Retrotransposon expression profiling: Unveiling the hidden SINE transcriptome through Next-Generation Sequencing data analysis

Of the ~1.7 million SINE elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which SINE transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq datasets and unique SINE DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual SINE elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ~1300 Alu loci and ~1100 MIR loci corresponding to detectable transcripts, with ~120 and ~60 respectively Alu and MIR loci expressed in at least three cell lines. In vitro transcription of selected SINEs did not reflect their in vivo expression properties, and required the native 5’-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for SINEs nested within Pol II-transcribed genes raising the possibility of an underlying mechanism for Pol II gene regulation by SINE transcriptional units. Moreover the application of our bioinformatic pipeline to both RNA-seq data of cells subjected to an in vitro pro-oncogenic stimulus and of in vivo matched tumor and non-tumor samples allowed us to detect increased Alu RNA expression as well as the source loci of such deregulation. The ability to investigate SINE transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant SINE RNAs and the assessment of SINE expression alteration under pathological conditions.

Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro

The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO 4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use. © Springer-Verlag 2006.

Meta-analysis of the population and phenotypic expression of CYP2C9/2C19 polymorphism on drug metabolism in different ethnicities

The term "pharmacogenetics" has been defined as the scientific study of inherited factors that affect the human drug response. Many pharmacogenetie studies have been published since 1995 and have focussed on the principal enzyme family involved in drug metabolism, the cytochrome P450 family, particularly cytochrome P4502C9 and 2C19. In order to investigate the pharmacogenetic aspect of pharmacotherapy, the relevant studies describing the association of pharmacogenetic factor(s) in drug responses must be retrieved from existing literature using a systematic review approach. In addition, the estimation of variant allele prevalence for the gene under study between different ethnic populations is important for pharmacogenetic studies. In this thesis, the prevalence of CYP2C9/2C19 alleles between different ethnicities has been estimated through meta-analysis and the population genetic principle. The clinical outcome of CYP2C9/2C19 allelic variation on the pharmacotherapy of epilepsy has been investigated; although many new antiepileptic drugs have been launched into the market, carbamazepine, phenobarbital and phenytoin are still the major agents in the pharmacotherapy of epilepsy. Therefore, phenytoin was chosen as a model AED and the effect of CYP2C9/2C19 genetic polymorphism on phenytoin metabolism was further examined.An estimation of the allele prevalence was undertaken for three CYP2C9/2C19 alleles respectively using a meta-analysis of studies that fit the Hardy-Weinberg equilibrium. The prevalence of CYP2C9*1 is approximately 81%, 96%, 97% and 94% in Caucasian, Chinese, Japanese, African populations respectively; the pooled prevalence of CYP2C19*1 is about 86%, 57%, 58% and 85% in these ethnic populations respectively. However, the studies of association between CYP2C9/2C19 polymorphism and phenytoin metabolism failed to achieve any qualitative or quantitative conclusion. Therefore, mephenytoin metabolism was examined as a probe drug for association between CYP2C19 polymorphism and mephenytoin metabolic ratio. Similarly, analysis of association between CYP2C9 polymorphism and warfarin dose requirement was undertaken.It was confirmed that subjects carrying two mutated CYP2C19 alleles have higher S/R mephenytoin ratio due to deficient CYP2C19 enzyme activity. The studies of warfarin and CYP2C9 polymorphism did not provide a conclusive result due to poor comparability between studies.The genetic polymorphism of drug metabolism enzymes has been studied extensively, however other genetic factors, such as multiple drug resistance genes (MDR) and genes encoding ion channels, which may contribute to variability in function of drug transporters and targets, require more attention in future pharmacogenetic studies of antiepileptic drugs.

