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1.
Cell ; 183(6): 1665-1681.e18, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33188776

RESUMO

We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-µm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.


Assuntos
Código de Barras de DNA Taxonômico , Genômica , Especificidade de Órgãos/genética , Animais , Automação , Encéfalo/embriologia , Análise por Conglomerados , DNA Complementar/genética , Embrião de Mamíferos/metabolismo , Olho/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Microfluídica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única , Transcriptoma/genética
2.
Mol Cell ; 82(6): 1107-1122.e7, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35303483

RESUMO

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Fator de Processamento U2AF , Grânulos de Estresse , Humanos , Leucemia Mieloide Aguda/genética , Mutação , Síndromes Mielodisplásicas/genética , Sítios de Splice de RNA , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Grânulos de Estresse/metabolismo
3.
Immunity ; 52(6): 1007-1021.e8, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32497523

RESUMO

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.


Assuntos
Adenosina/química , Hematopoese/genética , Hematopoese/imunologia , Imunidade Inata/genética , RNA de Cadeia Dupla/metabolismo , Animais , Biomarcadores , Transtornos da Insuficiência da Medula Óssea/etiologia , Transtornos da Insuficiência da Medula Óssea/metabolismo , Transtornos da Insuficiência da Medula Óssea/patologia , Diferenciação Celular/genética , Modelos Animais de Doenças , Epigênese Genética , Expressão Gênica , Células-Tronco Hematopoéticas , Imunofenotipagem , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , RNA de Cadeia Dupla/química
4.
Nature ; 609(7926): 375-383, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35978191

RESUMO

Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2-5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.


Assuntos
Montagem e Desmontagem da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Animais , Encéfalo/metabolismo , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Epigenômica , Perfilação da Expressão Gênica , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Tonsila Palatina/citologia , Tonsila Palatina/imunologia
5.
Nature ; 606(7914): 585-593, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35483404

RESUMO

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.


Assuntos
COVID-19 , Inflamassomos , Macrófagos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19/patologia , COVID-19/fisiopatologia , COVID-19/virologia , Humanos , Inflamassomos/metabolismo , Interleucina-1 , Interleucina-18 , Pulmão/patologia , Pulmão/virologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/metabolismo , Pneumonia/virologia , Piroptose , Receptores de IgG , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade
6.
Genome Res ; 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36109149

RESUMO

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

7.
Proc Natl Acad Sci U S A ; 119(43): e2121077119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36269862

RESUMO

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.


Assuntos
Neutrófilos , Receptores de Fator Estimulador de Colônias de Granulócitos , Humanos , Camundongos , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos/genética , Citocinas
8.
Mod Pathol ; : 100615, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39322118

RESUMO

Myelodysplastic neoplasms/syndromes (MDS) are a heterogeneous group of biologically distinct entities characterized by variable degrees of ineffective hematopoiesis. Recently, two classification systems (the 5th edition of the WHO Classification and the International Consensus Classification) further sub-characterized MDS into morphologic and genetically defined groups. Accurate diagnosis and subclassification of MDS require a multistep systemic approach. The International Consortium for MDS (icMDS) summarizes a contemporary, practical, and multimodal approach to MDS diagnosis and classification.

9.
Ann Hematol ; 103(1): 105-116, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38036712

RESUMO

Patients with myelodysplastic syndromes/neoplasms (MDS) or acute myeloid leukemia (AML) with hypomethylating agent failure have a poor prognosis. Myeloid-derived suppressor cells (MDSCs) can contribute to MDS progression and mediate resistance to anti-PD1 therapy. As histone deacetylase inhibitors (HDACi) decrease MDSCs in preclinical models, we conducted an investigator-initiated, NCI-Cancer Therapy Evaluation Program-sponsored, multicenter, dose escalation, and expansion phase Ib trial (NCT02936752) of the HDACi entinostat and the anti-PD1 antibody pembrolizumab. Twenty-eight patients (25 MDS and 3 AML) were enrolled. During dose escalation (n=13 patients), there was one dose-limiting toxicity (DLT) on dose level (DL) 1 (G5 pneumonia/bronchoalveolar hemorrhage) and two DLTs at DL 2 (G3 pharyngeal mucositis and G3 anorexia). Per the 3 + 3 dose escalation design, DL 1 (entinostat 8 mg PO days 1 and 15 + pembrolizumab 200 mg IV day 1 every 21 days) was expanded and another 15 patients were enrolled. Hematologic adverse events (AEs) were common. The most common non-hematologic ≥G3 AEs were infection (32%), hypoxia/respiratory failure (11%), and dyspnea (11%). There were no protocol-defined responses among the 28 patients enrolled. Two patients achieved a marrow complete remission (mCR). Using a systems immunology approach with mass cytometry and machine learning analysis, mCR patients had increased classical monocytes and macrophages but there was no significant change of MDSCs. In conclusion, combining entinostat with pembrolizumab in patients with advanced MDS and AML was associated with limited clinical efficacy and substantial toxicity. Absence of an effect on MDSCs could be a potential explanation for the limited efficacy of this combination. ClinicalTrial.gov Identifier: NCT02936752.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Inibidores de Histona Desacetilases/efeitos adversos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/etiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/etiologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos
10.
Nucleic Acids Res ; 50(9): 5299-5312, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35524551

RESUMO

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced UV-crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites, with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.


