther ; 21(4): 786-95, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23358186

RESUMO

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5α, chimeric human-rhesus TRIM5α, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.


Assuntos
Infecções por HIV/imunologia , Linfócitos T/virologia , Antígenos CD4/metabolismo , Linhagem Celular , Humanos , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo
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