Expression Of Many Transcripts Research Articles

RNA oxidation in chromatin modification and DNA-damage response following exposure to formaldehyde

Formaldehyde is an environmental and occupational chemical carcinogen implicated in the damage of proteins and nucleic acids. However, whether formaldehyde provokes modifications of RNAs such as 8-oxo-7,8-dihydroguanine (8-oxoG) and the role that these modifications play on conferring long-term adverse health effects remains unexplored. Here, we profile 8-oxoG modifications using RNA-immunoprecipitation and RNA sequencing (8-oxoG RIP-seq) to identify 343 RNA transcripts heavily enriched in oxidations in human bronchial epithelial BEAS-2B cell cultures exposed to 1 ppm formaldehyde for 2 h. RNA oxidation altered expression of many transcripts involved in chromatin modification and p53-mediated DNA-damage responses, two pathways that play key roles in sustaining genome integrity and typically deregulated in tumorigenesis. Given that these observations were identified in normal cells exhibiting minimal cell stress and death phenotypes (for example, lack of nuclear shrinkage, F-actin alterations or increased LDH activity); we hypothesize that oxidative modification of specific RNA transcripts following formaldehyde exposure denotes an early process occurring in carcinogenesis analogous to the oxidative events surfacing at early stages of neurodegenerative diseases. As such, we provide initial investigations of RNA oxidation as a potentially novel mechanism underlying formaldehyde-induced tumorigenesis.

De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis.

Saffron (Crocus sativus L.) is commonly known as world’s most expensive spice with rich source of apocarotenoids and possesses magnificent medicinal properties. To understand the molecular basis of apocarotenoid biosynthesis/accumulation, we performed transcriptome sequencing from five different tissues/organs of C. sativus using Illumina platform. After comprehensive optimization of de novo transcriptome assembly, a total of 105, 269 unique transcripts (average length of 1047 bp and N50 length of 1404 bp) were obtained from 206 million high-quality paired-end reads. Functional annotation led to the identification of many genes involved in various biological processes and molecular functions. In total, 54% of C. sativus transcripts could be functionally annotated using public databases. Transcriptome analysis of C. sativus revealed the presence of 16721 SSRs and 3819 transcription factor encoding transcripts. Differential expression analysis revealed preferential/specific expression of many transcripts involved in apocarotenoid biosynthesis in stigma. We have revealed the differential expression of transcripts encoding for transcription factors (MYB, MYB related, WRKY, C2C2-YABBY and bHLH) involved in secondary metabolism. Overall, these results will pave the way for understanding the molecular basis of apocarotenoid biosynthesis and other aspects of stigma development in C. sativus.

Lysosomal and phagocytic activity is increased in astrocytes during disease progression in the SOD1 (G93A) mouse model of amyotrophic lateral sclerosis.

Astrocytes are key players in the progression of amyotrophic lateral sclerosis (ALS). Previously, gene expression profiling of astrocytes from the pre-symptomatic stage of the SOD1G93A model of ALS has revealed reduced lactate metabolism and altered trophic support. Here, we have performed microarray analysis of symptomatic and late-stage disease astrocytes isolated by laser capture microdissection (LCM) from the lumbar spinal cord of the SOD1G93A mouse to complete the picture of astrocyte behavior throughout the disease course. Astrocytes at symptomatic and late-stage disease show a distinct up-regulation of transcripts defining a reactive phenotype, such as those involved in the lysosome and phagocytic pathways. Functional analysis of hexosaminidase B enzyme activity in the spinal cord and of astrocyte phagocytic ability has demonstrated a significant increase in lysosomal enzyme activity and phagocytic activity in SOD1G93A vs. littermate controls, validating the findings of the microarray study. In addition to the increased reactivity seen at both stages, astrocytes from late-stage disease showed decreased expression of many transcripts involved in cholesterol homeostasis. Staining for the master regulator of cholesterol synthesis, SREBP2, has revealed an increased localization to the cytoplasm of astrocytes and motor neurons in late-stage SOD1G93A spinal cord, indicating that down-regulation of transcripts may be due to an excess of cholesterol in the CNS during late-stage disease possibly due to phagocytosis of neuronal debris. Our data reveal that SOD1G93A astrocytes are characterized more by a loss of supportive function than a toxic phenotype during ALS disease progression and future studies should focus upon restorative therapies.

