Time-course RNA-seq Research Articles

Abstract Tu086: Repairing nuclear envelope ruptures to ameliorate Lamin-related cardiomyopathy

Heterozygous loss-of-function mutations in LMNA , encoding nuclear lamina protein Lamin A/C, cause severe adult-onset dilated cardiomyopathy. A prevailing hypothesis posits that LMNA insufficiency causes nuclear envelope structural defects that ultimately cause the disease. However, the mechanisms linking defective nuclear envelopes to cardiomyopathy remain undefined. To determine specific nuclear envelope defects and their consequences, we deleted Lmna in cardiomyocytes in adult mice ( Lmna CKO ). Strikingly, a modest (50%) reduction of Lamin A/C caused widespread localized ruptures of the nuclear envelope in cardiomyocytes (En et al. bioRxiv 2023). The nuclear envelope ruptures did not cause immediate cell death, but accompanied a strong inflammatory response in the heart, prior to fatal cardiomyopathy. We hypothesized that DNA leaked from ruptured nuclei might elicit the cGAS-STING cytosolic DNA sensing pathway of innate immunity. Contrary to this hypothesis, we did not observe cGAS-STING activation in Lmna CKO cardiomyocytes. This lack of cGAS-STING activation was likely due to the near absence of cGAS protein in adult cardiomyocytes. To investigate the mechanism underlying cardiac inflammation in Lmna CKO mice, we conducted time-course single-nucleus RNA-seq. This analysis nominated cardiac fibroblasts as the central mediator of the inflammatory response, receiving ECM-mediated signaling from Lmna CKO cardiomyocytes and recruiting immune cells to the mutant hearts. Finally, we found evidence suggesting that nuclear envelope repair activity counteracts nuclear envelope ruptures in Lmna CKO mice. We found that the envelope ruptured sites co-localized with the ESCRT-III membrane remodeling complex, previously implicated in nuclear envelope repair. We further found that overexpression of DNA-binding protein BANF1 promoted ESCRT-III recruitment to the ruptured sites. We are currently investigating whether facilitated nuclear envelope repair ameliorates cardiomyopathy in Lmna CKO mice. If it does, nuclear envelope repair may be a potential therapeutic strategy for LMNA -related dilated cardiomyopathy.

Circadian clock shapes diurnal gene expression patterns linked to glucose metabolic processes in Chinese cabbage

The plant circadian clock temporally drives gene expression through the day and coordinates various physiological process with diurnal environmental changes. It is essential to confer plant fitness and competitive advantage to survive and thrive under natural condition by circadian control of gene transcription. Chinese cabbage (Brassica rapa ssp. pekinensis) is an economically important vegetable crop worldwide; however, there is little information concerning its circadian clock system. Here we uncovered that gene expression patterns were affected by circadian oscillators at both transcriptional and post-transcriptional levels in Chinese cabbage. Time-course RNA-seq analyses were conducted on two short-period lines (SPcc-1 and SPcc-2) and two long-period lines (LPcc-1 and LPcc-2) under constant light. We showed that 32.7-50.5% of the genes were regulated by the circadian oscillator and the expression peak of cycling genes appeared even earlier in short-period lines compared to long-period lines. In addition, approximately 250 splicing events showed circadian regulation, of which intron retention (IR) accounted for a large proportion. Rhythmically spliced genes included the clock genes LATE ELONGATED HYOCOTYL (BrLHY), REVEILLE 2 (BrRVE2) and EARLY FLOWERING 3 (BrELF3). We also found that the circadian oscillator could notably influence the diurnal expression patterns of genes that are associated with glucose metabolism via photosynthesis, Calvin cycle and tricarboxylic acid (TCA) cycle at both transcriptional and post-transcriptional levels. Taken together, our results demonstrate that circadian-regulated physiological processes contribute to Chinese cabbage growth and development.

Identification of host gene regulation and resistance pathway dynamics at diverse infection stages of Rhizoctonia solani AG3-TB.

