Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
1.
Nature ; 619(7969): 348-356, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37344597

RESUMO

The role of B cells in anti-tumour immunity is still debated and, accordingly, immunotherapies have focused on targeting T and natural killer cells to inhibit tumour growth1,2. Here, using high-throughput flow cytometry as well as bulk and single-cell RNA-sequencing and B-cell-receptor-sequencing analysis of B cells temporally during B16F10 melanoma growth, we identified a subset of B cells that expands specifically in the draining lymph node over time in tumour-bearing mice. The expanding B cell subset expresses the cell surface molecule T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) and a unique transcriptional signature, including multiple co-inhibitory molecules such as PD-1, TIM-3, TIGIT and LAG-3. Although conditional deletion of these co-inhibitory molecules on B cells had little or no effect on tumour burden, selective deletion of Havcr1 in B cells both substantially inhibited tumour growth and enhanced effector T cell responses. Loss of TIM-1 enhanced the type 1 interferon response in B cells, which augmented B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion of tumour-specific effector T cells. Our results demonstrate that manipulation of TIM-1-expressing B cells enables engagement of the second arm of adaptive immunity to promote anti-tumour immunity and inhibit tumour growth.


Assuntos
Linfócitos B , Melanoma , Animais , Camundongos , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Linfocitária , Melanoma/imunologia , Melanoma/patologia , Melanoma/prevenção & controle , Linfócitos T/citologia , Linfócitos T/imunologia , Citometria de Fluxo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Linfonodos/citologia , Linfonodos/imunologia , Apresentação de Antígeno , Receptores de Antígenos de Linfócitos B/genética , Análise da Expressão Gênica de Célula Única , Carga Tumoral , Interferon Tipo I
2.
Nature ; 599(7886): 684-691, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34789882

RESUMO

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.


Assuntos
Encéfalo/citologia , Células/classificação , Montagem e Desmontagem da Cromatina , Cromatina/química , Cromatina/genética , Genes , Conformação Molecular , Animais , Sítios de Ligação , Células/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Família Multigênica/genética , Neurônios/classificação , Neurônios/metabolismo , Desnaturação de Ácido Nucleico , Fatores de Transcrição/metabolismo
3.
Nature ; 592(7852): 128-132, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33536623

RESUMO

Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines1. In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum-even at steady state-reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling.


Assuntos
Imunidade Inata/imunologia , Linfócitos/imunologia , Linfócitos/patologia , Psoríase/imunologia , Psoríase/patologia , Pele/imunologia , Pele/patologia , Animais , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Modelos Animais de Doenças , Feminino , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-23/imunologia , Análise de Classes Latentes , Linfócitos/classificação , Masculino , Camundongos , Psoríase/genética , RNA Citoplasmático Pequeno/genética , Reprodutibilidade dos Testes , Fatores de Tempo
4.
Mol Cell ; 73(5): 930-945.e4, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30709709

RESUMO

R-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.


Assuntos
Ilhas de CpG , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Conformação de Ácido Nucleico , Complexo Repressor Polycomb 1/genética , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/genética
5.
Mol Cell ; 65(3): 373-375, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28157500

RESUMO

Polycomb proteins are well-known epigenetic repressors with unexplained roles in chromatin folding. In this issue of Molecular Cell, Kundu et al. (2017) investigate the structures of PRC1-mediated domains in stem cells and probe their changes upon differentiation and in PRC knockouts.


Assuntos
Células-Tronco Embrionárias/citologia , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Humanos , Complexo Repressor Polycomb 1/genética , Domínios Proteicos
6.
Mol Syst Biol ; 13(10): 946, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038337

RESUMO

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells.


Assuntos
Neurônios Dopaminérgicos/citologia , Genes Controladores do Desenvolvimento , Células-Tronco Embrionárias Murinas/citologia , Proteínas do Grupo Polycomb/metabolismo , RNA Polimerase II/metabolismo , Animais , Diferenciação Celular , Neurônios Dopaminérgicos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Fatores de Transcrição/genética
7.
Science ; 376(6592): eabi8175, 2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-35482859

RESUMO

Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanócitos/metabolismo , Melanoma/patologia , Mutação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Microambiente Tumoral/genética
8.
Nat Med ; 27(3): 546-559, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33654293

RESUMO

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.


Assuntos
COVID-19/epidemiologia , COVID-19/genética , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/fisiologia , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Internalização do Vírus , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , COVID-19/virologia , Catepsina L/genética , Catepsina L/metabolismo , Conjuntos de Dados como Assunto/estatística & dados numéricos , Demografia , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Humanos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Análise de Sequência de RNA/métodos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única/métodos
9.
Cell Stem Cell ; 20(5): 573-575, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28475876

RESUMO

Enhancers exist in different epigenetic states: active, primed, or poised. However, it is not yet understood how the different enhancer states influence gene activation. In this issue of Cell Stem Cell, Cruz-Molina et al. (2017) unravel how poised enhancers activate anterior neural genes and the role of Polycomb proteins in enhancer-promoter contacts.


Assuntos
Elementos Facilitadores Genéticos/genética , Neurônios/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Epigênese Genética/genética , Humanos , Camundongos , Modelos Teóricos , Complexo Repressor Polycomb 2/genética
10.
Nat Commun ; 8(1): 36, 2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28652613

RESUMO

Polycomb repressive complexes (PRCs) are important histone modifiers, which silence gene expression; yet, there exists a subset of PRC-bound genes actively transcribed by RNA polymerase II (RNAPII). It is likely that the role of Polycomb repressive complex is to dampen expression of these PRC-active genes. However, it is unclear how this flipping between chromatin states alters the kinetics of transcription. Here, we integrate histone modifications and RNAPII states derived from bulk ChIP-seq data with single-cell RNA-sequencing data. We find that Polycomb repressive complex-active genes have greater cell-to-cell variation in expression than active genes, and these results are validated by knockout experiments. We also show that PRC-active genes are clustered on chromosomes in both two and three dimensions, and interactions with active enhancers promote a stabilization of gene expression noise. These findings provide new insights into how chromatin regulation modulates stochastic gene expression and transcriptional bursting, with implications for regulation of pluripotency and development.Polycomb repressive complexes modify histones but it is unclear how changes in chromatin states alter kinetics of transcription. Here, the authors use single-cell RNAseq and ChIPseq to find that actively transcribed genes with Polycomb marks have greater cell-to-cell variation in expression.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas do Grupo Polycomb/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular Tumoral , Marcadores Genéticos , Camundongos , Camundongos Knockout , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
11.
Nat Struct Mol Biol ; 24(6): 515-524, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28436944

RESUMO

Gene expression states influence the 3D conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features that define promoter activation states or binding sites for the transcription factor CTCF. Single-cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.


Assuntos
DNA/química , DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Loci Gênicos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Animais , Cromatina/metabolismo , Simulação por Computador , Imunofluorescência , Hibridização in Situ Fluorescente , Camundongos , Microscopia Confocal , Ligação Proteica , Análise de Célula Única , Fatores de Transcrição/metabolismo
12.
Elife ; 42015 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-26687004

RESUMO

Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.


Assuntos
Regulação da Expressão Gênica , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Consenso , Metilação , Camundongos , Células NIH 3T3 , Fosforilação , Serina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA