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1.
Cell ; 181(2): 382-395.e21, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32246942

RESUMO

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.


Assuntos
Comunicação Celular/genética , Doença/genética , Oligodendroglia/metabolismo , Animais , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/metabolismo , Neurônios/metabolismo , Oligodendroglia/fisiologia , Fatores de Risco
2.
Nature ; 522(7555): 216-20, 2015 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-25896324

RESUMO

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.


Assuntos
Clobetasol/farmacologia , Miconazol/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camadas Germinativas/patologia , Humanos , Lisofosfatidilcolinas , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Esclerose Múltipla/patologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores de Glucocorticoides/metabolismo , Regeneração/efeitos dos fármacos , Técnicas de Cultura de Tecidos
3.
Blood ; 139(6): 802-804, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-35142852
4.
J Neurovirol ; 22(3): 336-48, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26631080

RESUMO

Theiler's murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown. Here, we employed pluripotent stem cell technologies to generate pure populations of mouse OPCs to study the temporal and molecular effects of TMEV infection. Global transcriptome analysis of RNA sequencing data revealed that TMEV infection of OPCs caused significant up-regulation of 1926 genes, whereas 1853 genes were significantly down-regulated compared to uninfected cells. Pathway analysis revealed that TMEV disrupted many genes required for OPC growth and maturation. Down-regulation of Olig2, a transcription factor necessary for OPC proliferation, was confirmed by real-time PCR, immunofluorescence microscopy, and western blot analysis. Depletion of Olig2 was not found to be specific to viral strain and did not require expression of the leader (L) protein, which is a multifunctional protein important for persistence, modulation of gene expression, and cell death. These data suggest that direct infection of OPCs by TMEV may inhibit remyelination during the chronic phase of TMEV-induced demyelinating disease.


Assuntos
Doenças Desmielinizantes/virologia , Interações Hospedeiro-Patógeno , Células Precursoras de Oligodendrócitos/virologia , Fator de Transcrição 2 de Oligodendrócitos/genética , Células-Tronco Pluripotentes/virologia , Theilovirus/genética , Animais , Diferenciação Celular , Linhagem Celular , Cricetinae , Doenças Desmielinizantes/patologia , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Anotação de Sequência Molecular , Células Precursoras de Oligodendrócitos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/deficiência , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Theilovirus/metabolismo , Transcriptoma
5.
Nat Methods ; 8(11): 957-62, 2011 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-21946668

RESUMO

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.


Assuntos
Oligodendroglia/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Humanos
6.
Cell Genom ; 3(6): 100318, 2023 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-37388913

RESUMO

Although vast numbers of putative gene regulatory elements have been cataloged, the sequence motifs and individual bases that underlie their functions remain largely unknown. Here, we combine epigenetic perturbations, base editing, and deep learning to dissect regulatory sequences within the exemplar immune locus encoding CD69. We converge on a ∼170 base interval within a differentially accessible and acetylated enhancer critical for CD69 induction in stimulated Jurkat T cells. Individual C-to-T base edits within the interval markedly reduce element accessibility and acetylation, with corresponding reduction of CD69 expression. The most potent base edits may be explained by their effect on regulatory interactions between the transcriptional activators GATA3 and TAL1 and the repressor BHLHE40. Systematic analysis suggests that the interplay between GATA3 and BHLHE40 plays a general role in rapid T cell transcriptional responses. Our study provides a framework for parsing regulatory elements in their endogenous chromatin contexts and identifying operative artificial variants.

7.
Nat Commun ; 14(1): 448, 2023 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-36707513

RESUMO

Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.


Assuntos
Cromatina , Leucemia , Humanos , Cromatina/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Montagem e Desmontagem da Cromatina , Leucemia/tratamento farmacológico , Leucemia/genética , Histona Acetiltransferases/metabolismo
8.
Cell Genom ; 3(7): 100321, 2023 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-37492096

RESUMO

Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents.

