Global Gene Expression Responses Research Articles

PHOSPHATASE 2A dephosphorylates PHYTOCHROME INTERACTING FACTOR3 to modulate photomorphogenesis in Arabidopsis.

The phytochrome (phy) family of sensory photoreceptors modulates developmental programs in response to ambient light. Phys also control gene expression in part by directly interacting with the bHLH class of transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and inducing their rapid phosphorylation and degradation. Several kinases have been shown to phosphorylate PIFs and promote their degradation. However, the phosphatases that dephosphorylate PIFs are less understood. Here, we describe four regulatory subunits of the Arabidopsis (Arabidopsis thaliana) protein PHOSPHATASE 2A (PP2A) family (B'α, B'β, B''α and B''β) that interact with PIF3 in yeast two-hybrid, in vitro and in vivo assays. The pp2ab''αβ and b''αβ/b'αβ mutants displayed short hypocotyls, while the overexpression of the B subunits induced longer hypocotyls compared to the wild type under red light. The light-induced degradation of PIF3 was faster in the b''αβ/b'αβ quadruple mutant compared to in the wild type. Consistently, immunoprecipitated PP2A A and B subunits directly dephosphorylated PIF3-MYC in vitro. RNA-seq analyses showed that B''α and B''β alter global gene expression in response to red light. PIFs (PIF1, PIF3, PIF4 and PIF5) are epistatic to these B subunits in regulating hypocotyl elongation under red light. Collectively, these data show an essential function of PP2A in dephosphorylating PIF3 to modulate photomorphogenesis in Arabidopsis.

Cultured Renal Proximal Tubular Epithelial Cells Resemble a Stressed/Damaged Kidney While Supporting BK Virus Infection.

BK virus (BKV; human polyomavirus 1) infections are asymptomatic in most individuals, and the virus persists throughout life without harm. However, BKV is a threat to transplant patients and those with immunosuppressive disorders. Under these circumstances, the virus can replicate robustly in proximal tubule epithelial cells (PT). Cultured renal proximal tubule epithelial cells (RPTE) are permissive to BKV and have been used extensively to characterize different aspects of BKV infection. Recently, lines of hTERT-immortalized RPTE have become available, and preliminary studies indicate they support BKV infection as well. Our results indicate that BKV infection leads to a similar response in primary and immortalized RPTE. In addition, we examined the patterns of global gene expression of primary and immortalized RPTE and compared them with uncultured PT freshly dissociated from human kidney. As expected, PT isolated from the healthy kidney express a number of differentiation-specific genes that are associated with kidney function. However, the expression of most of these genes is absent or repressed in cultured RPTE. Rather, cultured RPTE exhibit a gene expression profile indicative of a stressed or injured kidney. Inoculation of cultured RPTE with BKV results in the suppression of many genes associated with kidney stress. In summary, this study demonstrated similar global gene expression patterns and responses to BKV infection between primary and immortalized RPTE. Moreover, results from bulk transcriptome sequencing (RNA-seq) and SCT experiments revealed distinct transcriptomic signatures representing cell injury and stress in primary RPTE in contrast to the uncultured, freshly dissociated PT from human kidney. IMPORTANCE Cultured primary human cells provide powerful tools for the study of viral infectious cycles and host virus interactions. In the case of BKV-associated nephropathy, viral replication occurs primarily in the proximal tubule epithelia in the kidney. Consequently, cultured primary and immortalized renal proximal tubule epithelial cells (RPTE) are widely used to study BKV infection. In this work, using bulk and single-cell transcriptomics, we found that primary and immortalized RPTE responded similarly to BKV infection. However, both uninfected primary and immortalized RPTE have gene expression profiles that are markedly different from healthy proximal tubule epithelia isolated directly from human kidney without culture. Cultured RPTE are in a gene expression state indicative of an injured or stressed kidney. These results raise the possibility that BKV replicates preferentially in injured or stressed kidney epithelial cells during nephropathy.

Diurnal transcriptome dynamics reveal the photoperiod response of Pyrus.