Rhizoctonia solani is a fungal pathogen that causes significant losses in agricultural production. Because of its rapid transmission and broad host range, the exploration of genes involved in defense responses to the infection of R. solani has become an important task. Here, we performed a time-course RNA-Seq experiment to explore crucial genes or pathways involved in host responses to R. solani AG3-TB infection at 6, 12, 24, 36, 48, and 72 hours post inoculation (hpi). GO and KEGG enrichment analysis revealed that most DEGs were enriched in the basal metabolism pathways, including carbohydrate metabolic processes and the biosynthesis of amino acids. Moreover, catalase (CAT) and superoxide dismutase (SOD) were up-regulated, and transcription factors (TFs) such as WRKY, AP2, and MYB were increased significantly compared to the control (0 hpi). Silencing of WRKY70 and catalase-3 exhibited elevated susceptibility to the fungal infection. To summarize, the TFs WRKY70 and WRKY75, genes involved in jasmonic acid (JA), salicylic acid (SA), and brassinosteroids (BR) signaling pathways, and defense-related enzymes may play crucial roles in the host responses to R. solani AG3-TB infection.

Time-course RNAseq data of murine AB1 mesothelioma and Renca renal cancer following immune checkpoint therapy

Time-critical transcriptional events in the immune microenvironment are important for response to immune checkpoint blockade (ICB), yet these events are difficult to characterise and remain incompletely understood. Here, we present whole tumor RNA sequencing data in the context of treatment with ICB in murine models of AB1 mesothelioma and Renca renal cell cancer. We sequenced 144 bulk RNAseq samples from these two cancer types across 4 time points prior and after treatment with ICB. We also performed single-cell sequencing on 12 samples of AB1 and Renca tumors an hour before ICB administration. Our samples were equally distributed between responders and non-responders to treatment. Additionally, we sequenced AB1-HA mesothelioma tumors treated with two sample dissociation protocols to assess the impact of these protocols on the quality transcriptional information in our samples. These datasets provide time-course information to transcriptionally characterize the ICB response and provide detailed information at the single-cell level of the early tumor microenvironment prior to ICB therapy.

Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer.

The estrogen receptor-α (ER) drives 75% of breast cancers. On activation, the ER recruits and assembles a 1-2 MDa transcriptionally active complex. These complexes can modulate tumour growth, and understanding the roles of individual proteins within these complexes can help identify new therapeutic targets. Here, we present the discovery of ER and ZMIZ1 within the same multi-protein assembly by quantitative proteomics, and validated by proximity ligation assay. We characterise ZMIZ1 function by demonstrating a significant decrease in the proliferation of ER-positive cancer cell lines. To establish a role for the ER-ZMIZ1 interaction, we measured the transcriptional changes in the estrogen response post-ZMIZ1 knockdown using an RNA-seq time-course over 24 h. Gene set enrichment analysis of the ZMIZ1-knockdown data identified a specific delay in the response of estradiol-induced cell cycle genes. Integration of ENCODE data with our RNA-seq results identified that ER and ZMIZ1 both bind the promoter of E2F2. We therefore propose that ER and ZMIZ1 interact to enable the efficient estrogenic response at subset of cell cycle genes via a novel ZMIZ1-ER-E2F2 signalling axis. Finally, we show that high ZMIZ1 expression is predictive of worse patient outcome, ER and ZMIZ1 are co-expressed in breast cancer patients in TCGA and METABRIC, and the proteins are co-localised within the nuclei of tumour cell in patient biopsies. In conclusion, we establish that ZMIZ1 is a regulator of the estrogenic cell cycle response and provide evidence of the biological importance of the ER-ZMIZ1 interaction in ER-positive patient tumours, supporting potential clinical relevance.

Ethylene inhibits photosynthesis via temporally distinct responses in tomato plants.