Assuntos
Nucleotídeos , Precursores de RNA , Fator de Processamento U2AF , Humanos , Nucleotídeos/metabolismo , RNA/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Fator de Processamento U2AF/metabolismo , Uridina/metabolismo
11.
Curr Opin Hematol ; 30(2): 70-77, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36602939

RESUMO

PURPOSE OF REVIEW: The aim of this study was to provide insight into how novel next-generation sequencing (NGS) techniques are set to revolutionize clinical practice. RECENT FINDINGS: Advances in sequencing technologies have focused on improved capture of mutations and reads and cellular resolution. Both short and long read DNA sequencing technology are being refined and combined in novel ways with other multiomic approaches to gain unprecedented biological insight into disease. Single-cell (sc)DNA-seq and integrated scDNA-seq with immunophenotyping provide granular information on disease composition such as clonal hierarchy, co-mutation status, zygosity, clonal diversity and genotype phenotype correlations. These and other techniques can identify rare cell populations providing the opportunity for increased sensitivity in measurable residual disease monitoring and precise characterization of residual clones permitting distinction of leukemic from pre/nonmalignant clones. SUMMARY: Increasing genetics-based mechanistic insights and classification of myeloid diseases along with a decrease in the cost of high-throughput NGS mean novel sequencing technologies are closer to being a reality in standard clinical practice. These technologies are poised to improve diagnostics, our ability to monitor treatment response and minimal residual disease and allow the study of premalignant conditions such as clonal haematopoiesis.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA/métodos , Mutação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estudos de Associação Genética
12.
Haematologica ; 108(2): 522-531, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35979721

RESUMO

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.


Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Animais , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Modelos Animais de Doenças , Leucemia Mieloide Aguda/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Apoptose , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Linhagem Celular Tumoral
13.
Blood ; 136(13): 1477-1486, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32640014

RESUMO

Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are among the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple-concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. In this study, we performed bulk and single-cell analyses of patients with myeloid malignancy who were harboring ≥2 splicing factor mutations, to understand the frequency and basis for the coexistence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4231 patients (q < .001), 0.85% harbored 2 concomitant bona fide splicing factor mutations, ∼50% of which were present in the same individual cells. However, the distribution of mutations in patients with double mutations deviated from that in those with single mutations, with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in those with double-mutations had reduced effects on RNA splicing and/or binding compared with the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating the molecular effects of splicing factor mutations as well as their cooccurrences/exclusivities with one another.


Assuntos
Epistasia Genética , Neoplasias Hematológicas/genética , Mutação , Fatores de Processamento de RNA/genética , Splicing de RNA , Alelos , Análise Mutacional de DNA , Genômica , Humanos , Leucemia Mieloide/genética , Análise de Célula Única
14.
Blood ; 134(18): 1547-1557, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31439541

RESUMO

The mechanisms underlying thrombocytosis in patients with iron deficiency anemia remain unknown. Here, we present findings that support the hypothesis that low iron biases the commitment of megakaryocytic (Mk)-erythroid progenitors (MEPs) toward the Mk lineage in both human and mouse. In MEPs of transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency anemia and thrombocytosis, we observed a Mk bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEPs. Bone marrow transplantation assays suggest that systemic iron deficiency, rather than a local role for Tmprss6-/- in hematopoietic cells, contributes to the MEP lineage commitment bias observed in Tmprss6-/- mice. Nontransgenic mice with acquired iron deficiency anemia also show thrombocytosis and Mk-biased MEPs. Gene expression analysis reveals that messenger RNAs encoding genes involved in metabolic, vascular endothelial growth factor, and extracellular signal-regulated kinase (ERK) pathways are enriched in Tmprss6-/- vs WT MEPs. Corroborating our findings from the murine models of iron deficiency anemia, primary human MEPs exhibit decreased proliferation and Mk-biased commitment after knockdown of transferrin receptor 2, a putative iron sensor. Signal transduction analyses reveal that both human and murine MEP have lower levels of phospho-ERK1/2 in iron-deficient conditions compared with controls. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEPs toward Mk lineage commitment.


Assuntos
Anemia Ferropriva/metabolismo , Diferenciação Celular/fisiologia , Células Progenitoras de Megacariócitos/metabolismo , Megacariócitos/metabolismo , Anemia Ferropriva/complicações , Animais , Proliferação de Células , Humanos , Ferro , Células Progenitoras de Megacariócitos/citologia , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Trombocitose/etiologia , Trombocitose/metabolismo
15.
Blood ; 129(18): 2465-2470, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28348147

RESUMO

Mutations in RNA splicing factors are the single most common class of genetic alterations in myelodysplastic syndrome (MDS) patients. Although much has been learned about how these mutations affect splicing at a global- and transcript-specific level, critical questions about the role of these mutations in MDS development and maintenance remain. Here we present the questions to be addressed in order to understand the unique enrichment of these mutations in MDS.