Photoperiod provides a key environmental signal that controls plant growth. Plants have evolved an integrated mechanism for sensing photoperiods with internal clocks to orchestrate physiological events. This mechanism has been identified to enable timely plant growth and improve fitness. Although the components and pathways underlying photoperiod regulation have been described in many species, diurnal patterns of gene expression at the genome-wide level under different photoperiods are rarely reported in perennial fruit trees. To explore the global gene expression in response to photoperiod, pear plants were cultured under long-day (LD) and short-day (SD) conditions. A time-series transcriptomic study was implemented using LD and SD samples collected at 4 h intervals over 2 days. We identified 13,677 rhythmic genes, of which 7639 were identified under LD and 10,557 under SD conditions. Additionally, 4674 genes were differentially expressed in response to photoperiod change. We also characterized the candidate homologs of clock-associated genes in pear. Clock genes were involved in the regulation of many processes throughout the day, including photosynthesis, stress response, hormone dynamics, and secondary metabolism. Strikingly, genes within photosynthesis-related pathways were enriched in both the rhythmic and differential expression analyses. Several key candidate genes were identified to be associated with regulating photosynthesis and improving productivity under different photoperiods. The results suggest that temporal variation in gene expression should not be ignored in pear gene function research. Overall, our work expands the understanding of photoperiod regulation of plant growth, particularly by extending the research to non-model trees.

Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host-Pathogen Response to Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host–pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host–pathogen interaction.

Regulation of cell cycle and differentiation markers by pathogenic, non-pathogenic and opportunistic skin bacteria

Skin is the first line of defense against the physical, chemical and the biological environment. It is an ideal organ for studying molecular responses to biological infections through a variety of skin cells that specialize in immune responses. Comparative analysis of skin response to pathogenic, non-pathogenic, and commensal bacteria would help in the identification of disease specific pathways for drug targets. In this study, we investigated human breast reduction skin responses to Cutibacterium acnes (C. acnes), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and TLR1/2 agonist using Affymetrix microarray chips. The Pam3CSK4 solution and bacterial cultures were prepared and inoculated in steel rings, that were placed on the acetone treated epidermis in a petri dish. After 24 h incubation, 8 mm punch biopsies were taken from the center of the ring, and RNA was extracted. The genome-wide expression was then analyzed using Affymetrix HG-133A gene chip microarray. We found that the C. acnes and S. aureus boosted the production of extracellular matrix components and attenuated the expression of differentiation markers. The above responses were mediated through the TLR2 pathway. Skin also responded to S. aureus and C. acnes by inducing the genes of the cell cycle machinery; this response was not TLR2-dependent. S. aureus induced, whereas C. acnes suppressed the genes associated with apoptosis; this was also not TLR2-dependent. Moreover, S. epidermis apparently did not lead to changes in gene expression. We conclude that the breast reduction skin is a very useful model to study the global gene expression in response to bacterial treatments.

Entangled gene regulatory networks with cooperative expression endow robust adaptive responses to unforeseen environmental changes

Living organisms must respond to environmental changes. Generally, accurate and rapid responses are provided by simple, unidirectional networks that connect inputs with outputs. Besides accuracy and speed, biological responses should also be robust to environmental or intracellular noise and mutations. Furthermore, cells must also respond to unforeseen environmental changes that have not previously been experienced, to avoid extinction prior to the evolutionary rewiring of their networks, which takes numerous generations. We have investigated gene regulatory networks that mutually activate or inhibit, and have demonstrated that complex entangled networks can make appropriate input-output relationships that satisfy the robust and adaptive responses required for unforeseen challenges. Such entangled networks function for sloppy and unreliable responses with low Hill coefficient reactions for the expression of each gene. To compensate for such sloppiness, several detours in the regulatory network exist. By taking advantage of the averaging over such detours, the network shows a higher robustness to environmental and intracellular noise as well as to mutations in the network, when compared to simple unidirectional circuits. Furthermore, the appropriate response to unforeseen challenges, allowing for functional outputs, is achieved as many genes exhibit similar dynamic expression responses, irrespective of inputs, as confirmed by applying dynamic time warping and dynamic mode decomposition. As complex entangled networks are common in gene regulatory networks and global gene expression responses are observed in microbial experiments, the present results provide a novel design principle for cellular networks.

Acetylation of the CspA family protein CspC controls the type III secretion system through translational regulation of exsA in Pseudomonas aeruginosa.

