RESUMEN
Population-scale single-cell genomics is a transformative approach for unraveling the intricate links between genetic and cellular variation. This approach is facilitated by cutting-edge experimental methodologies, including the development of high-throughput single-cell multiomics and advances in multiplexed environmental and genetic perturbations. Examining the effects of natural or synthetic genetic variants across cellular contexts provides insights into the mutual influence of genetics and the environment in shaping cellular heterogeneity. The development of computational methodologies further enables detailed quantitative analysis of molecular variation, offering an opportunity to examine the respective roles of stochastic, intercellular, and interindividual variation. Future opportunities lie in leveraging long-read sequencing, refining disease-relevant cellular models, and embracing predictive and generative machine learning models. These advancements hold the potential for a deeper understanding of the genetic architecture of human molecular traits, which in turn has important implications for understanding the genetic causes of human disease. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 25 is August 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
RESUMEN
Genetic regulation of gene expression is a complex process, with genetic effects known to vary across cellular contexts such as cell types and environmental conditions. We developed SURGE, a method for unsupervised discovery of context-specific expression quantitative trait loci (eQTLs) from single-cell transcriptomic data. This allows discovery of the contexts or cell types modulating genetic regulation without prior knowledge. Applied to peripheral blood single-cell eQTL data, SURGE contexts capture continuous representations of distinct cell types and groupings of biologically related cell types. We demonstrate the disease-relevance of SURGE context-specific eQTLs using colocalization analysis and stratified LD-score regression.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Sitios de Carácter Cuantitativo , Transcriptoma , Análisis de Secuencia de ARNRESUMEN
The host's gene expression and gene regulatory response to pathogen exposure can be influenced by a combination of the host's genetic background, the type of and exposure time to pathogens. Here we provide a detailed dissection of this using single-cell RNA-sequencing of 1.3M peripheral blood mononuclear cells from 120 individuals, longitudinally exposed to three different pathogens. These analyses indicate that cell-type-specificity is a more prominent factor than pathogen-specificity regarding contexts that affect how genetics influences gene expression (i.e., eQTL) and co-expression (i.e., co-expression QTL). In monocytes, the strongest responder to pathogen stimulations, 71.4% of the genetic variants whose effect on gene expression is influenced by pathogen exposure (i.e., response QTL) also affect the co-expression between genes. This indicates widespread, context-specific changes in gene expression level and its regulation that are driven by genetics. Pathway analysis on the CLEC12A gene that exemplifies cell-type-, exposure-time- and genetic-background-dependent co-expression interactions, shows enrichment of the interferon (IFN) pathway specifically at 3-h post-exposure in monocytes. Similar genetic background-dependent association between IFN activity and CLEC12A co-expression patterns is confirmed in systemic lupus erythematosus by in silico analysis, which implies that CLEC12A might be an IFN-regulated gene. Altogether, this study highlights the importance of context for gaining a better understanding of the mechanisms of gene regulation in health and disease.
Asunto(s)
Leucocitos Mononucleares , Lupus Eritematoso Sistémico , Regulación de la Expresión Génica , Humanos , Lectinas Tipo C/genética , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/genética , ARN/metabolismo , Receptores Mitogénicos/genética , Transducción de SeñalRESUMEN
The human immune system displays substantial variation between individuals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression. We subsequently show how eQTLs have dynamic allelic effects in B cells that are transitioning from naïve to memory states and demonstrate how commonly segregating alleles lead to interindividual variation in immune function. Finally, using a Mendelian randomization approach, we identify the causal route by which 305 risk loci contribute to autoimmune disease at the cellular level. This work brings together genetic epidemiology with scRNA-seq to uncover drivers of interindividual variation in the immune system.
Asunto(s)
Enfermedades Autoinmunes , Leucocitos Mononucleares , Alelos , Enfermedades Autoinmunes/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple , Células Precursoras de Linfocitos B , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ARNRESUMEN
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon-stimulated genes (ISGs) in monocytes, reduction of naïve CD4+ T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH+ CD8+ T cells. Cell type-specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type-specific cis-expression quantitative trait loci and to link SLE-associated variants to cell type-specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE.
Asunto(s)
Interferón Tipo I , Lupus Eritematoso Sistémico , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Humanos , Interferón Tipo I/metabolismo , Leucocitos Mononucleares , Lupus Eritematoso Sistémico/genética , RNA-Seq , Transcripción GenéticaRESUMEN
Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.
Asunto(s)
Saccharomyces cerevisiae/efectos de los fármacos , alfa-Sinucleína/toxicidad , Secuencia de Aminoácidos , Humanos , Mutación , Enfermedad de Parkinson/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.
Asunto(s)
Lentivirus/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN/métodos , Flujo de Trabajo , Secuencia de BasesRESUMEN
PURPOSE: Develop an automated exome analysis workflow that can produce a very small number of candidate variants yet still detect different numbers of deleterious variants between probands and unaffected siblings. METHODS: Ninety-seven outbred nuclear families from the Undiagnosed Diseases Program/Network included single probands and the corresponding unaffected sibling(s). Single-nucleotide polymorphism (SNP) chip and exome analyses were performed on all, with proband and unaffected sibling considered independently as the target. The total burden of candidate genetic variants was summed for probands and siblings over all considered disease models. RESULTS: Exome analysis workflow include automated programs for ethnicity-matched genotype calling, salvage pathway for Mendelian inconsistency, compound heterozygous recessive detection, BAM file regional curation, population frequency filtering, pedigree-aware BAM file noise evaluation, and exon deletion filtration. This workflow relied heavily on BAM file analysis. A greater average pathogenic variant number was found compared with unaffected siblings. This was significant (p < 0.05) when using published recommended thresholds, and implies that causal variants are retained in many probands' lists. CONCLUSION: Using Mendelian and non-Mendelian models, this agnostic exome analysis shows a difference between a small group of probands and their unaffected siblings. This workflow produces candidate lists small enough to pursue with laboratory validation.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Procesamiento Automatizado de Datos , Enfermedades Genéticas Congénitas/diagnóstico , Análisis de Secuencia de ADN , Exoma/genética , Exones/genética , Femenino , Enfermedades Genéticas Congénitas/genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Eliminación de Secuencia/genética , HermanosRESUMEN
Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Cromatina/genética , Polimorfismo de Nucleótido Simple , Adulto , Enfermedades Autoinmunes/genética , Femenino , Regulación de la Expresión Génica , Genotipo , Humanos , Masculino , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
BACKGROUND: In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. RESULTS: The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. CONCLUSIONS: We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.