Search

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates has been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRD). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identify genomic targets for the advanced investigation of the pathogenesis of DRRD.

Genome wide association studies of asthma have not explained environmental risk or variable clinical efficacy of glucocorticoids. Bidirectional enhancer RNA (eRNA) transcription is a widespread response to environmental signals and glucocorticoids. Therefore, we investigated whether single nucleotide polymorphisms (SNPs) within dynamically regulated enhancer RNA (eRNA)-transcribing regions contribute to genetic variation in asthma. Through applying machine-learning methods to a large clinical cohort, we identified novel associations between asthma and 36 SNPs located in eRNA-transcribing regions implicated in regulating diverse cellular processes relevant to asthma. Functional validation established thatrs258760(mean allele frequency = 0.35, asthma odds ratio 0.95; P

Stem cell (SC) exhaustion is a hallmark of aging. However, the process of SC depletion during aging has not been observed in live animals, and the underlying mechanism contributing to tissue deterioration remains obscure. We find that, in aged mice, epithelial cells escape from the hair follicle (HF) SC compartment to the dermis, contributing to HF miniaturization. Single-cell RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) reveal reduced expression of cell adhesion and extracellular matrix genes in aged HF-SCs, many of which are regulated by Foxc1 and Nfatc1. Deletion of Foxc1 and Nfatc1 recapitulates HF miniaturization and causes hair loss. Live imaging captures individual epithelial cells migrating away from the SC compartment and HF disintegration. This study illuminates a hitherto unknown activity of epithelial cells escaping from their niche as a mechanism underlying SC reduction and tissue degeneration. Identification of homeless epithelial cells in aged tissues provides a new perspective for understanding aging-associated diseases. Using live imaging, the study shows, in mice, that epithelial cells escape from the hair follicle stem cell compartment during aging. Stem cell escape is associated with reduced cell adhesion and extracellular matrix gene expression and leads to hair follicle miniaturization.

Background A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. Results Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ′ end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. Conclusions Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.

Nascent transcription assays, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have uncovered a myriad of unstable RNAs being actively produced from numerous sites genome-wide. These transcripts provide a more complete and immediate picture of the impact of regulatory events. Transcription factors recruit RNA polymerase II, effectively initiating the process of transcription; repressors inhibit polymerase recruitment. Efficiency of recruitment is dictated by sequence elements in and around the RNA polymerase loading zone. A combination of sequence elements and RNA binding proteins subsequently influence the ultimate stability of the resulting transcript. Some of these transcripts are capable of providing feedback on the process, influencing subsequent transcription. By monitoring RNA polymerase activity, nascent assays provide insights into every step of the regulated process of transcription.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 h and 18 h postinfection and observed widespread changes in metal transport, metal-dependent genes, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 45-fold at 12 h postinfection. Further, manipulation of zinc in the medium alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. The 129S6 macrophages leverage intricate means of manipulating zinc availability and distribution to limit the pathogen’s access to zinc, while simultaneously ensuring sufficient zinc to support the immune response.

The heterogeneity of respirable particulates and compounds complicates our understanding of transcriptional responses to air pollution. Here, we address this by applying precision nuclear run-on sequencing and the assay for transposase-accessible chromatin sequencing to measure nascent transcription and chromatin accessibility in airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to identify temporally distinct roles for ternary response factor–serum response factor complexes, the aryl hydrocarbon receptor (AHR), and NFκB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 min of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 min after exposure. The kinetics of AHR- and NFκB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFκB-mediated responses through 120 min of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFκB signaling in controlling proinflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.

Skeletal muscle cells are multinucleated syncytial cells arising from cell fusion, yet despite sharing a common cytoplasm individual myonuclei express distinct transcriptional programs. Whether individual myonuclei acquire heterogenous transcriptional states via differences in their progenitors, during differentiation, or once their anatomical position is acquired, is not known. We performed transcriptome and pseudotime analysis of single myogenic nuclei from uninjured and post-injury murine skeletal muscle to assess when myonuclear heterogeneity is acquired. Two distinct progenitors contribute to myonuclei, one a non-myogenic fibroblast subtype, and skeletal muscle stem cells the other. Both progenitors enter a single pseudotime trajectory that bifurcates as myonuclei mature into two branches segregated by myosin isoform expression and metabolic profiles, suggesting transcriptional heterogeneity is acquired as myonuclei mature. In aged skeletal muscle myogenic progenitor expansion is perturbed and nuclei from aged muscle display distinct pseudotemporal kinetics compared to nuclei from young mice. In aged mice, the inferred myogenic differentiation trajectory is delayed, altering the distribution of myogenic nuclei in pseudotime, suggesting that altered transcriptional dynamics in nuclei in aged mice may drive age-associated muscle deficits and bias myonuclei towards acquiring oxidative metabolic profiles.