Ethylene is a volatile plant hormone that regulates many developmental processes and responses toward (a)biotic stress. Studies have shown that high levels of ethylene repress vegetative growth in many important crops, including tomato (Solanum lycopersicum), possibly by inhibiting photosynthesis. We investigated the temporal effects of ethylene on young tomato plants using an automated ethylene gassing system to monitor the physiological, biochemical, and molecular responses through time course RNA-seq of a photosynthetically active source leaf. We found that ethylene evokes a dose-dependent inhibition of photosynthesis, which can be characterized by 3 temporally distinct phases. The earliest ethylene responses that marked the first phase and occurred a few hours after the start of the treatment were leaf epinasty and a decline in stomatal conductance, which led to lower light perception and CO2 uptake, respectively, resulting in a rapid decline of soluble sugar levels (glucose, fructose). The second phase of the ethylene effect was marked by low carbohydrate availability, which modulated plant energy metabolism to adapt by using alternative substrates (lipids and proteins) to fuel the TCA cycle. Long-term continuous exposure to ethylene led to the third phase, characterized by starch and chlorophyll breakdown, which further inhibited photosynthesis, leading to premature leaf senescence. To reveal early (3 h) ethylene-dependent regulators of photosynthesis, we performed a ChIP-seq experiment using anti-ETHYLENE INSENSITIVE 3-like 1 (EIL1) antibodies and found several candidate transcriptional regulators. Collectively, our study revealed a temporal sequence of events that led to the inhibition of photosynthesis by ethylene and identified potential transcriptional regulators responsible for this regulation.

A resource of single-cell gene expression profiles in a planarian Dugesia japonica.

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset.

Time-course RNA-Seq analysis defines immunological and inflammatory mechanisms influenced by bovine respiratory disease

Bovine respiratory disease (BRD) remains the leading disease in beef cattle. Host gene expression at facility arrival may indi­cate BRD risk, however, time-course RNA-Seq could define how BRD development influences immunological and inflammatory responses. Here, we evaluated blood transcriptomes of high-risk beef cattle at 3 timepoints to illustrate BRD-associated host response.

Phased grapevine genome sequence of an Rpv12 carrier for biotechnological exploration of resistance to Plasmopara viticola.

The downy mildew disease caused by the oomycete Plasmopara viticola is a serious threat for grapevine and can cause enormous yield losses in viticulture. The quantitative trait locus Rpv12, mediating resistance against P. viticola, was originally found in Asian Vitis amurensis. This locus and its genes were analyzed here in detail. A haplotype-separated genome sequence of the diploid Rpv12-carrier Gf.99-03 was created and annotated. The defense response against P. viticola was investigated in an infection time-course RNA-seq experiment, revealing approximately 600 upregulated Vitis genes during host-pathogen interaction. The Rpv12 regions of the resistance and the sensitivity encoding Gf.99-03 haplotype were structurally and functionally compared with each other. Two different clusters of resistance-related genes were identified within the Rpv12 locus. One cluster carries a set of four differentially expressed genes with three ACCELERATED CELL DEATH 6-like genes. The other cluster carries a set of six resistance gene analogs related to qualitative pathogen resistance. The Rpv12 locus and its candidate genes for P. viticola resistance provide a precious genetic resource for P. viticola resistance breeding. Newly developed co-segregating simple sequence repeat markers in close proximity to the R-genes enable its improved applicability in marker-assisted grapevine breeding.