The ability to fine tune global gene expression in response to host environment is critical for the virulence of pathogenic bacteria. The host temperature is exploited by the bacteria as a cue for triggering virulence gene expression. However, little is known about the mechanism employed by Pseudomonas aeruginosa to response to host body temperature. CspA family proteins are RNA chaperones that modulate gene expression. Here we explored the functions of P. aeruginosa CspA family proteins and found that CspC (PA0456) controls the bacterial virulence. Combining transcriptomic analyses, RNA-immunoprecipitation and high-throughput sequencing (RIP-Seq), we demonstrated that CspC represses the type III secretion system (T3SS) by binding to the 5′ untranslated region of the mRNA of exsA, which encodes the T3SS master regulatory protein. We further demonstrated that acetylation at K41 of the CspC reduces its affinity to nucleic acids. Shifting the culture temperature from 25°C to 37°C or infection of mouse lung increased the CspC acetylation, which derepressed the expression of the T3SS genes, resulting in elevated virulence. Overall, our results identified the regulatory targets of CspC and revealed a regulatory mechanism of the T3SS in response to temperature shift and host in vivo environment.

Firstde novotranscriptome analysis of the Antarctic springtailCryptopygus terranovus(Collembola:Isotomidae) following mid-term heat exposure

AbstractGlobal human activities, such as greenhouse emissions and pollution, are promoting global warming, environmental changes and biodiversity reduction. Pristine environments such as those of Antarctica are not immune to these phenomena, as is noticeable from the increasing pace of the temperature shift registered within the continent in recent decades. In this study, we describe the firstde novotranscriptome analysis of the endemic Antarctic springtail (= collembolan)Cryptopygus terranovusand we evaluate its global gene expression response following a mid-term exposure of 20 days to 18°C. Expression data are compared with wild specimens sampled from their native environment to outline the molecular mechanisms triggered by the thermal exposure. Although individual plasticity in transcript modulation is assessed, several pathways appear to be differentially modulated in springtails subjected to the heat treatmentvswild specimens. Through enrichment analysis, we show that protein catabolism, fatty acid metabolism and a sexual response characterized by spermatid development are induced, while carbohydrate consumption, lipid catabolism and tissue development are downregulated in treated samples compared to controls.

Transcriptomic Profiling of the Adaptive and Innate Immune Responses of Atlantic Salmon to Renibacterium salmoninarum Infection.

Bacterial Kidney Disease (BKD), which is caused by a Gram-positive, intracellular bacterial pathogen (Renibacterium salmoninarum), affects salmonids including Atlantic salmon (Salmo salar). However, the transcriptome response of Atlantic salmon to BKD remained unknown before the current study. We used a 44K salmonid microarray platform to characterise the global gene expression response of Atlantic salmon to BKD. Fish (~54 g) were injected with a dose of R. salmoninarum (H-2 strain, 2 × 108 CFU per fish) or sterile medium (control), and then head kidney samples were collected at 13 days post-infection/injection (dpi). Firstly, infection levels of individuals were determined through quantifying the R. salmoninarum level by RNA-based TaqMan qPCR assays. Thereafter, based on the qPCR results for infection level, fish (n = 5) that showed no (control), higher (H-BKD), or lower (L-BKD) infection level at 13 dpi were subjected to microarray analyses. We identified 6,766 and 7,729 differentially expressed probes in the H-BKD and L-BKD groups, respectively. There were 357 probes responsive to the infection level (H-BKD vs. L-BKD). Several adaptive and innate immune processes were dysregulated in R. salmoninarum-infected Atlantic salmon. Adaptive immune pathways associated with lymphocyte differentiation and activation (e.g., lymphocyte chemotaxis, T-cell activation, and immunoglobulin secretion), as well as antigen-presenting cell functions, were shown to be differentially regulated in response to BKD. The infection level-responsive transcripts were related to several mechanisms such as the JAK-STAT signalling pathway, B-cell differentiation and interleukin-1 responses. Sixty-five microarray-identified transcripts were subjected to qPCR validation, and they showed the same fold-change direction as microarray results. The qPCR-validated transcripts studied herein play putative roles in various immune processes including pathogen recognition (e.g., tlr5), antibacterial activity (e.g., hamp and camp), regulation of immune responses (e.g., tnfrsf11b and socs1), T-/B-cell differentiation (e.g., ccl4, irf1 and ccr5), T-cell functions (e.g., rnf144a, il13ra1b and tnfrsf6b), and antigen-presenting cell functions (e.g., fcgr1). The present study revealed diverse immune mechanisms dysregulated by R. salmoninarum in Atlantic salmon, and enhanced the current understanding of Atlantic salmon response to BKD. The identified biomarker genes can be used for future studies on improving the resistance of Atlantic salmon to BKD.