Analysis of Global Gene Expression in Brachypodium distachyon Reveals Extensive Network Plasticity in Response to Abiotic Stress

Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

Deciphering the role of micrornas in early stages of haematopoiesis

Early steps of development are crucial to the emergence and expansion of haematopoieitic stem cells (HSC) as these cells are responsible for the formation of all blood cells throughout life. This research project focuses on the identification of miRNAs that can act as modulators of HSC emergence and expansion during embryonic life. MiRNAs are small non-coding RNAs that modulate the expression of many transcripts simultaneously and their biological role can be cell-type specific. First, we generated a miRNA expression profile of tissues that are involved in HSC emergence and expansion (E11 AGM/Aorta-Gonad-Mesonephros, E11 Yolk Sac, E12 Placenta, E14 Fetal Liver), using Illumina deep sequencing technology.

The Oncogene eIF4E: Using Biochemical Insights to Target Cancer

The eukaryotic translation initiation factor eIF4E is overexpressed in many human malignancies where it is typically a harbinger of poor prognosis. eIF4E is positioned as a nexus in post-transcriptional gene expression. To carry out these functions, eIF4E needs to bind the m(7)G cap moiety on mRNAs. It plays critical roles in mRNA translation, mRNA export, and most likely in mRNA stability as well. Through these activities, eIF4E coordinately modulates the expression of many transcripts involved in proliferation and survival. eIF4E function is controlled by interactions with protein cofactors in concert with many signaling pathways, including Ras, Mnk, Erk, MAPK, PI3K, mTOR, and Akt. This review describes the eIF4E activity and provides several examples of cellular control mechanisms. Further, we describe some therapeutic strategies in preclinical and clinical development.

Analysis of Phakopsora pachyrhizi transcript abundance in critical pathways at four time-points during infection of a susceptible soybean cultivar using deep sequencing.

BackgroundPhakopsora pachyrhizi, the causal agent responsible for soybean rust, is among the top hundred most virulent plant pathogens and can cause soybean yield losses of up to 80% when appropriate conditions are met. We used mRNA-Seq by Illumina to analyze pathogen transcript abundance at 15 seconds (s), 7 hours (h), 48 h, and 10 days (d) after inoculation (ai) of susceptible soybean leaves with P. pachyrhizi to gain new insights into transcript abundance in soybean and the pathogen at specific time-points during the infection including the uredinial stage.ResultsOver three million five hundred thousand sequences were obtained for each time-point. Energy, nucleotide metabolism, and protein synthesis are major priorities for the fungus during infection and development as indicated by our transcript abundance studies. At all time-points, energy production is a necessity for P. pachyrhizi, as indicated by expression of many transcripts encoding enzymes involved in oxidative phosphorylation and carbohydrate metabolism (glycolysis, glyoxylate and dicarboxylate, pentose phosphate, pyruvate). However, at 15 sai, transcripts encoding enzymes involved in ATP production were highly abundant in order to provide enough energy for the spore to germinate, as observed by the expression of many transcripts encoding proteins involved in electron transport. At this early time-point, transcripts encoding proteins involved in RNA synthesis were also highly abundant, more so than transcripts encoding genes involved in DNA and protein synthesis. At 7 hai, shortly after germination during tube elongation and penetration, transcripts encoding enzymes involved in deoxyribonucleotide and DNA synthesis were highly abundant. At 48 hai, transcripts encoding enzymes involved in amino acid metabolism were highly abundant to provide for increased protein synthesis during haustoria maturation. During sporulation at 10 dai, the fungus still required carbohydrate metabolism, but there also was increased expression of transcripts encoding enzymes involved in fatty acid metabolism.ConclusionThis information provides insight into molecular events and their timing throughout the life cycle of the P. pachyrhizi, and it may be useful in the development of new methods of broadening resistance of soybean to soybean rust.