MultiWGCNA: an R package for deep mining gene co-expression networks in multi-trait expression data

BackgroundGene co-expression networks represent modules of genes with shared biological function, and have been widely used to model biological pathways in gene expression data. Co-expression networks associated with a specific trait can be constructed and identified using weighted gene co-expression network analysis (WGCNA), which is especially useful for the study of transcriptional signatures in disease. WGCNA networks are typically constructed using both disease and wildtype samples, so molecular pathways associated with disease are identified. However, it would be advantageous to study such co-expression networks in their disease context across spatiotemporal conditions, but currently there is no comprehensive software implementation for this type of analysis.ResultsHere, we introduce a WGCNA-based procedure, multiWGCNA, that is tailored to datasets with variable spatial or temporal traits. As well as constructing the combined network, multiWGCNA also generates a network for each condition separately, and subsequently maps these modules between and across designs, and performs relevant downstream analyses, including module-trait correlation and module preservation. When applied to astrocyte-specific RNA-sequencing (RNA-seq) data from various brain regions of mice with experimental autoimmune encephalitis, multiWGCNA resolved the de novo formation of the neurotoxic astrocyte transcriptional program exclusively in the disease setting. Using time-course RNA-seq from mice with tau pathology (rTg4510), we demonstrate how multiWGCNA can also be used to study the temporal evolution of pathological modules over the course of disease progression.ConclusionThe multiWGCNA R package can be applied to expression data with two dimensions, which is especially useful for the study of disease-associated modules across time or space. The source code and functions are freely available at: https://github.com/fogellab/multiWGCNA.

The RNA binding protein DND1 is elevated in a subpopulation of pro-spermatogonia and targets chromatin modifiers and translational machinery during late gestation.

DND1 is essential to maintain germ cell identity. Loss of Dnd1 function results in germ cell differentiation to teratomas in some inbred strains of mice or to somatic fates in zebrafish. Using our knock-in mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP), we distinguished two male germ cell (MGC) populations during late gestation cell cycle arrest (G0), consistent with recent reports of heterogeneity among MGCs. Most MGCs express lower levels of DND1-GFP (DND1-GFP-lo), but some MGCs express elevated levels of DND1-GFP (DND1-GFP-hi). A RNA-seq time course confirmed high Dnd1 transcript levels in DND1-GFP-hi cells along with 5-10-fold higher levels for multiple epigenetic regulators. Using antibodies against DND1-GFP for RNA immunoprecipitation (RIP)-sequencing, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0 including DNA methyltransferases (Dnmts), histone deacetylases (Hdacs), Tudor domain proteins (Tdrds), actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that in DND1-GFP-hi cells, DND1 hosts coordinating mRNA regulons that consist of functionally related and localized groups of epigenetic enzymes and translational components.

Novel context-specific genome-scale modelling explores the potential of triacylglycerol production by Chlamydomonas reinhardtii

Gene expression data of cell cultures is commonly measured in biological and medical studies to understand cellular decision-making in various conditions. Metabolism, affected but not solely determined by the expression, is much more difficult to measure experimentally. Finding a reliable method to predict cell metabolism for expression data will greatly benefit metabolic engineering. We have developed a novel pipeline, OVERLAY, that can explore cellular fluxomics from expression data using only a high-quality genome-scale metabolic model. This is done through two main steps: first, construct a protein-constrained metabolic model (PC-model) by integrating protein and enzyme information into the metabolic model (M-model). Secondly, overlay the expression data onto the PC-model using a novel two-step nonconvex and convex optimization formulation, resulting in a context-specific PC-model with optionally calibrated rate constants. The resulting model computes proteomes and intracellular flux states that are consistent with the measured transcriptomes. Therefore, it provides detailed cellular insights that are difficult to glean individually from the omic data or M-model alone. We apply the OVERLAY to interpret triacylglycerol (TAG) overproduction by Chlamydomonas reinhardtii, using time-course RNA-Seq data. We show that OVERLAY can compute C. reinhardtii metabolism under nitrogen deprivation and metabolic shifts after an acetate boost. OVERLAY can also suggest possible ‘bottleneck’ proteins that need to be overexpressed to increase the TAG accumulation rate, as well as discuss other TAG-overproduction strategies.

