RESUMO
DNA methylation comprises a cumulative record of lifetime exposures superimposed on genetically determined markers. Little is known about methylation dynamics in humans following an acute perturbation, such as infection. We characterized the temporal trajectory of blood epigenetic remodeling in 133 participants in a prospective study of young adults before, during, and after asymptomatic and mildly symptomatic SARS-CoV-2 infection. The differential methylation caused by asymptomatic or mildly symptomatic infections was indistinguishable. While differential gene expression largely returned to baseline levels after the virus became undetectable, some differentially methylated sites persisted for months of follow-up, with a pattern resembling autoimmune or inflammatory disease. We leveraged these responses to construct methylation-based machine learning models that distinguished samples from pre-, during-, and postinfection time periods, and quantitatively predicted the time since infection. The clinical trajectory in the young adults and in a diverse cohort with more severe outcomes was predicted by the similarity of methylation before or early after SARS-CoV-2 infection to the model-defined postinfection state. Unlike the phenomenon of trained immunity, the postacute SARS-CoV-2 epigenetic landscape we identify is antiprotective.
Assuntos
COVID-19 , Adulto Jovem , Humanos , COVID-19/genética , SARS-CoV-2/genética , Estudos Prospectivos , Metilação de DNA/genética , Processamento de Proteína Pós-TraducionalRESUMO
Although reduced representation bisulfite sequencing (RRBS) measures DNA methylation (DNAme) across CpG-rich genomic regions with high sensitivity, the assay can be time-consuming and prone to batch effects. Here, we present a high-throughput, automated RRBS protocol starting with DNA extraction from frozen rat tissues. We describe steps for RRBS library preparation, library quality control, and sequencing. We also detail an optimized pipeline for sequencing data processing. This protocol has been applied successfully to DNAme profiling across multiple rat tissues. For complete details on the use and execution of this protocol, please refer to Nair et al.1.
Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Sulfitos , Animais , Metilação de DNA/genética , Ratos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sulfitos/química , Análise de Sequência de DNA/métodos , DNA/genética , Ilhas de CpG/genética , Biblioteca GênicaRESUMO
Regular exercise has many physical and brain health benefits, yet the molecular mechanisms mediating exercise effects across tissues remain poorly understood. Here we analyzed 400 high-quality DNA methylation, ATAC-seq, and RNA-seq datasets from eight tissues from control and endurance exercise-trained (EET) rats. Integration of baseline datasets mapped the gene location dependence of epigenetic control features and identified differing regulatory landscapes in each tissue. The transcriptional responses to 8 weeks of EET showed little overlap across tissues and predominantly comprised tissue-type enriched genes. We identified sex differences in the transcriptomic and epigenomic changes induced by EET. However, the sex-biased gene responses were linked to shared signaling pathways. We found that many G protein-coupled receptor-encoding genes are regulated by EET, suggesting a role for these receptors in mediating the molecular adaptations to training across tissues. Our findings provide new insights into the mechanisms underlying EET-induced health benefits across organs.
Assuntos
Condicionamento Físico Animal , Transcriptoma , Animais , Condicionamento Físico Animal/fisiologia , Masculino , Ratos , Feminino , Metilação de DNA , Epigênese Genética , Epigenômica , Adaptação Fisiológica/genética , Especificidade de Órgãos , Ratos Sprague-DawleyRESUMO
Endurance exercise is an important health modifier. We studied cell-type specific adaptations of human skeletal muscle to acute endurance exercise using single-nucleus (sn) multiome sequencing in human vastus lateralis samples collected before and 3.5 hours after 40 min exercise at 70% VO2max in four subjects, as well as in matched time of day samples from two supine resting circadian controls. High quality same-cell RNA-seq and ATAC-seq data were obtained from 37,154 nuclei comprising 14 cell types. Among muscle fiber types, both shared and fiber-type specific regulatory programs were identified. Single-cell circuit analysis identified distinct adaptations in fast, slow and intermediate fibers as well as LUM-expressing FAP cells, involving a total of 328 transcription factors (TFs) acting at altered accessibility sites regulating 2,025 genes. These data and circuit mapping provide single-cell insight into the processes underlying tissue and metabolic remodeling responses to exercise.
RESUMO
Transcription factors (TFs) play a key role in regulating gene expression and responses to stimuli. We conducted an integrated analysis of chromatin accessibility, DNA methylation, and RNA expression across eight rat tissues following endurance exercise training (EET) to map epigenomic changes to transcriptional changes and determine key TFs involved. We uncovered tissue-specific changes and TF motif enrichment across all omic layers, differentially accessible regions (DARs), differentially methylated regions (DMRs), and differentially expressed genes (DEGs). We discovered distinct routes of EET-induced regulation through either epigenomic alterations providing better access for TFs to affect target genes, or via changes in TF expression or activity enabling target gene response. We identified TF motifs enriched among correlated epigenomic and transcriptomic alterations, DEGs correlated with exercise-related phenotypic changes, and EET-induced activity changes of TFs enriched for DEGs among their gene targets. This analysis elucidates the unique transcriptional regulatory mechanisms mediating diverse organ effects of EET.
