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1.
Br J Haematol ; 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38887897

RESUMO

The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

2.
Blood Adv ; 7(10): 2082-2093, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-36649572

RESUMO

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because patients with GATA2 haploinsufficiency have inborn mutations, prenatal defects are likely to influence disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos remain unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We showed that Gata2+/- HSPCs initiate, but cannot fully activate, hematopoietic programming during EHT. In addition, due to the reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress endothelial identity to complete maturation. Finally, we showed that hematopoietic-specific induction of gfi1b could restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates the pivotal role of Gata2 in the regulation of the transcriptional network governing HSPC identity throughout the EHT.


Assuntos
Deficiência de GATA2 , Peixe-Zebra , Gravidez , Feminino , Animais , Camundongos , Peixe-Zebra/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo
3.
Blood Adv ; 5(13): 2687-2700, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34170285

RESUMO

The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs. Gata2b-deficient zebrafish have a reduction in embryonic definitive hematopoietic stem and progenitor cell (HSPC) numbers, but are viable. This allows us to uniquely study the role of GATA2 in adult hematopoiesis. gata2b mutants have impaired myeloid lineage differentiation. Interestingly, this defect arises not in granulocyte-monocyte progenitors, but in HSPCs. Gata2b-deficient HSPCs showed impaired progression of the myeloid transcriptional program, concomitant with increased coexpression of lymphoid genes. This resulted in a decrease in myeloid-programmed progenitors and a relative increase in lymphoid-programmed progenitors. This shift in the lineage output could function as an escape mechanism to avoid a block in lineage differentiation. Our study helps to deconstruct the functions of GATA2 during hematopoiesis and shows that lineage differentiation flows toward a lymphoid lineage in the absence of Gata2b.


Assuntos
Células-Tronco Hematopoéticas , Peixe-Zebra , Animais , Diferenciação Celular , Fator de Transcrição GATA2/genética , Hematopoese , Camundongos , Monócitos , Proteínas de Peixe-Zebra
4.
Sci Rep ; 11(1): 7505, 2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33820917

RESUMO

Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon.


Assuntos
Cálcio/metabolismo , Desmina/genética , Técnicas de Inativação de Genes , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Peixe-Zebra/genética , Animais , Sequência de Bases , Desmina/metabolismo , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/ultraestrutura , Mutação/genética , Junção Neuromuscular/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/embriologia
5.
Commun Biol ; 3(1): 71, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054973

RESUMO

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2aΔi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2aΔi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.


Assuntos
Reprogramação Celular/genética , Sequência Conservada , Endotélio/metabolismo , Elementos Facilitadores Genéticos , Fator de Transcrição GATA2/genética , Hematopoese/genética , Deleção de Sequência , Fatores Etários , Animais , Sequência de Bases , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Loci Gênicos , Células-Tronco Hematopoéticas/metabolismo , Peixe-Zebra
6.
Front Genome Ed ; 2: 602182, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-34713225

RESUMO

Inherited bone marrow failure syndromes (IBMFS) are monogenetic disorders that result in a reduction of mature blood cell formation and predisposition to leukemia. In children with myeloid leukemia the gene most often mutated is Gata binding protein 2 (GATA2) and 80% of patients with GATA2 mutations develop myeloid malignancy before the age of forty. Although GATA2 is established as one of the key regulators of embryonic and adult hematopoiesis, the mechanisms behind the leukemia predisposition in GATA2 haploinsufficiencies is ambiguous. The only curative treatment option currently available is allogeneic hematopoietic stem cell transplantation (allo-SCT). However, allo-SCT can only be applied at a relatively late stage of the disease as its applicability is compromised by treatment related morbidity and mortality (TRM). Alternatively, autologous hematopoietic stem cell transplantation (auto-SCT), which is associated with significantly less TRM, might become a treatment option if repaired hematopoietic stem cells would be available. Here we discuss the recent literature on leukemia predisposition syndromes caused by GATA2 mutations, current knowledge on the function of GATA2 in the hematopoietic system and advantages and pitfalls of potential treatment options provided by genome editing.

7.
J Am Soc Nephrol ; 25(8): 1653-61, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24610927

RESUMO

Nephronophthisis (NPHP) is one of the most common genetic causes of CKD; however, the underlying genetic abnormalities have been established in <50% of patients. We performed genome-wide analysis followed by targeted resequencing in a Turkish consanguineous multiplex family and identified a canonic splice site mutation in ANKS6 associated with an NPHP-like phenotype. Furthermore, we identified four additional ANKS6 variants in a cohort of 56 unrelated patients diagnosed with CKD due to nephronophthisis, chronic GN, interstitial nephritis, or unknown etiology. Immunohistochemistry in human embryonic kidney tissue demonstrated that the expression patterns of ANKS6 change substantially during development. Furthermore, we detected increased levels of both total and active ß-catenin in precystic tubuli in Han:SPRD Cy/+ rats. Overall, these data indicate the importance of ANKS6 in human kidney development and suggest a mechanism by which mutations in ANKS6 may contribute to an NPHP-like phenotype in humans.


Assuntos
Doenças Renais Císticas/genética , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Mutação/genética , Proteínas Nucleares/genética , Fenótipo , Adolescente , Adulto , Criança , Estudos de Coortes , Feminino , Humanos , Lactente , Doenças Renais Císticas/complicações , Doenças Renais Císticas/patologia , Masculino , Pessoa de Meia-Idade , Linhagem , Turquia
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