Significance We analyzed data from saliva-based COVID-19 screening deployed on the University of Colorado Boulder campus. Our dataset is unique in that all SARS-CoV-2−positive individuals reported no symptoms at the time of saliva collection, and therefore were infected but asymptomatic or presymptomatic. We found that 1) the distribution of viral loads observed in our asymptomatic college population was indistinguishable from what has been reported in hospitalized populations; 2) regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the infection; and 3) just 2% of infected individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and likely also superspreaders., We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and possibly also superspreaders.

RATIONALE: Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known. OBJECTIVES: Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659. METHODS: We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages. RESULTS: The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice. CONCLUSION: Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.

Numerous reports have suggested that infectious agents could play a role in neurodegenerative diseases, but specific etiological agents have not been convincingly demonstrated. To search for candidate agents in an unbiased fashion, we have developed a bioinformatic pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences with no significant nucleotide similarity hits in GenBank. Effectiveness of the pipeline was tested using publicly available RNA-seq data and in a reconstruction experiment using synthetic data. We then applied this pipeline to a novel RNA-seq dataset generated from a cohort of 120 samples from amyotrophic lateral sclerosis patients and controls, and identified sequences corresponding to known bacteria and viruses, as well as novel virus-like sequences. The presence of these novel virus-like sequences, which were identified in subsets of both patients and controls, were confirmed by quantitative RT-PCR. We believe this pipeline will be a useful tool for the identification of potential etiological agents in the many RNA-seq datasets currently being generated.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron availability, increasing exposure to bactericidal copper, and manipulating zinc to restrict the growth of pathogens. Manipulation of zinc at the host-pathogen interface depends on both the pathogen’s identity and the nature of the host cell. Here we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella Typhimurium. Unlike Balb/c and C57BL/6 mice, 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 Hrs and 18 Hrs post-infection and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 36-fold 12 hrs post-infection. Further, manipulation of zinc in the media alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing our results to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response, with 129S6 macrophages transitioning from M1 to M2 polarization over the course of infection and showing signs of recovery. Our results reveal that functional Slc11a1 profoundly affects the transcriptional landscape upon infection. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. 129S6 macrophage leverage intricate means of manipulating zinc availability and distribution to limit the pathogen’s access to zinc while simultaneously ensuring sufficient zinc to support the immune response.Author summaryMetal ions play an important role in influencing how immune cells such as macrophages respond to infection by pathogens. Because metal ions are both essential to survival, as well toxic when present is excessive amounts, the host and the pathogen have evolved diverse strategies to regulate metal acquisition and availability. Here, we show that the metal transporter slc11a1 plays a critical role in defining the host response to Salmonella infection. Infection causes widespread changes in expression of metal regulatory genes to limit the pathogen’s access to iron, increase its exposure to copper, and remodel zinc to ensure increased zinc in the cytosol and limited zinc for the pathogen. Macrophages expressing functional slc11a1 have a different profile of metal regulation and vastly different outcomes compared to immune compromised macrophage, demonstrating significantly different nutritional immune responses in immune competent versus immune compromised macrophages.

Heterogeneity of respirable particulates and compounds complicates understanding transcriptional responses to air pollution. Here, we address this problem through applying precision nuclear run-on sequencing (PRO-seq) to measure nascent transcription and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to agnostically identify temporally distinct roles for the TCF/SRF family, the aryl hydrocarbon receptor (AHR), and NFkB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 minutes of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 minutes. The kinetics of AHR- and NFkB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFkB responses through 120 minutes of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFkB signaling in controlling pro-inflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.