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Concomitant profiling of transcriptome and chromatin accessibility in isolated nuclei can reveal gene regulatory control mechanisms in health and disease. We report a single nucleus multi-omics analysis protocol optimized for frozen archived postmortem human pituitaries that is also effective for frozen ovine and murine pituitaries and human skeletal muscle biopsies. Its main advantages are that (1) it is not limited to fresh tissue, (2) it avoids tissue dissociation-induced transcriptional changes, and (3) it includes a novel, automated quality control pipeline. For complete details on the use and execution of this protocol, please refer to Ruf-Zamojski et al. (2021) and Zhang et al. (2022).
Assuntos
Cromatina , Transcriptoma , Animais , Núcleo Celular , Congelamento , Humanos , Camundongos , Ovinos/genética , Núcleo SolitárioRESUMO
Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.
Assuntos
Cromatina , Transcriptoma , Idoso , Criança , Sequenciamento de Cromatina por Imunoprecipitação , Feminino , Humanos , Masculino , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Male sex is a major risk factor for SARS-CoV-2 infection severity. To understand the basis for this sex difference, we studied SARS-CoV-2 infection in a young adult cohort of United States Marine recruits. Among 2,641 male and 244 female unvaccinated and seronegative recruits studied longitudinally, SARS-CoV-2 infections occurred in 1,033 males and 137 females. We identified sex differences in symptoms, viral load, blood transcriptome, RNA splicing, and proteomic signatures. Females had higher pre-infection expression of antiviral interferon-stimulated gene (ISG) programs. Causal mediation analysis implicated ISG differences in number of symptoms, levels of ISGs, and differential splicing of CD45 lymphocyte phosphatase during infection. Our results indicate that the antiviral innate immunity set point causally contributes to sex differences in response to SARS-CoV-2 infection. A record of this paper's transparent peer review process is included in the supplemental information.
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COVID-19 , Imunidade Inata , Caracteres Sexuais , Feminino , Humanos , Masculino , Adulto Jovem , COVID-19/imunologia , Interferons , Proteômica , SARS-CoV-2RESUMO
To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.
Assuntos
Cromatina/genética , Metilação de DNA , Redes Reguladoras de Genes , Hipófise/metabolismo , Regulon/genética , Transcriptoma/genética , Animais , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Modelos Genéticos , Hipófise/citologia , Regiões Promotoras Genéticas/genética , Fatores SexuaisRESUMO
Exercise has multi-systemic benefits and attenuates the physiological impairments associated with aging. Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. However, the impact of regular exercise and acute exercise on circulating exosomal microRNAs (exomiRs) in older populations remains unknown. In the present study, we analyzed circulating exomiR expression in endurance-trained elderly men (n = 5) and age-matched sedentary males (n = 5) at baseline (Pre), immediately after a forty minute bout of aerobic exercise on a cycle ergometer (Post), and three hours after this acute exercise (3hPost). Following the isolation and enrichment of exosomes from plasma, exosome-enriched preparations were characterized and exomiR levels were determined by sequencing. The effect of regular exercise on circulating exomiRs was assessed by comparing the baseline expression levels in the trained and sedentary groups. The effect of acute exercise was determined by comparing baseline and post-training expression levels in each group. Regular exercise resulted in significantly increased baseline expression of three exomiRs (miR-486-5p, miR-215-5p, miR-941) and decreased expression of one exomiR (miR-151b). Acute exercise altered circulating exomiR expression in both groups. However, exomiRs regulated by acute exercise in the trained group (7 miRNAs at Post and 8 at 3hPost) were distinct from those in the sedentary group (9 at Post and 4 at 3hPost). Pathway analysis prediction and reported target validation experiments revealed that the majority of exercise-regulated exomiRs are targeting genes that are related to IGF-1 signaling, a pathway involved in exercise-induced muscle and cardiac hypertrophy. The immediately post-acute exercise exomiR signature in the trained group correlates with activation of IGF-1 signaling, whereas in the sedentary group it is associated with inhibition of IGF-1 signaling. While further validation is needed, including measurements of IGF-1/IGF-1 signaling in blood or skeletal muscle, our results suggest that training status may counteract age-related anabolic resistance by modulating circulating exomiR profiles both at baseline and in response to acute exercise.