Evidence for the Involvement of Vernalization-related Genes in the Regulation of Cold-induced Ripening in \u2018D\u2019Anjou\u2019 and \u2018Bartlett\u2019 Pear Fruit

European pear (Pyrus communis L.) cultivars require a genetically pre-determined duration of cold-temperature exposure to induce autocatalytic system 2 ethylene biosynthesis and subsequent fruit ripening. The physiological responses of pear to cold-temperature-induced ripening have been well characterized, but the molecular mechanisms underlying this phenomenon continue to be elucidated. This study employed previously established cold temperature conditioning treatments for ripening of two pear cultivars, ‘D’Anjou’ and ‘Bartlett’. Using a time-course transcriptomics approach, global gene expression responses of each cultivar were assessed at four stages of developmental during the cold conditioning process. Differential expression, functional annotation, and gene ontology enrichment analyses were performed. Interestingly, evidence for the involvement of cold-induced, vernalization-related genes and repressors of endodormancy release was found. These genes have not previously been described to play a role in fruit during the ripening transition. The resulting data provide insight into cultivar-specific mechanisms of cold-induced transcriptional regulation of ripening in European pear, as well as a unique comparative analysis of the two cultivars with very different cold conditioning requirements.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

SUN-LB53 Role of the Glucocorticoid Receptor Phosphorylation in Neural Development

Synthetic glucocorticoid (sGC) administration in pregnancy has greatly reduced the risk of respiratory distress, intraventricular hemorrhage and necrotizing enterocolitis in premature infants. Significant evidence has accumulated in human and animal models that prenatal exposure to sGCs can lead to adverse side effects such as reduced birthweight, increased risk for hypertension, cardiovascular, metabolic, and neurological problems later in life. Phosphorylation of the glucocorticoid receptor (GR) has been shown to play a significant role in a cells response to sGC administration, altering target gene activation versus repression, the magnitude and duration of the response. The GR receptor is phosphorylated on three sites (S203, S211, S226) in the N-terminal. An increased in the ratio of phosphorylation on S211 to S226 is associated with enhanced transcriptional activation. Furthermore, changes in S221/S226 ratio are associated with distinct neurological disorders in humans. We have previously shown that in-utero exposure to a single dose of dexamethasone (Dex) reduces proliferation in cerebral cortical and hypothalamic neural stem cells (NSCs), alters neuronal differentiation, neuronal morphology and adult behavior. To investigate the role of receptor phosphorylation on NSCs biology and brain development, mice with a serine (S211) to alanine (S211A) knockin were generated. NSCs were isolated from the mouse E14.5 cerebral cortex and the transcriptional and biological response of cells were examined in response to sGC or vehicle stimulation. Affymetrix complete genome arrays were used to identify changes in global gene expression in response to 4 hours of 10-7 M Dex exposure. Basally, 2651 genes were >1.5 fold (p < 0.05) differentially regulated in S211A versus wildtype, with 929 distinct upregulated and 1722 downregulated. Sex specific differences were observed basally, with 382 upregulated and 824 down regulated in females compared to 1191 upregulated and 1353 downregulated in males. Ingenuity pathway analysis (IPA) revealed that the only significant pathways that were altered basally in S211A versus wildtype were valine and isoleucine degradation, fatty acid beta oxidation and glutathione redox reaction I, all with negative Z scores (Z scores -2.1 to -3.16, P < 1.3E-01 to 1.3E-06). In response to a 4-hour Dex stimulation, 473 and 657 genes were upregulated and 782 and 996 genes were downregulated in females versus male respectively in S211A compared to wildtype. IPA analysis revealed that only one significant pathway with a Z score >2 that was altered in S211A versus wildtype in response to dex was of activation LPS/IL1 mediated inhibition of RXR function (Z = 2.82, p <3.08E-03). Some of the most significant genes changed basally in S211A versus wildtype include genes involved in the cell cycle. To determine if these transcriptional changes led to a distinct biological response, proliferation and differentiation studies were performed. Basally, S211A cells exhibit enhanced proliferation compared to wildtype cells in vitro. These findings were validated by in-vivo findings demonstrated by increased expression of TBR2, an immediate progenitor cell marker in the cerebral cortex at E17.5. These studies identify distinct pathways and developmental neurological processes that are sensitive to phosphorylation of GR on S211 basally and in response to sGC exposure.

