The diverse health benefits of exercise are associated with multi-organ molecular responses. Alternative RNA splicing (AS) is an important determinant of transcriptome and proteome diversity. We profiled the temporal effects of acute endurance and resistance exercise on the AS landscape of human skeletal muscle, adipose tissue, and blood, and studied regulatory mechanisms through integrated multi-omic analyses. We identified 5102 distinct differential AS (DAS) events, with the majority modifying protein-coding sequence (89%) and being independent of altered RNA expression (67%). Endurance and resistance exercise induced differing patterns of AS alterations with divergent temporal trajectories. We inferred the DAS-associated RNA-binding and DNA-binding proteins. In skeletal muscle, where DAS events were the most abundant, DAS genes were enriched for muscle structure- and RNA splicing-related processes, and splicing machinery components were regulated at the protein phosphorylation, RNA, and AS levels. These findings implicate AS regulation as a major mediator of the responses to exercise.

Summary/Abstract The estrous cycle transcriptional and chromatin dynamics in pituitary cell types have not been investigated. We report single-nucleus multiomics assays in 18 adult mouse pituitaries (102,069 post quality-control nuclei) across 6 cycle time points. Differential analysis revealed cycle stage-dependent epigenetic and transcriptional remodeling across the major pituitary cell populations. In gonadotropes and lactotropes, we identified temporal patterns of differential gene expression linked to various biological processes, notably neuronal and synaptic-related ontologies. Pseudotime trajectory analysis was consistent with a rapid transition of individual gonadotropes through different cellular states. In gonadotropes and lactotropes, we uncovered gene regulatory circuits whose activity varies between consecutive cycle time points. We experimentally validated a gonadotrope ETS2-driven circuit that differentially regulates Fshb gene expression between 2 and 9 am on estrus. Our data and analyses, available at https://rstudio-connect.hpc.mssm.edu/snpit_estrous_browser/ , provide a window into gene regulatory mechanisms underlying estrous cycle stage transitions. Highlights Pituitary cells show chromatin and transcriptome plasticity across the estrous cycle We identify stage-modulated gene regulatory circuits across pituitary cell types Gonadotrope DEGs form distinct pathway-annotated temporal trajectory clusters We validate a gonadotrope ETS2-driven circuit regulating the Fshb gene eTOC blurb Zhang et al. conduct a single-nucleus multiomics analysis of mouse pituitaries in vivo across the estrous cycle. They reveal an epigenetic and transcriptomic plasticity in pituitary cell populations. In gonadotropes and lactotropes, they demonstrate that DEGs clustered by temporal trajectories are enriched for distinct biological processes and identify stage-modulated gene regulatory circuits. They experimentally validate a gonadotrope ETS2-driven circuit regulating Fshb expression. Their dynamic molecular atlas of the cycling pituitary captures key cis -regulatory mechanisms underlying estrous cycle stage transitions.

Transcription factors play a key role in regulating gene expression. We conduct an integrated analysis of chromatin accessibility, DNA methylation, mRNA expression, protein abundance and phosphorylation across eight tissues in fifty rats of equally represented sexes following endurance exercise training to identify coordinated epigenomic and transcriptional changes and determine key transcription factors involved. We uncover tissue-specific endurance exercise training associated changes and transcription factor motif enrichment across differentially expressed genes, accessible regions, and methylated regions. We discover distinct routes of training-induced regulation through either epigenomic alterations providing better access for transcription factors to affect target genes, or via changes in transcription factor expression or activity enabling target gene responses. We identify transcription factor motifs enriched among correlated epigenomic and transcriptomic alterations, differentially expressed genes correlated with exercise-related phenotypic and cell type composition changes, and training-induced activity changes of transcription factors whose target genes are enriched for differentially expressed genes. This analysis elucidates the unique gene regulatory mechanisms mediating diverse transcriptional responses to training across tissues.

Long non-coding RNAs (lncRNAs) regulate multiple cellular processes. However, knowledge of the responses and regulatory functions of lncRNAs in physical exercise and training remains limited. As part of the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we conducted a comprehensive analysis of lncRNA expression patterns in 18 tissues after an 8-week progressive endurance training program in rats. The lncRNA expression pattern was largely tissue-specific. In total, 759 unique lncRNAs were found to be differentially expressed across all tissues, generally displaying lower abundance, shorter transcript length, and reduced GC content compared with protein-coding genes. The most pronounced changes were observed in white and brown adipose tissues, the hypothalamus, and the adrenal gland. In the two skeletal muscle tissues investigated, only two lncRNAs were commonly differentially expressed. White and brown adipose tissues revealed a correlation between upregulated differentially expressed lncRNAs and coding genes associated with immune regulation. We identified substantial sex differences in the lncRNA regulatory landscape in response to exercise training. This comprehensive tissue-specific characterization of exercise-responsive lncRNAs opens new avenues for understanding exercise as molecular medicine and may inform the development of lncRNA-targeted therapeutics that harness the beneficial effects of exercise.

