RESUMEN
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.
Asunto(s)
Linfocitos B , Melanoma , Animales , Ratones , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Activación de Linfocitos , Melanoma/inmunología , Melanoma/patología , Melanoma/prevención & control , Linfocitos T/citología , Linfocitos T/inmunología , Citometría de Flujo , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Presentación de Antígeno , Receptores de Antígenos de Linfocitos B/genética , Análisis de Expresión Génica de una Sola Célula , Carga Tumoral , Interferón Tipo IRESUMEN
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.
Asunto(s)
Encéfalo/citología , Células/clasificación , Ensamble y Desensamble de Cromatina , Cromatina/química , Cromatina/genética , Genes , Conformación Molecular , Animales , Sitios de Unión , Células/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Familia de Multigenes/genética , Neuronas/clasificación , Neuronas/metabolismo , Desnaturalización de Ácido Nucleico , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Inmunidad Innata/inmunología , Linfocitos/inmunología , Linfocitos/patología , Psoriasis/inmunología , Psoriasis/patología , Piel/inmunología , Piel/patología , Animales , Diferenciación Celular , Linaje de la Célula , Cromatina/genética , Modelos Animales de Enfermedad , Femenino , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-23/inmunología , Análisis de Clases Latentes , Linfocitos/clasificación , Masculino , Ratones , Psoriasis/genética , ARN Citoplasmático Pequeño/genética , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
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.
Asunto(s)
Islas de CpG , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/fisiología , Complejo Represivo Polycomb 1/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Sitios de Unión , Línea Celular , Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Conformación de Ácido Nucleico , Complejo Represivo Polycomb 1/genética , Unión Proteica , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Relación Estructura-Actividad , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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.
Asunto(s)
Células Madre Embrionarias/citología , Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/metabolismo , Animales , Diferenciación Celular , Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Humanos , Complejo Represivo Polycomb 1/genética , Dominios ProteicosRESUMEN
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.
Asunto(s)
Neuronas Dopaminérgicas/citología , Genes del Desarrollo , Células Madre Embrionarias de Ratones/citología , Proteínas del Grupo Polycomb/metabolismo , ARN Polimerasa II/metabolismo , Animales , Diferenciación Celular , Neuronas Dopaminérgicas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Melanocitos/metabolismo , Melanoma/patología , Mutación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Microambiente Tumoral/genéticaRESUMEN
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.
Asunto(s)
COVID-19/epidemiología , COVID-19/genética , Interacciones Huésped-Patógeno/genética , SARS-CoV-2/fisiología , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/estadística & datos numéricos , Internalización del Virus , Adulto , Anciano , Anciano de 80 o más Años , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/patología , COVID-19/virología , Catepsina L/genética , Catepsina L/metabolismo , Conjuntos de Datos como Asunto/estadística & datos numéricos , Demografía , Femenino , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Pulmón/metabolismo , Pulmón/virología , Masculino , Persona de Mediana Edad , Especificidad de Órganos/genética , Sistema Respiratorio/metabolismo , Sistema Respiratorio/virología , Análisis de Secuencia de ARN/métodos , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
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.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Neuronas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas/genética , Animales , Epigénesis Genética/genética , Humanos , Ratones , Modelos Teóricos , Complejo Represivo Polycomb 2/genéticaRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas del Grupo Polycomb/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Marcadores Genéticos , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
ADN/química , ADN/metabolismo , Células Madre Embrionarias/fisiología , Sitios Genéticos , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Animales , Cromatina/metabolismo , Simulación por Computador , Técnica del Anticuerpo Fluorescente , Hibridación Fluorescente in Situ , Ratones , Microscopía Confocal , Unión Proteica , Análisis de la Célula Individual , Factores de Transcripción/metabolismoRESUMEN
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.