9.
Nat Genet ; 54(10): 1504-1513, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36195755

RESUMO

Epigenomic maps identify gene regulatory elements by their chromatin state. However, prevailing short-read sequencing methods cannot effectively distinguish alleles, evaluate the interdependence of elements in a locus or capture single-molecule dynamics. Here, we apply targeted nanopore sequencing to profile chromatin accessibility and DNA methylation on contiguous ~100-kb DNA molecules that span loci relevant to development, immunity and imprinting. We detect promoters, enhancers, insulators and transcription factor footprints on single molecules based on exogenous GpC methylation. We infer relationships among dynamic elements within immune loci, and order successive remodeling events during T cell stimulation. Finally, we phase primary sequence and regulatory elements across the H19/IGF2 locus, uncovering primate-specific features. These include a segmental duplication that stabilizes the imprinting control region and a noncanonical enhancer that drives biallelic IGF2 expression in specific contexts. Our study advances emerging strategies for phasing gene regulatory landscapes and reveals a mechanism that overrides IGF2 imprinting in human cells.


Assuntos
Impressão Genômica , RNA Longo não Codificante , Alelos , Animais , Cromatina/genética , DNA/metabolismo , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Humanos , Fator de Crescimento Insulin-Like II/genética , RNA Longo não Codificante/genética , Fatores de Transcrição/genética
10.
Cell Chem Biol ; 26(4): 593-599.e4, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30773481

RESUMO

Small molecules that promote oligodendrocyte formation have been identified in "drug repurposing" screens to nominate candidate therapeutics for diseases in which myelin is lost, including multiple sclerosis. We recently reported that many such molecules enhance oligodendrocyte formation not by their canonical targets but by inhibiting a narrow range of enzymes in cholesterol biosynthesis. Here we identify enhancers of oligodendrocyte formation obtained by screening a structurally diverse library of 10,000 small molecules. Identification of the cellular targets of these validated hits revealed a majority inhibited the cholesterol biosynthesis enzymes CYP51, TM7SF2, or EBP. In addition, evaluation of analogs led to identification of CW3388, a potent EBP-inhibiting enhancer of oligodendrocyte formation poised for further optimization.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Oligodendroglia/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Esteroide Isomerases/antagonistas & inibidores , Inibidores de 14-alfa Desmetilase/química , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Camundongos , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Oxirredutases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Esteroide Isomerases/metabolismo
11.
Nat Biotechnol ; 36(2): 179-189, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29251726

RESUMO

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.


Assuntos
Sistemas CRISPR-Cas/genética , Epistasia Genética/genética , Testes Genéticos , RNA Guia de Cinetoplastídeos/genética , Apoptose/genética , Técnicas de Inativação de Genes , Marcação de Genes , Humanos , Aprendizado de Máquina , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Transdução de Sinais/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Proteína Supressora de Tumor p53/genética
12.
Cell Stem Cell ; 20(2): 233-246.e7, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-27989769

RESUMO

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.


Assuntos
Montagem e Desmontagem da Cromatina , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Acetilação/efeitos dos fármacos , Sequência de Bases , Biomarcadores Tumorais/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Elementos Facilitadores Genéticos/genética , Glioblastoma/metabolismo , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Metilação/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
13.
Methods Mol Biol ; 1074: 1-13, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23975801

RESUMO

Mouse epiblast stem cells (EpiSCs) are pluripotent embryonic cells that can be used to interrogate developmental transitions that occur during gastrulation. EpiSCs can also be robustly differentiated into functional somatic and germ cell derivatives making them a useful tool for studying development and regenerative medicine. Typically, mouse EpiSCs are isolated from the early postimplantation epiblast around 5.5 days post coitum (dpc). This chapter describes the methods for isolation of mouse EpiSCs from preimplantation blastocyst-stage mouse embryos (3.5 dpc). This technique enables the routine ability to derive EpiSC lines as it is much less labor intensive than isolation of EpiSCs from the postimplantation epiblast. We also detail relevant assays used to characterize new EpiSC lines and distinguish them from mouse embryonic stem cells.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Animais , Diferenciação Celular , Linhagem Celular , Gastrulação/genética , Células Germinativas/citologia , Camundongos
14.
Nat Biotechnol ; 31(5): 426-33, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23584611

RESUMO

Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to induced oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies.


Assuntos
Fibroblastos/citologia , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Fibroblastos/fisiologia , Melhoramento Genético/métodos , Camundongos , Transplante de Células-Tronco/métodos
15.
Cell Stem Cell ; 8(3): 318-25, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21362571

RESUMO

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.


Assuntos
Blastocisto/citologia , Separação Celular/métodos , Camadas Germinativas/citologia , Células-Tronco/citologia , Animais , Sequência de Bases , Blastocisto/metabolismo , Diferenciação Celular/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Dados de Sequência Molecular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco/metabolismo
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