Previous single-cell profiling studies of the pituitary gland have yielded minimally reproducible insights due to their low statistical power and methodological inconsistencies. To address this, we generate the uniformly pre-processed Consensus Pituitary Atlas (CPA) using all 283 existing mouse pituitary single-cell datasets (∼1.3 million high-quality cells). The CPA reveals cell typing and lineage markers, including low-expression transcripts that previous analyses could not detect. Leveraging the scale of the CPA, we develop machine learning models to automate and standardize cell type annotation and doublet identification for future studies. Utilizing the curated metadata, we identify sex-biased and age-dependent gene expression patterns at cell type resolution. To uncover drivers of cell fates, first we determine consensus cell communication patterns. Second, we use RNA sequencing and chromatin accessibility data to identify transcription factors associated with cell fates across modalities. The epitome platform provides a user-friendly interface with the CPA and allows streamlined analyses.

Regular physical activity represents one of the greatest mechanisms for maintaining human health, yet the underlying molecular transducers of these benefits remain incompletely understood. Multi-omic assays now provide new opportunities to study the coordinated molecular responses of body tissues to different exercise modalities. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this need by creating a molecular map of the response to physical activity. Described here is the first human cohort of MoTrPAC: sedentary adults enrolled prior to study suspension during the COVID-19 pandemic (N=175) randomized to either endurance or resistance exercise, or non-exercise control. From these participants, we detail their global acute molecular response in skeletal muscle, adipose tissue, and blood, integrated at multiple levels: tissue, exercise modality, timepoint, and omic category. These analyses characterize key molecular pathways, identify central regulators, and implicate novel candidate exerkines in mediating multi-organ exercise effects.

Exercise induces widespread health benefits across multiple tissues, yet the acute molecular responses in human adipose tissue remain poorly defined. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) profiled temporal molecular changes in abdominal subcutaneous adipose tissue (ASAT) following a single bout of exercise. Healthy sedentary adults were randomized to endurance (EE), resistance (RE), or control (CON) groups. ASAT biopsies were collected pre-exercise and at 45min, 4hr, and 24hr post-exercise, followed by transcriptomic, proteomic, phosphoproteomic, and metabolomic analyses. EE and RE elicited distinct, time-resolved molecular programs involving angiogenesis, extracellular matrix remodeling, mitochondrial metabolism, substrate utilization, and circadian regulation. Phosphoproteomics revealed acute changes in cytoskeletal and branched-chain amino acid metabolism proteins associated with glycemic control. Temporal metabolomic shifts were cell-type-specific. Finally, we identified candidate adipose-derived exerkines with predicted endocrine actions. This multi-omic map of acute ASAT responses offers insight into adipose-specific mechanisms by which exercise promotes metabolic health.

Exercise benefits numerous organ systems and tissues, however limited knowledge exists about its underlying molecular pathways. Identifying the exercise-induced biochemical changes that occur in the circulation may provide further insights into how exercise confers systemic health changes. Here, we perform large-scale plasma proteomic, metabolomic, and whole blood transcriptional profiling in sedentary human participants undergoing acute endurance exercise (EE), resistance exercise (RE), or a non-exercise control (CON) in up to 7 timepoints over a 24 hour period. We observe 7066 transcript, 189 protein, and 448 metabolite changes in response to EE or RE compared to CON. Our analyses reveal numerous shared biochemical responses between EE and RE modes, but also differences in immune cell responses, lipid metabolism, and pathways reflective of tissue repair and angiogenesis. Taken together, our findings highlight novel temporal and exercise mode-specific blood-based molecular responses to acute exercise, and provide a new resource for the scientific community.

Repair of acutely injured skeletal muscle relies on an adequate inflammatory response predominated by monocyte/macrophage infiltration. The process requires injured muscles to produce C-C chemokine ligand 2 (CCL2). The present study identified fibro/adipogenic progenitors (FAPs) as the primary source of CCL2 in acutely injured muscle, where the pro-inflammatory subcluster of FAPs expanded rapidly and expressed the highest level of CCL2. FAP-specific deletion of Ccl2 largely abolished CCL2 production by acutely injured muscle, reducing monocyte/macrophage infiltration and impairing muscle regeneration. In vitro, the CCL2 expression by both mouse and human FAPs was induced by danger signal-containing muscle homogenates through Toll-like receptor signaling. The CCL2 expression by mouse FAPs was also induced by infiltrating neutrophils, partly through their secretion of pro-inflammatory cytokines. Our findings suggest an important immune sentinel role for FAPs, as they sense muscle damage, produce CCL2 to recruit inflammatory monocytes, and promote injury repair.

