RESUMO
Cells require coordinated control over gene expression when responding to environmental stimuli. Here we apply scATAC-seq and single-cell RNA sequencing (scRNA-seq) in resting and stimulated human blood cells. Collectively, we generate ~91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of the immunological response across cell types, stimuli, and time. Advancing tools to integrate multi-omics data, we develop functional inference of gene regulation (FigR), a framework to computationally pair scA-TAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene-regulatory networks (GRNs) to identify candidate transcription factor (TF) regulators. Utilizing these paired multi-omics data, we define domains of regulatory chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility and gene expression at timescales of minutes. Construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues.
RESUMO
Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2+ macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Análise de Célula Única , Transcriptoma/genética , Linfócitos B/imunologia , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Endoteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Proteínas de Membrana/genética , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Sequência de RNA , Microambiente Tumoral , Proteínas Supressoras de Tumor/genéticaRESUMO
Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.
Assuntos
Processamento Alternativo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/metabolismo , Córtex Pré-Frontal/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Animais Recém-Nascidos , Biologia Computacional , Feminino , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Camundongos , Modelos Animais , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Análise de Célula Única/métodos , Análise EspacialRESUMO
Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (105 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.
Assuntos
Cromatina/química , Epigenômica/métodos , Microfluídica/métodos , Análise de Célula Única/métodos , Animais , Encéfalo/citologia , Linhagem Celular , Sobrevivência Celular , Cromatina/metabolismo , Técnicas de Química Combinatória , Desoxirribonucleases/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , CamundongosRESUMO
Androgenic hormones regulate many aspects of animal social behavior, including the elaborate display routines on which many species rely for advertisement and competition. One way that this might occur is through peripheral effects of androgens, particularly on skeletal muscles that control complex movements and postures of the body and its limbs. However, the specific contribution of peripheral androgen-muscle interactions to the performance of elaborate behavioral displays in the natural world has never been examined. We study this issue in one of the only natural physiological models of animal acrobatics: the golden-collared manakin (Manacus vitellinus). In this tropical bird, males compete with each other and court females by producing firecracker-like wing- snaps and by rapidly dancing among saplings over the forest floor. To test how activation of peripheral androgen receptors (AR) influences this display, we treat reproductively active adult male birds with the peripherally selective antiandrogen bicalutamide (BICAL) and observe the effects of this manipulation on male display performance. We not only validate the peripheral specificity of BICAL in this species, but we also show that BICAL treatment reduces the frequency with which adult male birds perform their acrobatic display maneuvers and disrupts the overall structure and fine-scale patterning of these birds' main complex wing-snap sonation. In addition, this manipulation has no effect on the behavioral metrics associated with male motivation to display. Together, our findings help differentiate the various effects of peripheral and central AR on the performance of a complex sociosexual behavioral phenotype by indicating that peripheral AR can optimize the motor skills necessary for the production of an elaborate animal display.