Elizabethkingia anophelis: Physiologic and Transcriptomic Responses to Iron Stress.

In this study, we investigated the global gene expression responses of Elizabethkingia anophelis to iron fluxes in the midgut of female Anopheles stephensi mosquitoes fed sucrose or blood, and in iron-poor or iron-rich culture conditions. Of 3,686 transcripts revealed by RNAseq technology, 218 were upregulated while 112 were down-regulated under iron-poor conditions. Hemolysin gene expression was significantly repressed when cells were grown under iron-rich or high temperature (37°C) conditions. Furthermore, hemolysin gene expression was down-regulated after a blood meal, indicating that E. anophelis cells responded to excess iron and its associated physiological stress by limiting iron loading. By contrast, genes encoding respiratory chain proteins were up-regulated under iron-rich conditions, allowing these iron-containing proteins to chelate intracellular free iron. In vivo studies showed that growth of E. anophelis cells increased 3-fold in blood-fed mosquitoes over those in sucrose-fed ones. Deletion of siderophore synthesis genes led to impaired cell growth in both iron-rich and iron-poor media. Mutants showed more susceptibility to H2O2 toxicity and less biofilm formation than did wild-type cells. Mosquitoes with E. anophelis experimentally colonized in their guts produced more eggs than did those treated with erythromycin or left unmanipulated, as controls. Results reveal that E. anophelis bacteria respond to varying iron concentration in the mosquito gut, harvest iron while fending off iron-associated stress, contribute to lysis of red blood cells, and positively influence mosquito host fecundity.

Fungal elicitor-induced transcriptional changes of genes related to branched-chain amino acid metabolism in Streptomyces natalensis HW-2.

Natamycin is a polyene macrolide antibiotic and widely used as a natural food preservative. Fungal elicitor had positive effects on the natamycin biosynthesis in Streptomyces natalensis HW-2. However, the global gene expression in response to fungal elicitor is not still reported. In the study, RNA-Seq was used to check the change of transcriptome by fungal elicitor in S. natalensis HW-2. The results showed that there were 1265 differential expression genes (DEGs) at 40h and 2196 DEGs at 80h. Most of the genes involved in natamycin biosynthesis were upregulated. KEGG pathway analysis showed that fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid (BCAA) metabolism. There were 23 upregulated or downregulated DEGs involved in BCAA biosynthesis and degradation at 40h and 80h. To confirm whether the improvement of BCAA biosynthesis could produce more natamycin, metabolic engineering was used to homologously overexpress the gene ilvH which encoded the regulatory subunit of acetolactate synthase (ALS) in S. natalensis. The results showed that overexpression of ilvH in S. natalensis HW-2 increased natamycin production to 1.25g/L in the flask, which was a 32% increase compared with that of the parent strain. Real-time quantitative PCR analysis showed that the transcriptional level of ilvH in mutant strain S. natalensis ZS101 was significantly increased. Acetyl-CoA content was also raised. The results suggested that the fungal elicitor enhanced natamycin biosynthesis by improving precursor supply via BCAA metabolism. This study will open a new avenue for enhancing natamycin production by metabolic engineering and adding fungal elicitor. KEY POINTS: • The fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid metabolism by RNA-Seq. • The homologous overexpression of gene ilvH increased natamycin production by 32% and acetyl-CoA content was raised in mutant strain S. natalensis ZS101.

Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis.

Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

Acute sprint exercise transcriptome in human skeletal muscle.

