RESUMO
Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Infecções/etiologia , Análise de Célula Única , Animais , Biomarcadores , Imunoprecipitação da Cromatina , Epigênese Genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Memória Imunológica , Infecções/metabolismo , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Fatores de Tempo , TranscriptomaRESUMO
Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.
Assuntos
Hematopoese/fisiologia , Megacariócitos/patologia , Mielofibrose Primária/sangue , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Megacariócitos/fisiologia , Pessoa de Meia-Idade , Mutação , Receptores Imunológicos/genética , Análise de Célula Única/métodosRESUMO
Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
RESUMO
Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.
Assuntos
Células-Tronco Pluripotentes Induzidas , Atrofia Muscular Espinal , Humanos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
BACKGROUND: Epigenetic modification of chromatin plays a pivotal role in regulating gene expression during cell differentiation. The scale and complexity of epigenetic data pose significant challenges for biologists to identify the regulatory events controlling cell differentiation. RESULTS: To reduce the complexity, we developed a package, called Snapshot, for clustering and visualizing candidate cis-regulatory elements (cCREs) based on their epigenetic signals during cell differentiation. This package first introduces a binarized indexing strategy for clustering the cCREs. It then provides a series of easily interpretable figures for visualizing the signal and epigenetic state patterns of the cCREs clusters during the cell differentiation. It can also use different hierarchies of cell types to highlight the epigenetic history specific to any particular cell lineage. We demonstrate the utility of Snapshot using data from a consortium project for ValIdated Systematic IntegratiON (VISION) of epigenomic data in hematopoiesis. CONCLUSION: The package Snapshot can identify all distinct clusters of genomic locations with unique epigenetic signal patterns during cell differentiation. It outperforms other methods in terms of interpreting and reproducing the identified cCREs clusters. The package of Snapshot is available at GitHub: https://github.com/guanjue/Snapshot .
Assuntos
Cromatina , Epigenômica , Epigenômica/métodos , Diferenciação Celular/genética , Epigênese Genética , Análise por ConglomeradosRESUMO
Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.
Assuntos
Proteínas de Transporte/genética , Morfogênese/genética , Deficiência de Vitamina B 12/genética , Vitamina B 12/genética , Proteínas de Peixe-Zebra/genética , Animais , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Camundongos , Mutação/genética , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/patologia , Oxirredutases/genética , Retina/crescimento & desenvolvimento , Retina/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Follicular T helper (Tfh) cells provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated protein (SAP) prevent GC formation because of defective T cell-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally competent "Tfh-like" cells that expressed interleukin-21, Tfh cell markers, and Bcl6 and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wild-type, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2, and Th17 cells could be reprogrammed to obtain Tfh cell characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21, Gata3, and Rorc in Tfh-like and ex vivo Tfh cells and on Bcl6 in non-Tfh cells, supporting the concept of plasticity between Tfh and other Th cell populations.
Assuntos
Diferenciação Celular , Epigênese Genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Interleucinas/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-6 , Proteína Associada à Molécula de Sinalização da Ativação LinfocitáriaRESUMO
Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor GATA1 Erythroid cells from patients with DBA have not been well characterized, and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an ex vivo culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation in comparison with controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translation is a common feature of DBA caused by both RP and GATA1 mutations. This trial was registered at www.clinicaltrials.gov as #NCT00106015.
Assuntos
Anemia de Diamond-Blackfan/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Criança , Pré-Escolar , Células Eritroides/metabolismo , Células Eritroides/patologia , Eritropoese/genética , Feminino , Fator de Transcrição GATA1/genética , Genes Dominantes , Genes Ligados ao Cromossomo X , Humanos , Masculino , Modelos Genéticos , Mutação , Proteínas Ribossômicas/genética , Transcriptoma , Adulto JovemRESUMO
T helper 17 (Th17) cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 cell differentiation is relatively unknown. We demonstrate that CD4(+) T cells lacking Itk, a tyrosine kinase required for full TCR-induced phospholipase C-gamma (PLC-gamma1) activation, exhibit decreased interleukin-17A (IL-17A) expression in vitro and in vivo, despite relatively normal expression of retinoic acid receptor-related orphan receptor-gammaT (ROR-gammaT) and IL-17F. IL-17A expression was rescued by pharmacologically induced Ca(2+) influx or constitutively activated nuclear factor of activated T cells (NFAT). Conversely, decreased TCR stimulation or calcineurin inhibition preferentially reduced IL-17A expression. We further found that the promoter of Il17a but not Il17f has a conserved NFAT binding site that bound NFATc1 in wild-type but not Itk-deficient cells, even though both exhibited open chromatin conformations. Finally, Itk(-/-) mice also showed differential regulation of IL-17A and IL-17F in vivo. Our results suggest that Itk specifically couples TCR signaling to Il17a expression and the differential regulation of Th17 cell cytokines through NFATc1.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Interleucina-17/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Cálcio/imunologia , Cálcio/metabolismo , Citocinas/metabolismo , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Fosfolipase C gama/imunologia , Fosfolipase C gama/metabolismo , Regiões Promotoras Genéticas/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores do Ácido Retinoico/imunologia , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/imunologia , Receptores dos Hormônios Tireóideos/metabolismo , Transdução de Sinais/imunologiaRESUMO
Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramide-laden macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67phox and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67phox to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease.