Time-course RNA-seq analysis provides an improved understanding of genetic regulation in response to cold stress from white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is an important legume forage, which is widely distributed in cool-season regions. Therefore, cold stress is a major environmental factor limiting its growth and production, while little is known about the its cold tolerance at molecular level. In the present study, we performed time-course RNA-seq analysis under cold stress. RNA-seq results suggested that genes associated with “oxidoreductase activity” and “transcription regulator activity” are more likely related to the response of white clover to cold stress. To identify the specific gene modules and the hub genes of white clover in response to cold stress, we applied weighted gene co-expression network analyses (WGCNA) to transcriptome data. We also found that gene modules that focus on protein kinase activity, DNA-binding transcription factor activity and oxidoreductase activity, are more likely to be involved in the response of white clover to cold stress. Especially, we identified several AP2/ERF TF genes and CDPK genes as pivotal genes in white clover in response to cold stress, which would provide helpful insights into the molecular mechanisms underlying the response of white clover to cold stress.

Characterization of the genetic and regulatory networks associated with sugar and acid metabolism in apples via an integrated strategy.

Although sugars and acids have a substantial influence on the taste of apple fruits, the genetic and regulatory networks underlying their metabolism in fruit remain insufficiently determined. To fully decipher the genetic basis of the accumulation of sugars and acids in apple fruits, we adopted an integrated strategy that included time-course RNA-seq, QTL mapping, and whole-genome sequencing to examine two typical cultivars ('HanFu' and 'Huahong') characterized by distinctive flavors. Whole-genome sequencing revealed substantial genetic variation between the two cultivars, thereby providing an indication of the genetic basis of the distinct phenotypes. Constructed co-expression networks yielded information regarding the intra-relationships among the accumulation of different types of metabolites, and also revealed key regulatory nodes associated with the accumulation of sugars and acids, including the genes MdEF2, MdPILS5, and MdGUN8. Additionally, on the basis of QTL mapping using a high-density genetic map, we identified a series of QTLs and functional genes underlying vital traits, including sugar and acid contents. Collectively, our methodology and observations will provide an important reference for further studies focusing on the flavor of apples.

Chronic cAMP activation induces adipocyte browning through discordant biphasic remodeling of transcriptome and chromatin accessibility

ObjectiveAdipose tissue thermogenesis has been suggested as a new therapeutic target to promote energy metabolism for obesity and metabolic disease. Cold-inducible thermogenic adipocytes, called beige adipocytes, have attracted significant attention for their potent anti-obesity activity in adult humans. In this study, we identified the mechanisms underlying beige adipocyte recruitment, so-called adipocyte browning, by different stimuli. MethodsWe generated a new adipocyte cell line with enhanced browning potentials and determined its transcriptomic and epigenomic responses following cAMP (forskolin, FSK) versus PPARγ activation (rosiglitazone). We performed time-course RNA-seq and compared the treatments and in vivo adipocyte browning. We also developed an improved protocol for Assay for Transposase Accessible Chromatin-sequencing (ATAC-seq) and defined changes in chromatin accessibility in a time course. The RNA-seq and ATAC-seq data were integrated to determine the kinetics of their coordinated regulation and to identify a transcription factor that drives these processes. We conducted functional studies using pharmacological and genetic approaches with specific inhibitors and shRNA-mediated knockdown, respectively. ResultsFSK, not rosiglitazone, resulted in a biphasic transcriptomic response, resembling the kinetics of in vivo cold-induced browning. FSK promoted tissue remodeling first and subsequently shifted energy metabolism, concluding with a transcriptomic profile similar to that induced by rosiglitazone. The thermogenic effects of FSK were abolished by PPARγ antagonists, indicating PPARγ as a converging point. ATAC-seq uncovered that FSK leads to a significant chromatin remodeling that precedes or persists beyond transcriptomic changes, whereas rosiglitazone induces minimal changes. Motif analysis identified nuclear factor, interleukin 3 regulated (NFIL3) as a transcriptional regulator connecting the biphasic response of FSK-induced browning, as indicated by disrupted thermogenesis with NFIL3 knockdown. ConclusionsOur findings elucidated unique dynamics of the transcriptomic and epigenomic remodeling in adipocyte browning, providing new mechanistic insights into adipose thermogenesis and molecular targets for obesity treatment.

Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production

Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.

High temporal resolution RNA-seq time course data reveals widespread synchronous activation between mammalian lncRNAs and neighboring protein-coding genes.

The advent of massively parallel sequencing revealed extensive transcription beyond protein-coding genes, identifying tens of thousands of long noncoding RNAs (lncRNAs). Selected functional examples raised the possibility that lncRNAs, as a class, may maintain broad regulatory roles. Expression of lncRNAs is strongly linked with adjacent protein-coding gene expression, suggesting potential cis-regulatory functions. A more detailed understanding of these regulatory roles may be obtained through careful examination of the precise timing of lncRNA expression relative to adjacent protein-coding genes. Despite the diversity of reported lncRNA regulatory mechanisms, where causal cis-regulatory relationships exist, lncRNA transcription is expected to precede changes in target gene expression. Using a high temporal resolution RNA-seq time course, we profiled the expression dynamics of several thousand lncRNAs and protein-coding genes in synchronized, transitioning human cells. Our findings reveal that lncRNAs are expressed synchronously with adjacent protein-coding genes. Analysis of lipopolysaccharide-activated mouse dendritic cells revealed the same temporal relationship observed in transitioning human cells. Our findings suggest broad-scale cis-regulatory roles for lncRNAs are not common. The strong association between lncRNAs and adjacent genes may instead indicate an origin as transcriptional by-products from active protein-coding gene promoters and enhancers.

200-LB: Persistent cAMP Signaling Induces Adipocyte Browning through Biphasic Transcriptional and Epigenetic Remodeling

Beige adipocytes are inducible thermogenic cells formed in response to cold exposure and other environmental cues within white adipose tissue. Their capacity to produce heat would provide metabolic benefits against obesity and type 2 diabetes; however, the mechanisms underlying beige adipocyte recruitment, so-called adipocyte browning, induced by different stimuli remain unclear. Using our new adipocyte cell line with improved browning potentials, we compared adipocyte browning responses induced by cAMP (forskolin) versus PPARɣ activation (rosiglitazone) . Time-course RNA-seq revealed that chronic activation of cAMP but not PPARɣ resulted in a biphasic response. The cAMP signaling promoted tissue remodeling at the early phase and subsequently activated energy metabolism while inhibiting cell cycle, which resembles the kinetics of in vivo cold-induced browning. Those late-phase changes were highly correlated with those by PPARɣ activation. Consistently, the cAMP-driven effects were abolished by treatment with PPARɣ antagonists, suggesting PPARɣ as a converging point of its thermogenic actions. Assay for transposase-accessible chromatin sequencing (ATAC-seq) revealed unique dynamics of chromatin landscape distinct from transcriptional changes during cAMP-induced browning. Chromatin at early-induced genes opened acutely and stayed open until the late phase despite the rapid return of gene expression. The chromatin regions at late-induced genes also opened early, preceding transcriptional activation. Using a motif discovery method, we identified NFIL3 that was enriched in both early and late phases of cAMP-induced browning. Reduced thermogenic gene expression and mitochondrial respiration by NFIL3 knockdown suggest NFIL3 as a putative transcription factor mediating the biphasic response of adipocyte browning. These findings define new mechanisms that orchestrate transcriptional and epigenetic remodeling underlying beige adipocyte formation. Disclosure J. So: None. S. Taleb: None. J. Wann: None. O. Strobel: None. H. Roh: None. Funding American Diabetes Association (7-21-JDF-056) , National Institute of Health (DK129289)

Temporal transcriptomic analysis using TrendCatcher identifies early and persistent neutrophil activation in severe COVID-19.

Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there are not enough methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic data sets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy, to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal data sets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk and single-cell RNA-Seq time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways, as well as impaired type I IFN (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS-CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets.