RESUMO
Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Medula Espinal , Animais , Humanos , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Camundongos , Análise da Expressão Gênica de Célula Única , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Neuroglia/metabolismo , Neuroglia/patologiaRESUMO
Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1-5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.
Assuntos
Cromatina , Epigenoma , Mamíferos , Transcriptoma , Animais , Humanos , Camundongos , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Epigenômica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Mamíferos/genética , Histonas/química , Histonas/metabolismo , Análise de Célula Única , Especificidade de Órgãos , Encéfalo/embriologia , Encéfalo/metabolismo , Envelhecimento/genéticaRESUMO
Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2-5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.
Assuntos
Montagem e Desmontagem da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Animais , Encéfalo/metabolismo , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Epigenômica , Perfilação da Expressão Gênica , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Tonsila Palatina/citologia , Tonsila Palatina/imunologiaRESUMO
DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.
Assuntos
Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Apoptose/genética , Sobrevivência Celular/genética , Dano ao DNA/genética , Células HEK293 , Humanos , RNA Longo não Codificante/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
BACKGROUND: Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations. RESULTS: We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS. CONCLUSIONS: Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.
Assuntos
Bainha de Mielina , Oligodendroglia , Axônios/fisiologia , Diferenciação Celular/genética , Linhagem da Célula , Sistema Nervoso Central/fisiologia , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismoRESUMO
Transcription of HIV provirus is a key step of the viral cycle, and depends on the recruitment of the cellular positive transcription elongation factor b (P-TEFb) to the HIV promoter. The viral transactivator Tat can displace P-TEFb from the 7SK small nuclear ribonucleoprotein, where it is bound and inactivated by HEXIM1, and bring it to TAR, which allows the stalled RNA polymerase II to transition to successful transcription elongation. In this study, we designed a chimeric inhibitor of HIV transcription by combining functional domains from HEXIM1 and Tat. The chimera (HT1) potently inhibited gene expression from the HIV promoter, by competing with Tat for TAR and P-TEFb binding, while keeping the latter inactive. HT1 inhibited spreading infection as well as viral reactivation in lymphocyte T cell line models of HIV latency, with little effect on cellular transcription and metabolism. This proof-of-concept study validates an innovative approach to interfering with HIV transcription via peptide mimicry and competition for RNA-protein interactions. HT1 represents a new candidate for HIV therapy, or HIV cure via the proposed block and lock strategy.
Assuntos
Infecções por HIV/terapia , HIV-1/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Replicação Viral/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV , Soropositividade para HIV , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Células Jurkat , Provírus/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição , Latência Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag-RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.
RESUMO
In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.
Assuntos
Encéfalo , Linhagem da Célula , Oligodendroglia , Animais , Oligodendroglia/fisiologia , Camundongos , Encéfalo/citologia , Encéfalo/embriologia , Linhagem da Célula/fisiologia , Diferenciação Celular/fisiologia , Camundongos Transgênicos , Bainha de Mielina/metabolismo , Bainha de Mielina/fisiologiaRESUMO
Spatial omics emerged as a new frontier of biological and biomedical research. Here, we present spatial-CUT&Tag for spatially resolved genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding, and next-generation sequencing. Spatially resolved chromatin states in mouse embryos revealed tissue-type-specific epigenetic regulations in concordance with ENCODE references and provide spatial information at tissue scale. Spatial-CUT&Tag revealed epigenetic control of the cortical layer development and spatial patterning of cell types determined by histone modification in mouse brain. Single-cell epigenomes can be derived in situ by identifying 20-micrometer pixels containing only one nucleus using immunofluorescence imaging. Spatial chromatin modification profiling in tissue may offer new opportunities to study epigenetic regulation, cell function, and fate decision in normal physiology and pathogenesis.
Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Cromatina/metabolismo , Epigênese Genética , Código das Histonas , Histonas/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Epigenoma , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Microfluídica , Neurônios/citologia , Análise de Célula ÚnicaRESUMO
Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.
Assuntos
Oligodendroglia , Prosencéfalo , Animais , Diferenciação Celular/fisiologia , Humanos , Camundongos , Oligodendroglia/metabolismo , Transcriptoma/genéticaRESUMO
Mature oligodendrocytes (MOLs) show transcriptional heterogeneity, the functional consequences of which are unclear. MOL heterogeneity might correlate with the local environment or their interactions with different neuron types. Here, we show that distinct MOL populations have spatial preference in the mammalian central nervous system (CNS). We found that MOL type 2 (MOL2) is enriched in the spinal cord when compared to the brain, while MOL types 5 and 6 (MOL5/6) increase their contribution to the OL lineage with age in all analyzed regions. MOL2 and MOL5/6 also have distinct spatial preference in the spinal cord regions where motor and sensory tracts run. OL progenitor cells (OPCs) are not specified into distinct MOL populations during development, excluding a major contribution of OPC intrinsic mechanisms determining MOL heterogeneity. In disease, MOL2 and MOL5/6 present different susceptibility during the chronic phase following traumatic spinal cord injury. Our results demonstrate that the distinct MOL populations have different spatial preference and different responses to disease.