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1.
Cell Rep ; 27(11): 3228-3240.e7, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31189107

RESUMO

Human erythropoiesis serves as a paradigm of physiologic cellular differentiation. This process is also of considerable interest for better understanding anemias and identifying new therapies. Here, we apply deep transcriptomic and accessible chromatin profiling to characterize a faithful ex vivo human erythroid differentiation system from hematopoietic stem and progenitor cells. We reveal stage-specific transcriptional states and chromatin accessibility during various stages of erythropoiesis, including 14,260 differentially expressed genes and 63,659 variably accessible chromatin peaks. Our analysis suggests differentiation stage-predominant roles for specific master regulators, including GATA1 and KLF1. We integrate chromatin profiles with common and rare genetic variants associated with erythroid cell traits and diseases, finding that variants regulating different erythroid phenotypes likely act at variable points during differentiation. In addition, we identify a regulator of terminal erythropoiesis, TMCC2, more broadly illustrating the value of this comprehensive analysis to improve our understanding of erythropoiesis in health and disease.

2.
Elife ; 82019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31070582

RESUMO

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.

3.
Nat Genet ; 51(4): 683-693, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858613

RESUMO

Widespread linkage disequilibrium and incomplete annotation of cell-to-cell state variation represent substantial challenges to elucidating mechanisms of trait-associated genetic variation. Here we perform genetic fine-mapping for blood cell traits in the UK Biobank to identify putative causal variants. These variants are enriched in genes encoding proteins in trait-relevant biological pathways and in accessible chromatin of hematopoietic progenitors. For regulatory variants, we explore patterns of developmental enhancer activity, predict molecular mechanisms, and identify likely target genes. In several instances, we localize multiple independent variants to the same regulatory element or gene. We further observe that variants with pleiotropic effects preferentially act in common progenitor populations to direct the production of distinct lineages. Finally, we leverage fine-mapped variants in conjunction with continuous epigenomic annotations to identify trait-cell type enrichments within closely related populations and in single cells. Our study provides a comprehensive framework for single-variant and single-cell analyses of genetic associations.


Assuntos
Hematopoese/genética , Polimorfismo de Nucleotídeo Único/genética , Linhagem da Célula/genética , Cromatina/genética , Mapeamento Cromossômico/métodos , Epigenômica/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Desequilíbrio de Ligação/genética , Fenótipo , Locos de Características Quantitativas/genética , Sequências Reguladoras de Ácido Nucleico/genética
4.
J Exp Med ; 216(5): 1050-1060, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-30914438

RESUMO

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

5.
Cell ; 176(6): 1325-1339.e22, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30827679

RESUMO

Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.

7.
Am J Hum Genet ; 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

8.
Cell ; 173(1): 90-103.e19, 2018 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-29551269

RESUMO

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.

10.
Am J Med Genet C Semin Med Genet ; 172(4): 332-348, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27792859

RESUMO

Congenital anemias comprise a group of blood disorders characterized by a reduction in the number of peripherally circulating erythrocytes. Various genetic etiologies have been identified that affect diverse aspects of erythroid physiology and broadly fall into two main categories: impaired production or increased destruction of mature erythrocytes. Current therapies are largely focused on symptomatic treatment and are often based on transfusion of donor-derived erythrocytes and management of complications. Hematopoietic stem cell transplantation represents the only curative option currently available for the majority of congenital anemias. Recent advances in gene therapy and genome editing hold promise for the development of additional curative strategies for these blood disorders. The relative ease of access to the hematopoietic stem cell compartment, as well as the possibility of genetic manipulation ex vivo and subsequent transplantation in an autologous manner, make blood disorders among the most amenable to cellular therapies. Here we review cell-based and gene therapy approaches, and discuss the limitations and prospects of emerging avenues, including genome editing tools and the use of pluripotent stem cells, for the treatment of congenital forms of anemia. © 2016 Wiley Periodicals, Inc.


Assuntos
Anemia/congênito , Anemia/terapia , Anemia/genética , Transplante de Células/métodos , Transplante de Células/tendências , Edição de Genes , Terapia Genética/métodos , Terapia Genética/tendências , Transplante de Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes/transplante
11.
Proc Natl Acad Sci U S A ; 113(16): 4434-9, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27044088

RESUMO

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.


Assuntos
Anemia de Diamond-Blackfan/metabolismo , Fator de Transcrição GATA1/metabolismo , Mutação , Elementos de Resposta , Transcrição Genética , Anemia de Diamond-Blackfan/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas , Fator de Transcrição GATA1/genética , Humanos , Células K562 , Motivos de Nucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T
12.
Cell Stem Cell ; 18(1): 73-78, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26607381

RESUMO

Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.


Assuntos
Eritrócitos/citologia , Células-Tronco/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Citocinas/metabolismo , Células-Tronco Embrionárias/citologia , Sangue Fetal/citologia , Técnicas Genéticas , Variação Genética , Genoma Humano , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/análise , Humanos , Mutação , Células-Tronco Pluripotentes/citologia , Medicina Regenerativa/métodos
13.
J Clin Invest ; 125(4): 1665-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25705881

RESUMO

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.


Assuntos
5-Aminolevulinato Sintetase/genética , Anemia Diseritropoética Congênita/genética , Anemia Macrocítica/genética , Eritropoese/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto , Mutação Puntual , 5-Aminolevulinato Sintetase/metabolismo , Adulto , Células Cultivadas , Exoma/genética , Feminino , Genes Dominantes , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Hemorragia/etiologia , Humanos , Sobrecarga de Ferro/etiologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Gravidez , Complicações Hematológicas na Gravidez/genética , Ligação Proteica , Conformação Proteica , Transtornos Puerperais/etiologia , Fosfato de Piridoxal/metabolismo , RNA Mensageiro/genética , Reticulócitos/metabolismo , Inativação do Cromossomo X
14.
Am J Hematol ; 90(5): 386-91, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25615569

RESUMO

Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.


Assuntos
Ciclina A2/genética , Citocinese/genética , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Genoma , RNA Mensageiro/genética , Animais , Diferenciação Celular , Tamanho Celular , Ciclina A2/antagonistas & inibidores , Ciclina A2/metabolismo , Células Precursoras Eritroides/citologia , Seguimentos , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Camundongos , Cultura Primária de Células , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
15.
Nat Med ; 20(7): 748-53, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24952648

RESUMO

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.


Assuntos
Anemia de Diamond-Blackfan/genética , Fator de Transcrição GATA1/genética , Biossíntese de Proteínas , Humanos , Mutação , RNA Mensageiro/genética , Proteínas Ribossômicas/genética
16.
Genes Dev ; 26(18): 2075-87, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22929040

RESUMO

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components-a finding resulting from the biological follow-up of unbiased human genetic studies.


Assuntos
Ciclo Celular/fisiologia , Diferenciação Celular , Ciclina D3/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Animais , Contagem de Células , Tamanho Celular , Células Cultivadas , Ciclina D3/genética , Eritropoese/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Camundongos , Camundongos Knockout
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