AimTo examine global gene expression response to profound metabolic and hormonal stress induced by acute sprint exercise.MethodsHealthy men and women (n = 14) performed three all-out cycle sprints interspersed by 20 min recovery. Muscle biopsies were obtained before the first, and 2h and 20 min after last sprint. Microarray analysis was performed to analyse acute gene expression response and repeated blood samples were obtained.ResultsIn skeletal muscle, a set of immediate early genes, FOS, NR4A3, MAFF, EGR1, JUNB were markedly upregulated after sprint exercise. Gene ontology analysis from 879 differentially expressed genes revealed predicted activation of various upstream regulators and downstream biofunctions. Gene signatures predicted an enhanced turnover of skeletal muscle mass after sprint exercise and some novel induced genes such as WNT9A, FZD7 and KLHL40 were presented. A substantial increase in circulating free fatty acids (FFA) was noted after sprint exercise, in parallel with upregulation of PGC-1A and the downstream gene PERM1 and gene signatures predicting enhanced lipid turnover. Increase in growth hormone and insulin in blood were related to changes in gene expressions and both hormones were predicted as upstream regulators.ConclusionThis is the first study reporting global gene expression in skeletal muscle in response to acute sprint exercise and several novel findings are presented. First, in line with that muscle hypertrophy is not a typical finding after a period of sprint training, both hypertrophy and atrophy factors were regulated. Second, systemic FFA and hormonal and exposure might be involved in the sprint exercise-induced changes in gene expression.

Identification of zinc and Zur-regulated genes in Corynebacterium diphtheriae.

Corynebacterium diphtheriae is a Gram-positive bacterial pathogen and the causative agent of diphtheria, a severe disease of the upper respiratory tract of humans. Factors required for C. diphtheriae to survive in the human host are not well defined, but likely include the acquisition of essential metals such as zinc. In C. diphtheriae, zinc-responsive global gene regulation is controlled by the Zinc Uptake Regulator (Zur), a member of the Fur-family of transcriptional regulators. In this study, we use transcriptomics to identify zinc-regulated genes in C. diphtheriae by comparing gene expression of a wild-type strain grown without and with zinc supplementation. Zur-regulated genes were identified by comparing wild-type gene expression with that of an isogenic zur mutant. We observed zinc repression of several putative surface proteins, the heme efflux system hrtBA, various ABC transporters, and the non-ribosomal peptide synthetase/polyketide synthase cluster sidAB. Furthermore, increased gene expression in response to zinc was observed for the alcohol dehydrogenase, adhA. Zinc and Zur regulation were confirmed for several genes by complementing the zur deletion and subsequent RT-qPCR analysis. We used MEME to predict Zur binding sites within the promoter regions of zinc- and Zur-regulated genes, and verified Zur binding by electrophoretic mobility shift assays. Additionally, we characterized cztA (dip1101), which encodes a putative cobalt/zinc/cadmium efflux family protein. Deletion of cztA results in increased sensitivity to zinc, but not to cobalt or cadmium. This study advances our knowledge of changes to Zur-dependent global gene expression in response to zinc in C. diphtheriae. The identification of zinc-regulated ABC transporters herein will facilitate future studies to characterize zinc transport in C. diphtheriae.

Transcriptional network regulation of the brassinosteroid signaling pathway by the BES1-TPL-HDA19 co-repressor complex.

The brassinosteroid-related BES1 and BZR1 transcription factors dynamically modulate downstream gene networks via the TPL-HDA19 co-repressor complex in BR-signaling pathways in Arabidopsis thaliana. Brassinosteroids (BRs) are plant steroid hormones that are essential for diverse growth and developmental processes across the whole life cycle of plants. In Arabidopsis thaliana, the BR-related transcription factors BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINAZOLE-RESISTANT 1 (BZR1) regulate a range of global gene expression in response to BR and several external signaling cues; however, the molecular mechanisms by which they mediate the reprogramming of downstream transcription remain unclear. We here report that formation of a protein complex between BES1 and BZR1 and Histone Deacetylase 19 (HDA19) via the conserved ERF-associated amphiphilic repression (EAR) motif proved essential for regulation of BR-signaling-related gene expression. Defects in BR-related functions of BES1 and BZR1 proteins containing a mutated EAR motif were completely rescued by artificial fusion with EAR-repression domain (SRDX), TOPLESS (TPL), or HDA19 proteins. RNA-sequencing analysis of Arabidopsis plants over-expressing bes1-DmEAR or bes1-DmEAR-HDA19 revealed an essential role for HDA19 activity in regulation of BES1/BZR1-mediated BR signaling. In addition to BR-related gene expression, the BES1-HDA19 transcription factor complex was important for abiotic stress-related drought stress tolerance and organ boundary formation. These results suggested that integrating activation of BR-signaling pathways with the formation of the protein complex containing BES1/BZR1 and TPL-HDA19 via the EAR motif was important in fine-tuning BR-related gene networks in plants.