Aging is accompanied by a general decline in the function of many cellular pathways. However, whether these are causally or functionally interconnected remains elusive. Here, we study the effect of mitochondrial–nuclear communication on stem cell aging. We show that aged mesenchymal stem cells exhibit reduced chromatin accessibility and lower histone acetylation, particularly on promoters and enhancers of osteogenic genes. The reduced histone acetylation is due to impaired export of mitochondrial acetyl-CoA, owing to the lower levels of citrate carrier (CiC). We demonstrate that aged cells showed enhanced lysosomal degradation of CiC, which is mediated via mitochondrial-derived vesicles. Strikingly, restoring cytosolic acetyl-CoA levels either by exogenous CiC expression or via acetate supplementation, remodels the chromatin landscape and rescues the osteogenesis defects of aged mesenchymal stem cells. Collectively, our results establish a tight, age-dependent connection between mitochondrial quality control, chromatin and stem cell fate, which are linked together by CiC. Aged mesenchymal stem cells exhibit decreased osteogenesis. Pouikli et al. link impaired MSC differentiation to histone hypoacetylation caused by lower mitochondrial acetyl-CoA export due to enhanced lysosomal degradation of the citrate carrier Slc25a1. Restoring histone acetylation to youthful levels rescues osteogenesis.

The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this –3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the –3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.

We have been using the Inbred Long- and Short-Sleep mouse strains (ILS, ISS) and a recombinant inbred panel derived from them, the LXS, to investigate the genetic underpinnings of acute ethanol tolerance which is considered to be a risk factor for alcohol use disorders (AUDs). Here, we have used RNA-seq to examine the transcriptome of whole brain in 40 of the LXS strains 8 hours after a saline or ethanol "pretreatment" as in previous behavioral studies. Approximately 1/3 of the 14,184 expressed genes were significantly heritable and many were unique to the pretreatment. Several thousand cis- and trans-eQTLs were mapped; a portion of these also were unique to pretreatment. Ethanol pretreatment caused differential expression (DE) of 1,230 genes. Gene Ontology (GO) enrichment analysis suggested involvement in numerous biological processes including astrocyte differentiation, histone acetylation, mRNA splicing, and neuron projection development. Genetic correlation analysis identified hundreds of genes that were correlated to the behaviors. GO analysis indicated that these genes are involved in gene expression, chromosome organization, and protein transport, among others. The expression profiles of the DE genes and genes correlated to AFT in the ethanol pretreatment group (AFT-Et) were found to be similar to profiles of HDAC inhibitors. Hdac1, a cis-regulated gene that is located at the peak of a previously mapped QTL for AFT-Et, was correlated to 437 genes, most of which were also correlated to AFT-Et. GO analysis of these genes identified several enriched biological process terms including neuron-neuron synaptic transmission and potassium transport. In summary, the results suggest widespread genetic effects on gene expression, including effects that are pretreatment-specific. A number of candidate genes and biological functions were identified that could be mediating the behavioral responses. The most prominent of these was Hdac1 which may be regulating genes associated with glutamatergic signaling and potassium conductance.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a “master regulator” role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Government regulators and others concerned about toxic chemicals in the environment hold that a mechanistic, causal explanation of toxicity is strongly preferred over a statistical or machine learning-based prediction by itself. Elucidating a mechanism of toxicity is, however, a costly and time-consuming process that requires the participation of specialists from a variety of fields, often relying on animal models. We present an innovative mechanistic inference framework (MechSpy), which can be used as a hypothesis generation aid to narrow the scope of mechanistic toxicology analysis. MechSpy generates hypotheses of the most likely mechanisms of toxicity, by combining a semantically-interconnected knowledge representation of human biology, toxicology and biochemistry with gene expression time series on human tissue. Using vector representations of biological entities, MechSpy seeks enrichment in a manually-curated list of high-level mechanisms of toxicity, represented as biochemically- and causally-linked ontology concepts. Besides predicting the canonical mechanism of toxicity for many well-studied compounds, we experimentally validated some of our predictions for other chemicals without an established mechanism of toxicity. This framework can be modified to include additional mechanisms of toxicity, and is generalizable to other types of mechanisms of human biology.Author summarySeveral recent computational methods have displayed excellent performance in predicting toxicity outcomes [1–3] of chemicals. Yet, to our knowledge, there is to date no computational approach to generate mechanistic hypotheses to answer why these chemicals elicit a toxic response. There is great value in understanding the mechanism of toxicity for a chemical that appears to elicit an adverse response. Novel small molecule development is one example, where a chemical that failed initial toxicological screenings could be assessed to evaluate the actual mechanism of toxicity, greatly reducing research time and expenses on subsequent ones. The value of a mechanistic awareness of toxicity also applies to pharmacovigilance, when researching rare adverse effects of a drug in subsets of the population. The development of oncological chemotherapeutics is another example, where certain mechanisms of cytotoxicity can actually be desirable to eliminate different types of tumor cells. More importantly, the costs, time expenditure, and ethical concerns of toxicity animal models, make in vitro and in silico approaches an enticing alternative. We present a solution that uses a combination of gene expression assays and biomedical knowledge to address the gap of answering the why question.

Ageing is accompanied by a general decline in the function of many cellular pathways, with metabolic alterations, epigenetic modifications, and stem cell exhaustion representing three important hallmarks of the ageing process. However, whether these pathways are causally or functionally related at a molecular level remains poorly understood. Here, we use bone marrow-derived mesenchymal stem cells (MSCs) isolated from young and old mice to address how age-dependent changes in metabolism and epigenetics are linked and how they impact on the ageing transcriptome and differentiation potential. Given that MSCs maintain specific age-associated properties even under prolonged culture conditions, such as the age-dependent decrease in osteogenic differentiation, they are an excellent model to investigatein vitrothe connection of ageing hallmarks on a mechanistic level. In this study, we demonstrate that upon ageing, osteogenic potential of MSCs declines as a consequence of deregulated mito-nuclear communication, mediated by decreased levels of the citrate carrier (CiC). Age-dependent down-regulation of CiC results in acetyl-CoA trapping within mitochondria, hypo-acetylation of histones and chromatin compaction. Together, these changes lead to an altered transcriptional output and are responsible for the reduced differentiation capacity into osteoblasts. Strikingly, short-term supplementation of aged cells with acetate, an exogenous source for cytosolic acetyl-CoA production, rescues not only the age-associated reduction of histone acetylation, but also the osteogenesis defect, representing a potential target forin vitroMSC rejuvenation.

Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother’s milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

1AbstractDetecting differential activation of transcription factors (TFs) in response to perturbation provides insight into cellular processes. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects differential activity of hundreds of TFs given any set of perturbation data. TFEA draws inspiration from GSEA and detects positional motif enrichment within a list of ranked regions of interest (ROIs). As ROIs are typically inferred from the data, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent (eg. PRO-Seq), CAGE, ChIP-Seq, and accessibility (e.g. ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.

The assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) is an inexpensive protocol for measuring open chromatin regions. ATAC-seq is also relatively simple and requires fewer cells than many other high-throughput sequencing protocols. Therefore, it is tractable in numerous settings where other high throughput assays are challenging to impossible. Hence it is important to understand the limits of what can be inferred from ATAC-seq data. In this work, we leverage ATAC-seq to predict the presence of nascent transcription. Nascent transcription assays are the current gold standard for identifying regions of active transcription, including markers for functional transcription factor (TF) binding. We combine mapped short reads from ATAC-seq with the underlying peak sequence, to determine regions of active transcription genome-wide. We show that a hybrid signal/sequence representation classified using recurrent neural networks (RNNs) can identify these regions across different cell types.

The Δ40p53 isoform heterotetramerizes with WTp53 to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with knock-in cell lines that generated homogeneous Δ40p53:WTp53 tetramers from the native TP53 locus, and examined with transcriptomics (PRO-seq, RNA-seq), metabolomics, and other methods. Although phenotypically similar to WTp53 under normal conditions, Δ40p53:WTp53 failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of eRNA transcription and subsequent failure to induce mRNA, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil) also showed Δ40p53:WTp53 defects in mRNA induction; however, other stimulus-specific TFs could then drive the response, yielding similar outcomes vs. WTp53. Our results define how Δ40p53 alters WTp53 function and identify an eRNA requirement for mRNA biogenesis. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest human p53 evolved as a tetramer to support eRNA transcription.