Exploring ant-based algorithms for gene expression data analysis

Objective: Recently, much research has been proposed using nature inspired algorithms to perform complex machine learning tasks. Ant colony optimization (ACO) is one such algorithm based on swarm intelligence and is derived from a model inspired by the collective foraging behavior of ants. Taking advantage of the ACO in traits such as self-organization and robustness, this paper investigates ant-based algorithms for gene expression data clustering and associative classification. Methods and material: An ant-based clustering (Ant-C) and an ant-based association rule mining (Ant-ARM) algorithms are proposed for gene expression data analysis. The proposed algorithms make use of the natural behavior of ants such as cooperation and adaptation to allow for a flexible robust search for a good candidate solution. Results: Ant-C has been tested on the three datasets selected from the Stanford Genomic Resource Database and achieved relatively high accuracy compared to other classical clustering methods. Ant-ARM has been tested on the acute lymphoblastic leukemia (ALL)/acute myeloid leukemia (AML) dataset and generated about 30 classification rules with high accuracy. Conclusions: Ant-C can generate optimal number of clusters without incorporating any other algorithms such as K-means or agglomerative hierarchical clustering. For associative classification, while a few of the well-known algorithms such as Apriori, FP-growth and Magnum Opus are unable to mine any association rules from the ALL/AML dataset within a reasonable period of time, Ant-ARM is able to extract associative classification rules.

Microarray analysis of expression of cell death-associated genes in spinal cord cells with cyclic tensile strain

Previous studies have described alterations in gene expression following spinal cord injury, but this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile strain on cultured spinal cord cells from E15 Sprague-Dawley rats. Microarray analysis of gene expression and categorization of identified genes were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. The application of cyclic tensile strain reduced the viability of cultured spinal cord cells significantly in a dose- and time-dependent manner. GO analysis identified candidate genes related to apoptosis (44) and to response to stimulus (17). KEGG analysis identified changes in the expression levels of 12 genes of the mitogen-activated protein kinase (MAPK) signaling pathway, which were confirmed to be upregulated and validated by RT-PCR analysis. Spinal cord cells undergo cell death in response to cyclic tensile strain, which were dose- and time-dependent, with upregulation of various genes, in particular of the MAPK pathway.

Preimplantation genetic diagnosis: Analysis of gene expression, nuclear DNA and cytoplasmic DNA

Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy, these results suggest that the degradation of these messages in aging oocytes may be involved with inappropriate chromosome separation during meiosis. ^ In order to investigate the cause of embryonic rescue observed following clinical cytoplasmic transfer procedures and with the objective of developing a diagnostic tool, mtDNA concentrations in polar bodies and subcellular components were evaluated. First, the typical concentration of mtDNA in human and mouse oocytes was determined by fluorescent rapid cycle PCR. Some disparity was noted between the copy numbers of individual cytoplasmic samples which may limit the use of the current methodology for the clinical assessment of the corresponding oocyte. ^

Analysis of the Expression Pattern of Wnt Receptor Frizzled 3

Wnt signaling plays a vital role in many developmental processes. Wnt signaling has been implicated in neural crest induction and cell differentiation among other functions. In mice Wnts comprise a family of nineteen glycoproteins that bind to Frizzled (Fzd) receptors and LRP5/6 co-receptors. This activates beta-catenin, which translocates into the nucleus and acts as a transcription factor, resulting in differential gene expression. Specifically, Fzd 3 enhances Wnt 1 signaling. Wnt 1 and Fzd 3 are involved in neural crest induction and in neural crest-derived melanocyte development. We analyzed the expression pattern ofFzd 3 and the LRP 5/6 by in situ hybridization inmouse embryos. Our data suggests a role for these genes in neural crest induction and in melanocyte differentiation in the murine system. Results show Fzd 3 expression in the anterior part of the neural tube and in the hindbrain, while LRP 5 is expressed in the anterior part of the neural tube, in the hindbrain, and in the eye. We conclude that Fzd 3 and LRP 5 are expressed in the neural crest. In addition, Fzd 3 might act as the receptor while LRP 5 might act as the co-receptor for Wntl signaling in the murine system.