Photosynthesis and yield response to elevated CO2, C4 plant foxtail millet behaves similarly to C3 species

Foxtail millet (Setaria italica) is a nutrient-rich food source traditionally grown in arid and semi-arid areas, as it is well adapted to drought climate. Yet there is limited information as how the crop responses to the changing climate. In order to investigate the response of foxtail millet to elevated [CO2] and the underlying mechanism, the crop was grown at ambient [CO2] (400 μmol mol−1) and elevated [CO2] (600 μmol mol−1) in an open-top chamber (OTC) experimental facility in North China. The changes in leaf photosynthesis, chlorophyll fluorescence, biomass, yield and global gene expression in response to elevated [CO2] were determined. Despite foxtail millet being a C4 photosynthetic crop, photosynthetic rates (PN) and intrinsic water-use efficiency (WUEi), were increased under elevated [CO2]. Similarly, grain yield and above-ground biomass also significantly increased (P < 0.05) for the two years of experimentation under elevated [CO2]. Increases in seeds and tiller number, spike and stem weight were the main contributors to the increased grain yield and biomass. Using transcriptomic analyses, this study further identified some genes which play a role in cell wall reinforcement, shoot initiation, stomatal conductance, carbon fixation, glycolysis / gluconeogenesis responsive to elevated [CO2]. Changes in these genes reduced plant height, increased stem diameters, and promote CO2 fixation. Higher photosynthetic rates at elevated [CO2] demonstrated that foxtail millet was not photosynthetically saturated at elevated [CO2] and its photosynthesis response to elevated [CO2] were analogous to C3 plants.

Inhibition of GGPPS1 attenuated LPS-induced acute lung injury and was associated with NLRP3 inflammasome suppression.

Inhibition of the mevalonate pathway using statins has been shown to be beneficial in the treatment of acute lung injury (ALI). Here, we investigated whether partial inhibition of this pathway by targeting geranylgeranyl pyrophosphate synthase large subunit 1 (GGPPS1), a catalase downstream of the mevalonate pathway, was effective at treating lung inflammation in ALI. Lipopolysaccharide (LPS) was intratracheally instilled to induce ALI in lung-specific GGPPS1-knockout and wild-type mice. Expression of GGPPS1 in lung tissues and alveolar epithelial cells was examined. The severity of lung injury and inflammation was determined in lung-specific GGPPS1 knockout and wild-type mice by measuring alveolar exudate, neutrophil infiltration, lung injury, and cell death. Change in global gene expression in response to GGPPS1 depletion was measured using mRNA microarray and verified in vivo and in vitro. We found that GGPPS1 levels increased significantly in lung tissues and alveolar epithelial cells in LPS-induced ALI mice. Compared with wild-type and simvastatin treated mice, the specific deletion of pulmonary GGPPS1 attenuated the severity of lung injury by inhibiting apoptosis of AECs. Furthermore, deletion of GGPPS1 inhibited LPS-induced inflammasome activation, in terms of IL-1β release and pyroptosis, by downregulating NLRP3 expression. Finally, downregulation of GGPPS1 reduced the membrane expression of Ras-related protein Rab10 and Toll-like receptor 4 (TLR4) and inhibited the phosphonation of IκB. This effect might be attributed to the downregulation of GGPP levels. Our results suggested that inhibition of pulmonary GGPPS1 attenuated LPS-induced ALI predominantly by suppressing the NLRP3 inflammasome through Rab10-mediated TLR4 replenishment.