Assuntos
Doença de Gaucher/genética , Doença de Gaucher/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Biomarcadores , Citofagocitose/genética , Citofagocitose/imunologia , Genótipo , Glucosilceramidase/genética , Humanos , Imuno-Histoquímica , Mutação , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/genética , Explosão Respiratória/imunologiaRESUMO
BACKGROUND: Laminins are heterotrimeric complexes, consisting of α, ß and γ subunits that form a major component of basement membranes and extracellular matrix. Laminin complexes have different, but often overlapping, distributions and functions. METHODS: Under our clinical protocol, NCT00068224, we have performed extensive clinical and neuropsychiatric phenotyping, neuroimaging and molecular analysis in patients with laminin α1 (LAMA1)-associated lamininopathy. We investigated the consequence of mutations in LAMA1 using patient-derived fibroblasts and neuronal cells derived from neuronal stem cells. RESULTS: In this paper we describe individuals with biallelic mutations in LAMA1, all of whom had the cerebellar dysplasia, myopia and retinal dystrophy, in addition to obsessive compulsive traits, tics and anxiety. Patient-derived fibroblasts have impaired adhesion, reduced migration, abnormal morphology and increased apoptosis due to impaired activation of Cdc42, a member of the Rho family of GTPases that is involved in cytoskeletal dynamics. LAMA1 knockdown in human neuronal cells also showed abnormal morphology and filopodia formation, supporting the importance of LAMA1 in neuronal migration, and marking these cells potentially useful tools for disease modelling and therapeutic target discovery. CONCLUSION: This paper broadens the phenotypes associated with LAMA1 mutations. We demonstrate that LAMA1 deficiency can lead to alteration in cytoskeletal dynamics, which may invariably lead to alteration in dendrite growth and axonal formation. Estimation of disease prevalence based on population studies in LAMA1 reveals a prevalence of 1-20 in 1â 000â 000. TRIAL REGISTRATION NUMBER: NCT00068224.
Assuntos
Doenças Cerebelares/metabolismo , Laminina/genética , Mutação , Miopia/metabolismo , Transtorno Obsessivo-Compulsivo/metabolismo , Adulto , Adesão Celular , Movimento Celular , Doenças Cerebelares/genética , Doenças Cerebelares/fisiopatologia , Criança , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Masculino , Miopia/genética , Miopia/fisiopatologia , Neurônios/metabolismo , Neurônios/fisiologia , Transtorno Obsessivo-Compulsivo/genética , Transtorno Obsessivo-Compulsivo/fisiopatologia , Linhagem , Distrofias Retinianas/genética , Distrofias Retinianas/metabolismo , Distrofias Retinianas/fisiopatologia , Síndrome , Transtornos de Tique/genética , Transtornos de Tique/metabolismo , Transtornos de Tique/fisiopatologia , Adulto Jovem , Proteína cdc42 de Ligação ao GTPRESUMO
Mammals express thousands of long noncoding (lnc) RNAs, a few of which are known to function in tissue development. However, the entire repertoire of lncRNAs in most tissues and species is not defined. Indeed, most lncRNAs are not conserved, raising questions about function. We used RNA sequencing to identify 1109 polyadenylated lncRNAs expressed in erythroblasts, megakaryocytes, and megakaryocyte-erythroid precursors of mice, and 594 in erythroblasts of humans. More than half of these lncRNAs were unannotated, emphasizing the opportunity for new discovery through studies of specialized cell types. Analysis of the mouse erythro-megakaryocytic polyadenylated lncRNA transcriptome indicates that ~75% arise from promoters and 25% from enhancers, many of which are regulated by key transcription factors including GATA1 and TAL1. Erythroid lncRNA expression is largely conserved among 8 different mouse strains, yet only 15% of mouse lncRNAs are expressed in humans and vice versa, reflecting dramatic species-specificity. RNA interference assays of 21 abundant erythroid-specific murine lncRNAs in primary mouse erythroid precursors identified 7 whose knockdown inhibited terminal erythroid maturation. At least 6 of these 7 functional lncRNAs have no detectable expression in human erythroblasts, suggesting that lack of conservation between mammalian species does not predict lack of function.
Assuntos
Eritropoese/genética , RNA Longo não Codificante/genética , Trombopoese/genética , Animais , Linhagem da Célula/genética , Sequência Conservada , Elementos Facilitadores Genéticos , Eritroblastos/metabolismo , Humanos , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Interferência de RNA , RNA Longo não Codificante/metabolismo , Especificidade da Espécie , Fatores de Transcrição/metabolismoRESUMO
Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.