Molecular and physiological mechanisms associated with root exposure to mercury in barley

Mercury (Hg) is a toxic metal that affects plant growth. Here the effect of Hg exposure on plant growth and leaf gas-exchange together with gene expression in roots is reported for barley. Hg was mainly accumulated in roots and only very small amounts were found in the shoots. Chlorophyll fluorescence and net photosynthesis were not affected by Hg. Nevertheless exposure to Hg reduced shoot and root growth, the shoot to root ratio, stomatal conductance, carbon isotope discrimination and expression of an aquaporin transcript, whereas abscisic acid related transcripts were over-expressed. These results suggested some degree of limitation to water uptake causing a moderate water stress when plants are exposed to Hg. Microarray (MapMan) analysis revealed changes in the transcription of genes involved in nitrogen metabolism, which were accompanied by decreased nitrogen concentrations in the shoots, together with an increase in transcripts associated with secondary metabolism, stress, inhibition of DNA synthesis/chromatin structure and cell organization elements. Moreover, Hg induced the expression of many transcripts known to be involved in the uptake, accumulation, transport and general responses to other heavy metals. It is concluded that barley is able to accumulate high amounts of Hg in roots through several transcriptional, metabolic and physiological adjustments.

The IHF regulon of exponentially growing Pseudomonas putida cells

Integration host factor (IHF) sites are largely absent from intergenic regions of ORFs encoding central metabolic functions in Pseudomonas putida mt-2. To gain an insight into this unequal distribution of otherwise abundant IHF-binding sequences, the transcriptome of IHF-plus and IHF-minus cells growing exponentially on glucose as sole carbon source was examined. In parallel, the cognate metabolic fluxes of the wild-type P. putida strain and its ihfA derivative were determined by culturing cells to a steady-state physiological regime with (13)C-labelled glucose. While expression of many transcripts was altered by the lack of IHF, flux balance analysis revealed that the ihfA mutation did not influence central carbon metabolism. Identification of multiple IHF sites adjacent to genes responsive to the factor allowed a refinement of the consensus and the mapping of the preferred binding positions for activation or repression of associated promoters. That few (if any) of the genes affected by IHF involved core pathways suggested that the central carbon metabolism tolerates the loss of the factor. Instead, IHF controlled various cell surface-related functions and downregulated genes encoding ribosomal proteins, the alpha subunit of RNA polymerase and components of the ATP synthase. These results were confirmed with lacZ fusions to a suite of promoters detected in the transcriptome as affected by IHF. Taken together, the data suggest that IHF plays a role in the physiological shift that sets P. putida for entering stationary phase.

Genome‐wide identification of vitamin D receptor (VDR) binding sites in DNA from prostate epithelial cells (PEC) by chromatin immunoprecipitation‐sequencing (ChIP‐seq)

The 1,25 dihydroxyvitamin D (1,25D) activates the VDR to bind DNA and initiate gene transcription. While microarray studies show 1,25D regulates expression of many transcripts, few of these genes have been examined for VDR binding. Using ChIP‐seq, we mapped VDR binding sites in DNA from the human PEC line, RWPE1. Cells were treated with vehicle or 1,25D (10 nM, 3 h) and subjected to ChIP using either VDR antibody or IgG. Using IgG as the reference, 1236 and 1175 VDR binding peaks were found in 1,25D and vehicle‐treated cells, respectively (0.1 FDR, 200 bp window, Cisgenome). Genes with known VDREs were confirmed e.g. CYP24 and IGFBP3. Correlation between ChIP‐seq peaks and a previous microarray study found that only 39% were associated with 1,25D‐regulated transcripts, including novel peaks close to the transcription factors (TF) JunB, FOS and TBP. Also only 5% of VDR peaks were found within 5 kb of the transcription start site of a gene and the known DR3 VDR binding motif was found in just 15% of the peaks. However, several other DNA‐binding motifs were enriched under the peaks suggesting indirect DNA association of VDR through other TF. Our data suggest the relationship between VDR binding and transcription is more complex that previously reported.Grant Funding Source: Showalter Trust to JCF