Assuntos
Epigênese Genética , Síndromes Mielodisplásicas , Fosfoproteínas , Fatores de Processamento de RNA , Splicing de RNA , RNA Mensageiro , Humanos , Síndromes Mielodisplásicas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Nucleic Acids Res ; 45(3): 1281-1296, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28003475

RESUMO

Molecular changes underlying stem cell differentiation are of fundamental interest. scRNA-seq on murine hematopoietic stem cells (HSC) and their progeny MPP1 separated the cells into 3 main clusters with distinct features: active, quiescent, and an un-characterized cluster. Induction of anemia resulted in mobilization of the quiescent to the active cluster and of the early to later stage of cell cycle, with marked increase in expression of certain transcription factors (TFs) while maintaining expression of interferon response genes. Cells with surface markers of long term HSC increased the expression of a group of TFs expressed highly in normal cycling MPP1 cells. However, at least Id1 and Hes1 were significantly activated in both HSC and MPP1 cells in anemic mice. Lineage-specific genes were differently expressed between cells, and correlated with the cell cycle stages with a specific augmentation of erythroid related genes in the G2/M phase. Most lineage specific TFs were stochastically expressed in the early precursor cells, but a few, such as Klf1, were detected only at very low levels in few precursor cells. The activation of these factors may correlate with stages of differentiation. This study reveals effects of cell cycle progression on the expression of lineage specific genes in precursor cells, and suggests that hematopoietic stress changes the balance of renewal and differentiation in these homeostatic cells.


Assuntos
Perfilação da Expressão Gênica/métodos , Células-Tronco Hematopoéticas/fisiologia , Análise de Célula Única/métodos , Anemia/genética , Animais , Linhagem da Célula/genética , Eritropoese/genética , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA/métodos , Fatores de Transcrição HES-1/genética , Fatores de Transcrição/genética
17.
Curr Opin Hematol ; 30(2): 29, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36752625
18.
Blood ; 128(14): 1829-1833, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27543436

RESUMO

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) can reconstitute a human hemato-lymphoid system when transplanted into immunocompromised mice. Although fetal liver-derived and cord blood-derived CD34+ cells lead to high engraftment levels, engraftment of mobilized, adult donor-derived CD34+ cells has remained poor. We generated so-called MSTRG and MISTRG humanized mice on a Rag2-/-Il2rg-/- background carrying a transgene for human signal regulatory protein α (SIRPα) and human homologs of the cytokine macrophage colony-stimulating factor, thrombopoietin, with or without interleukin-3 and granulocyte-macrophage colony-stimulating factor under murine promoters. Here we transplanted mobilized peripheral blood (PB) CD34+ cells in sublethally irradiated newborn and adult recipients. Human hematopoietic engraftment levels were significantly higher in bone marrow (BM), spleen, and PB in newborn transplanted MSTRG/MISTRG as compared with nonobese diabetic/severe combined immunodeficient Il2rg-/- or human SIRPα-transgenic Rag2-/-Il2rg-/- recipients. Furthermore, newborn transplanted MSTRG/MISTRG mice supported higher engraftment levels of human phenotypically defined HSPCs in BM, T cells in the thymus, and myeloid cells in nonhematopoietic organs such as liver, lung, colon, and skin, approximating the levels in the human system. Similar results were obtained in adult recipient mice. Thus, human cytokine knock-in mice might open new avenues for personalized studies of human pathophysiology of the hematopoietic and immune system in vivo.


Assuntos
Antígenos CD34/metabolismo , Citocinas/metabolismo , Técnicas de Introdução de Genes , Animais , Animais Recém-Nascidos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Tecido Linfoide/metabolismo , Camundongos Transgênicos
19.
J Immunol ; 197(3): 910-22, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27342846

RESUMO

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted E-twenty-six (ETS) factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Granulócitos/citologia , Receptores Citoplasmáticos e Nucleares/biossíntese , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Receptor de Lamina B
20.
Blood ; 126(4): 520-30, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-25964668

RESUMO

The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3(-/-) embryos, which are embryonic lethal by E18.5. Tmod3(-/-) embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3(-/-) fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3(-/-) MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3(-/-) MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3(-/-) MKs exhibit F-actin disturbances, and Tmod3(-/-) MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3(-/-) MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing.


Assuntos
Citoesqueleto de Actina/patologia , Plaquetas/patologia , Membrana Celular/patologia , Embrião de Mamíferos/patologia , Hemorragia/etiologia , Megacariócitos/patologia , Tropomodulina/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Apoptose , Plaquetas/metabolismo , Western Blotting , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Citoplasma/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Imunofluorescência , Hematopoese/fisiologia , Hemorragia/metabolismo , Hemorragia/patologia , Imunoprecipitação , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Ploidias , Polimerização
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