Assuntos
Mutação em Linhagem Germinativa , Leucemia Mielomonocítica Juvenil/genética , Mutação de Sentido Incorreto , Linhagem , Proteínas Proto-Oncogênicas c-cbl/genética , Ubiquitinação/genética , Adolescente , Adulto , Criança , Pré-Escolar , Exoma , Feminino , Seguimentos , Humanos , Lactente , Masculino , Modelos Moleculares , Penetrância , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-cbl/químicaRESUMO
DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and erythroblasts (ERYs). Methyl binding domain protein 2 (MBD) enrichment of DNA followed by massively parallel sequencing (MBD-seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSCs, with a 40% reduction in CMPs, and a 67% reduction in ERYs. Only 3% of peaks arise during differentiation, demonstrating a genome-wide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20%-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSCs and CMPs. Elevated genome-wide DNA methylation in HSCs and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSCs. Using de novo motif discovery, we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPA and ELF1, are overrepresented in methylated regions. Our genome-wide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Imunoprecipitação da Cromatina , Mapeamento Cromossômico/métodos , Ilhas de CpG , Proteínas de Ligação a DNA/genética , Eritroblastos/citologia , Eritroblastos/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Proteínas Nucleares/genética , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética , TranscriptomaRESUMO
KIT mutations are the most common secondary mutations in inv(16) acute myeloid leukemia (AML) patients and are associated with poor prognosis. It is therefore important to verify that KIT mutations cooperate with CBFB-MYH11, the fusion gene generated by inv(16), for leukemogenesis. Here, we transduced wild-type and conditional Cbfb-MYH11 knockin (KI) mouse bone marrow (BM) cells with KIT D816V/Y mutations. KIT transduction caused massive BM Lin(-) cell death and fewer colonies in culture that were less severe in the KI cells. D816Y KIT but not wild-type KIT enhanced proliferation in Lin(-) cells and led to more mixed lineage colonies from transduced KI BM cells. Importantly, 60% and 80% of mice transplanted with KI BM cells expressing D816V or D816Y KIT, respectively, died from leukemia within 9 months, whereas no control mice died. Results from limiting dilution transplantations indicate higher frequencies of leukemia-initiating cells in the leukemia expressing mutated KIT. Signaling pathway analysis revealed that p44/42 MAPK and Stat3, but not AKT and Stat5, were strongly phosphorylated in the leukemia cells. Finally, leukemia cells carrying KIT D816 mutations were sensitive to the kinase inhibitor PKC412. Our data provide clear evidence for cooperation between mutated KIT and CBFB-MYH11 during leukemogenesis.
Assuntos
Leucemia/genética , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Western Blotting , Transplante de Medula Óssea , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Progressão da Doença , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.
RESUMO
Knowledge of locations and activities of cis -regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our V al i dated S ystematic I ntegrati on (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
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Mutations in individuals with the lysosomal storage disorder Niemann-Pick disease, type C1 (NPC1) are heterogeneous, not localized to specific protein domains, and not correlated to time of onset or disease severity. We demonstrate direct correlation of the time of neurological symptom onset with the severity of lysosomal defects in NPC1 patient-derived fibroblasts. This is a novel assay for NPC1 individuals that may be predictive of NPC1 disease progression and broadly applicable to other lysosomal disorders.
Assuntos
Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/genética , Adolescente , Adulto , Transporte Biológico/genética , Células Cultivadas , Criança , Pré-Escolar , Progressão da Doença , Feminino , Fibroblastos , Humanos , Lactente , Recém-Nascido , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/genética , Lisossomos/patologia , Masculino , Glicoproteínas de Membrana/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Estrutura Terciária de ProteínaRESUMO
Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency characterized by high incidence of autoantibody-mediated autoimmune complications. Such a feature has been associated with defective suppressor activity of WAS protein-deficient, naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells on responder T cells. However, it remains to be established whether the altered B-cell tolerance reported in WAS patients and Was knockout (WKO) mice is secondary to abnormalities in the direct suppression of B-cell function by nTreg cells or to impaired regulation of T-helper function. Because activated nTreg cells are known to induce granzyme B-mediated B-cell killing, we decided to evaluate the regulatory capabilities of WKO nTregs on B lymphocytes. We found that preactivated WKO nTreg cells failed to effectively suppress B-cell proliferation and that such a defect was associated with reduced killing of B cells and significantly decreased degranulation of granzyme B. Altogether, these results provide additional mechanistic insights into the loss of immune tolerance in WAS.
Assuntos
Linfócitos B/fisiologia , Proliferação de Células , Linfócitos T Reguladores/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Animais , Linfócitos B/metabolismo , Morte Celular/genética , Morte Celular/imunologia , Degranulação Celular/genética , Degranulação Celular/imunologia , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Granzimas/metabolismo , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map trans-factor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1.