The epigenetic landscape and tumor microenvironment (TME) interactions of non-functioning pituitary adenomas (NFPAs), benign tumors with high morbidity and recurrence rates, are not well characterized. We completed single-nucleus (sn) multiomics assays on 4 gonadotrope NFPAs (34,819 cells) and 11 non-diseased postmortem control pituitaries (51,535 cells), finding decreased proportions of tumor-associated endothelial cells and pericytes and increased proportions of macrophages. We identified bidirectional tumor-macrophage crosstalk comprising nine ligand-receptor interactions and experimentally validated the macrophage-initiated SFRP1-FZD6 interaction, whose predicted target genes CCND1, CDK6, SGK1, and TGFBR2 were linked to tumorigenesis. We uncovered coordinated gene expression and chromatin accessibility programs, which distinguished adenoma cells from gonadotropes. Integrated transcriptome-chromatin modeling revealed gene regulatory circuits (GRCs) that showed altered activity in adenoma cells and were regulated by transcription factors (TFs), including PBX3 and MEF2C. Our study provides insight into the altered epigenetic gene control landscape and TME processes of the NFPA tumor phenotype. Our data are freely available at https://rstudio-connect.hpc.mssm.edu/nfpa_browser/.

The goal of the Molecular Transducers of Physical Activity Consortium (MoTrPAC) is to examine the physiological and molecular basis for health benefits in response to acute and chronic exercise. Prior to COVID-19 suspension, healthy, sedentary participants (N=206, 18-74y) were randomized to endurance exercise (N=80), resistance exercise (N=81), or non-exercise control (N=45) interventions. The prescribed vigorous acute endurance and resistance exercise bouts induced physiological and metabolic perturbations relative to resting homeostasis. The supervised chronic (3d/wk, 12wk) endurance or resistance training programs robustly improved several physiological parameters (i.e., VO2peak, muscular strength). Temporal biospecimen (blood, muscle, and adipose) collections and processing coupled to the acute exercise bouts were highly successful. In most cases, over 90% success was achieved for blood, muscle, and adipose samples. Endurance and resistance exercise induced distinct acute and chronic physiological responses, which provide a framework to interrogate the molecular basis for health adaptations to these two popular exercise modalities.

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.

Endurance exercise induces multisystem adaptations that improve performance and benefit health. Gene regulatory circuit responses within individual skeletal muscle cell types, which are key mediators of exercise effects, have not been studied. Here, we map transcriptome, chromatin, and regulatory circuit responses to acute endurance exercise in muscle using same-cell RNA-seq/ATAC-seq multiome assays. High-quality data were obtained from 37,154 nuclei comprising 14 cell types in vastus lateralis samples collected before and 3.5 h after either 40 min cycling exercise at 70% VO2max or 40 min supine rest. Both shared and cell-type-specific regulatory programs were identified. Differential gene expression and accessibility sites are largely distinct within nuclei for each cell type and muscle fiber, with the largest numbers of regulatory events observed in the three muscle fiber types (slow, fast, and intermediate) and lumican (LUM)-expressing fibro-adipogenic progenitor cells. Single-cell regulatory circuit triad reconstruction (transcription factor, chromatin interaction site, regulated gene) also identifies largely distinct gene regulatory circuits modulated by exercise in the three muscle fiber types and LUM-expressing fibro-adipogenic progenitor cells, involving a total of 328 transcription factors acting at chromatin sites regulating 2025 genes. This web-accessible single-cell data set and regulatory circuitry map serve as a resource for understanding the molecular underpinnings of the metabolic and physiological effects of exercise and for guiding interpretation of the exercise response literature in bulk tissue.

Monocytes/macrophages promote the repair of acutely injured muscle while contributing to dystrophic changes in chronically injured muscle in Duchenne muscular dystrophy (DMD) patients and animal models including mdx and mdx5cv mice. To elucidate the molecular mechanisms underlying this functional difference, we compared the transcriptomes of intramuscular monocytes/macrophages from wild-typed (WT) uninjured muscles, WT acutely injured muscles, and mdx5cv dystrophic muscles, using single cell-based RNA sequencing (scRNA-seq) analysis. Our study identified multiple transcriptomically diverse monocyte/macrophage subclusters, which appear to be induced by the intramuscular microenvironment. They expressed feature genes differentially involved in muscle inflammation, regeneration, and extracellular matrix (ECM) remodeling, but none of them conform to strict M1 or M2 activation. The Gpnmb+Spp1+ macrophage subcluster, an injury-associated subcluster that features the signature genes of reported scar-associated macrophages (SAMs) involved in ECM remodeling and fibrosis, is present transiently in acutely injured muscle and persistently in chronically injured dystrophic muscle, along with the persistence of monocytes. Our findings suggest that the persistent monocyte/macrophage infiltration and activation induced by continuous injury may underlie the pathogenic roles of macrophages in mdx5cv muscles. Controlling muscle injury and subsequent macrophage infiltration and activation may be important to the treatment of DMD.

Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training (ExT) and sex on its molecular landscape is not fully established. Utilizing an integrative multi-omics approach, and leveraging data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we show profound sexual dimorphism in the scWAT of sedentary rats and in the dynamic response of this tissue to ExT. Specifically, the scWAT of sedentary females displays -omic signatures related to insulin signaling and adipogenesis, whereas the scWAT of sedentary males is enriched in terms related to aerobic metabolism. These sex-specific -omic signatures are preserved or amplified with ExT. Integration of multi-omic analyses with phenotypic measures identifies molecular hubs predicted to drive sexually distinct responses to training. Overall, this study underscores the powerful impact of sex on adipose tissue biology and provides a rich resource to investigate the scWAT response to ExT. Using a multi-omics approach, the authors examine the molecular drivers of sexual dimorphism in the subcutaneous adipose tissue from sedentary and endurance-trained rats. These data provide a valuable resource for adipose tissue-related research.

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.

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.

Fibrosis is a prominent pathological feature of skeletal muscle in Duchenne muscular dystrophy (DMD). The commonly used disease mouse model, mdx5cv , displays progressive fibrosis in the diaphragm but not limb muscles. We use single-cell RNA sequencing to determine the cellular expression of the genes involved in extracellular matrix (ECM) production and degradation in the mdx5cv diaphragm and quadriceps. We find that fibro/adipogenic progenitors (FAPs) are not only the primary source of ECM but also the predominant cells that express important ECM regulatory genes, including Ccn2, Ltbp4, Mmp2, Mmp14, Timp1, Timp2, and Loxs. The effector and regulatory functions are exerted by diverse FAP clusters which are different between diaphragm and quadriceps, indicating their activation by different tissue microenvironments. FAPs are more abundant in diaphragm than in quadriceps. Our findings suggest that the development of anti-fibrotic therapy for DMD should target not only the ECM production but also the pro-fibrogenic regulatory functions of FAPs.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Abstract Disclosure: F.M. Ruf-Zamojski: None. W. Cheng: None. Z. Zhang: None. M. Zamojski: None. G.R. Smith: None. X. Chen: None. N. Mendelev: None. G. Strupinsky: None. C.A. Alonso: None. L. Ongaro Gambino: None. X. Zhou: None. E. Brule: None. M.S. Amper: None. P. Hanna: None. V.D. Nair: None. C.L. Andoniadou: None. J.L. Turgeon: None. O. Troyanskaya: None. E. Zaslavsky: None. D.J. Bernard: None. S.C. Sealfon: None. Single cell multi-omics datasets provide an unparalleled power to resolve gene regulatory circuits underlying cellular function in complex tissues such as the pituitary gland [1, 2]. To better understand cellular plasticity and dynamics in the mouse pituitary during the estrous cycle in vivo, we performed same-cell single nucleus (sn) multi-omics for gene expression and chromatin accessibility on individual pituitaries collected from mice at 9am on each day of the cycle, as well as at 6pm and 11pm on proestrus and at 2am on estrus to capture surge events. Cycle stage was determined by vaginal cytology and post-mortem measurement of serum LH and FSH levels. In total, 102,069 cells passed rigorous quality control (QC, [2]), with over 5,000 cells analyzed per sample, ∼2,000 genes/cell, ∼15,000 ATAC median high-quality fragments, and Transcription Start Site (TSS) enrichment scores above 9 for all samples. We identified 13 well-separated clusters in the snRNAseq and 10 in the snATACseq datasets representing the pituitary cell types that were followed over time. We integrated the gene expression and chromatin accessibility datasets and analyzed changes in cell type proportions, gene expression, and chromatin accessibility through time. We detected major differential gene expression changes in the gonadotropes and lactotropes, which we further investigated using pseudotime trajectory analyses. Several upstream Fshb loci showed dynamic changes during the estrous cycle. Additionally, we uncovered regulatory components of major pituitary genes over time using linkage data analysis.To our knowledge, this is the first study to present detailed and comprehensive data on gene expression and chromatin structure changes at sn resolution in all pituitary cell types during a dynamic physiological process. Thus, it provides critical new resources to the field of endocrinology. References:[1] Nat Comm, 2020, 12(2677), PMID:33976139.[2] Cell Reports, 2022, 38(10): 110467, PMID:35263594. Presentation: Thursday, June 15, 2023