Analysis of expression quantitative trait loci in D. melanogaster

Expression Quantitative Trait Loci (eQTL) analysis allows for the identification of genetic variation associated with variation in gene expression. It is often unclear however, which of the associated variants are causal, and by what mechanism. Integrating functional genomic data with eQTL data can provide insight into the impact of natural variation in the population, and the nature of the transcriptional machinery itself. In this thesis, I integrate functional genomic data with eQTL data derived from both 5’ CAGE and 3’ TagXseq expression assays, in developing embryos. I first use both datasets to analyse the transcription landscape in embryonic D., melanogaster, and then carry out an analysis of sequence motifs associated with transcription factor binding sites, promoters, and 3’ polyadenylation sites. Finally, I integrate functional genomic data, including these novel sequence motifs, to shed light on the mechanisms of gene expression variation in D.,melanogaster. I am able to demonstrate that some variants effecting gene regulation in Drosophila are found within haplotypes which buffer their effects.

Whole Genome Analysis of Gene Expression Reveals Coordinated Activation of Signaling and Metabolic Pathways during Pollen-Pistil Interactions in Arabidopsis

Plant reproduction depends on the concerted activation of many genes to ensure correct communication between pollen and pistil. Here, we queried the whole transcriptome of Arabidopsis (Arabidopsis thaliana) in order to identify genes with specific reproductive functions. We used the Affymetrix ATH1 whole genome array to profile wild-type unpollinated pistils and unfertilized ovules. By comparing the expression profile of pistils at 0.5, 3.5, and 8.0 h after pollination and applying a number of statistical and bioinformatics criteria, we found 1,373 genes differentially regulated during pollen-pistil interactions. Robust clustering analysis grouped these genes in 16 time-course clusters representing distinct patterns of regulation. Coregulation within each cluster suggests the presence of distinct genetic pathways, which might be under the control of specific transcriptional regulators. A total of 78% of the regulated genes were expressed initially in unpollinated pistil and/or ovules, 15% were initially detected in the pollen data sets as enriched or preferentially expressed, and 7% were induced upon pollination. Among those, we found a particular enrichment for unknown transcripts predicted to encode secreted proteins or representing signaling and cell wall-related proteins, which may function by remodeling the extracellular matrix or as extracellular signaling molecules. A strict regulatory control in various metabolic pathways suggests that fine-tuning of the biochemical and physiological cellular environment is crucial for reproductive success. Our study provides a unique and detailed temporal and spatial gene expression profile of in vivo pollen-pistil interactions, providing a framework to better understand the basis of the molecular mechanisms operating during the reproductive process in higher plants.

Recombinant expression of Aryl Hydrocarbon Receptor for quantitative ligand-binding analysis

Recombinant expression of the Aryl Hydrocarbon Receptor (AhR) yields small amounts of ligand- binding competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding competent AhR was sensitive to the protein concentration of Sf9 extract, and co-expression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20- fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding competent AhR, and sufficient AhR for quantitative analysis of ligand-binding.

POPLAR GENE EXPRESSION DATA ANALYSIS PIPELINES

Analyzing large-scale gene expression data is a labor-intensive and time-consuming process. To make data analysis easier, we developed a set of pipelines for rapid processing and analysis poplar gene expression data for knowledge discovery. Of all pipelines developed, differentially expressed genes (DEGs) pipeline is the one designed to identify biologically important genes that are differentially expressed in one of multiple time points for conditions. Pathway analysis pipeline was designed to identify the differentially expression metabolic pathways. Protein domain enrichment pipeline can identify the enriched protein domains present in the DEGs. Finally, Gene Ontology (GO) enrichment analysis pipeline was developed to identify the enriched GO terms in the DEGs. Our pipeline tools can analyze both microarray gene data and high-throughput gene data. These two types of data are obtained by two different technologies. A microarray technology is to measure gene expression levels via microarray chips, a collection of microscopic DNA spots attached to a solid (glass) surface, whereas high throughput sequencing, also called as the next-generation sequencing, is a new technology to measure gene expression levels by directly sequencing mRNAs, and obtaining each mRNA’s copy numbers in cells or tissues. We also developed a web portal (http://sys.bio.mtu.edu/) to make all pipelines available to public to facilitate users to analyze their gene expression data. In addition to the analyses mentioned above, it can also perform GO hierarchy analysis, i.e. construct GO trees using a list of GO terms as an input.