Gene Expression Profiles of Mouse Striatum in Control and Maneb + Paraquat-induced Parkinson’s Disease Phenotype: Validation of Differentially Expressed Energy Metabolizing Transcripts

The present study was undertaken to investigate the gene expression patterns of the striatum of control and maneb + paraquat-induced Parkinson's disease (PD) phenotype in mouse to identify the differentially expressed transcripts. The animals were treated with and without maneb (30 mg/kg, i.p.) + paraquat (10 mg/kg, i.p.), twice a week, for 3, 6, and 9 weeks. The RNA was isolated from control and treated mouse striatum and reverse transcribed, and equal quantities of labeled cDNA were mixed and hybridized with mouse 15 k arrays. Comparative transcription patterns showed the time of exposure dependent alteration in the expression of several transcripts associated with various pathways. RT-PCR reconfirmed the differential expression of some energy metabolizing transcripts. The study provides maneb + paraquat-induced differential expression of many transcripts using high-density microarray approach. Few transcripts, which were previously not reported to be associated with neuronal degeneration, were also identified. The results obtained thus suggest that maneb + paraquat induce neurotoxicity in the striatum in a time of exposure dependent manner via multiple pathways and defective energy metabolism could play a critical role.

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Beyond Our Pages

IL-13 and Epidermal Growth Factor Receptor Have Critical but Distinct Roles in Epithelial Cell Mucin Production

Overproduction of mucus is a central feature of asthma. The cytokine, IL-13, epidermal growth factor receptor (EGFR), and transcription factor, FOXA2, have each been implicated in mucus production, but the mechanistic relationships between these molecules are not yet well understood. To address this, we established a primary normal human bronchial epithelial cell culture system with IL-13-induced mucus production and gene transcript expression changes similar to those seen in vivo in mice. IL-13 did not stimulate release of the EGFR ligand, transforming growth factor (TGF)-alpha. However, there was constitutive release of TGF-alpha from normal human bronchial epithelial cells, and inhibition of TGF-alpha or EGFR reduced both constitutive and IL-13-induced mucin production. Microarray analysis revealed that IL-13 and the EGFR pathway appear to have almost completely independent effects on transcript expression. IL-13 induced a relatively small set of transcripts, including several novel transcripts that might play a role in pathogenesis of allergic airway disease. In contrast, EGFR activity had extensive effects, including altered expression of many transcripts associated with cell metabolism, survival, transcription, and differentiation. One of the few common effects of IL-13 and EGFR signaling was decreased expression of FOXA2, which is known to prevent mucus production. We conclude that the IL-13 and EGFR pathways make critical but quite distinct contributions to gene regulation in airway epithelial cells, and that both pathways affect expression of the key transcription factor, FOXA2, a known regulator of mucus production.

Early transcriptional effects of aldosterone in a mouse inner medullary collecting duct cell line.

The mineralocorticoid aldosterone is a major regulator of Na+ and acid-base balance and control of blood pressure. Although the long-term effects of aldosterone have been extensively studied, the early aldosterone-responsive genes remain largely unknown. Using DNA array technology, we have characterized changes in gene expression after 1 h of exposure to aldosterone in a mouse inner medullary collecting duct cell line, mIMCD-3. Results from three independent microarray experiments revealed that the expression of many transcripts was affected by aldosterone treatment. Northern blot analysis confirmed the upregulation of four distinct transcripts identified by the microarray analysis, namely, the serum and glucose-regulated kinase sgk, connective tissue growth factor, period homolog, and preproendothelin. Immunoblot analysis for preproendothelin demonstrated increased protein expression. Following the levels of the four transcripts over time showed that each had a unique pattern of expression, suggesting that the cellular response to aldosterone is complex. The results presented here represent a novel list of early aldosterone-responsive transcripts and provide new avenues for elucidating the mechanism of acute aldosterone action in the kidney.