Genome-wide Gene Expression Differences Research Articles

Personalized identification and characterization of genome-wide gene expression differences between patient-matched intracranial and extracranial melanoma metastasis pairs

Melanoma is the most serious type of skin cancer that frequently spreads to other organs of the human body. Especially melanoma metastases to the brain (intracranial metastases) are hard to treat and a major cause of death of melanoma patients. Little is known about molecular alterations and altered mechanisms that distinguish intra- from extracranial melanoma metastases. So far, almost all existing studies compared intracranial metastases from one set of patients to extracranial metastases of an another set of melanoma patients. This neglects the important facts that each melanoma is highly individual and that intra- and extracranial melanoma metastases from the same patient are more similar to each other than to melanoma metastases from other patients in the same organ. To overcome this, we compared the gene expression profiles of 16 intracranial metastases to their corresponding 21 patient-matched extracranial metastases in a personalized way using a three-state Hidden Markov Model (HMM) to identify altered genes for each individual metastasis pair. This enabled three major findings by considering the predicted gene expression alterations across all patients: (i) most frequently altered pathways include cytokine-receptor interaction, calcium signaling, ECM-receptor interaction, cAMP signaling, Jak-STAT and PI3K/Akt signaling, (ii) immune-relevant signaling pathway genes were downregulated in intracranial metastases, and (iii) intracranial metastases were associated with a brain-like phenotype gene expression program. Further, the integration of all differentially expressed genes across the patient-matched melanoma metastasis pairs led to a set of 103 genes that were consistently down- or up-regulated in at least 11 of the 16 of the patients. This set of genes contained many genes involved in the regulation of immune responses, cell growth, cellular signaling and transport processes. An analysis of these genes in the TCGA melanoma cohort showed that the expression behavior of 11 genes was significantly associated with survival. Moreover, a comparison of the 103 genes to three closely related melanoma metastasis studies revealed a core set of eight genes that were consistently down- or upregulated in intra- compared to extracranial metastases in at least two of the three related studies (down: CILP, DPT, FGF7, LAMP3, MEOX2, TMEM119; up: GLDN, PMP2) including FGF7 that was also significantly associated with survival. Our findings contribute to a better characterization of genes and pathways that distinguish intra- from extracranial melanoma metastasis and provide important hints for future experimental studies to identify potential targets for new therapeutic approaches.

The ability to induce heat shock transcription factor-regulated genes in response to lethal heat stress is associated with thermotolerance in tomato cultivars.

Heat stress is a severe challenge for plant production, and the use of thermotolerant cultivars is critical to ensure stable production in high-temperature-prone environments. However, the selection of thermotolerant cultivars is difficult due to the complex nature of heat stress and the time and space needed for evaluation. In this study, we characterized genome-wide differences in gene expression between thermotolerant and thermosensitive tomato cultivars and examined the possibility of selecting gene expression markers to estimate thermotolerance among different tomato cultivars. We selected one thermotolerant and one thermosensitive cultivar based on physiological evaluations and compared heat-responsive gene expression in these cultivars under stepwise heat stress and acute heat shock conditions. Transcriptomic analyses reveled that two heat-inducible gene expression pathways, controlled by the heat shock element (HSE) and the evening element (EE), respectively, presented different responses depending on heat stress conditions. HSE-regulated gene expression was induced under both conditions, while EE-regulated gene expression was only induced under gradual heat stress conditions in both cultivars. Furthermore, HSE-regulated genes showed higher expression in the thermotolerant cultivar than the sensitive cultivar under acute heat shock conditions. Then, candidate expression biomarker genes were selected based on the transcriptome data, and the usefulness of these candidate genes was validated in five cultivars. This study shows that the thermotolerance of tomato is correlated with its ability to maintain the heat shock response (HSR) under acute severe heat shock conditions. Furthermore, it raises the possibility that the robustness of the HSR under severe heat stress can be used as an indicator to evaluate the thermotolerance of crop cultivars.

A Transcriptome Sequencing Study on Genome-Wide Gene Expression Differences of Lung Cancer Cells Modulated by Fucoidan.

Fucoidan has received increasing attention in anti-(lung) tumors. However, the effect of fucoidan on the gene changes of lung cancer cells (LCCs) has not been examined systematically. Herein, we investigate the effect of fucoidan on the phenotypes of LCCs and their gene expression by transcriptome sequencing analysis. The phenotypes of LCCs are significantly inhibited by fucoidan. Importantly, compared to LCCs, 1 mg/ml fucoidan has no effect on the phenotypes of normal cells. Further, 6,930 differentially expressed genes (DEGs) in the transcriptome of LCCs (3,501 up-regulated and 3,429 down-regulated genes) are detected via RNA-sequencing between the fucoidan and control groups. Gene Ontology analysis confirms that DEGs are reflected in DNA replication, cell-substrate junction, regulation of cell cycle phase transition, apoptosis, focal adhesion, cadherin binding, and cell adhesion molecule binding. Thus, our findings on the transcriptomic level highlight the therapeutic potential of fucoidan for lung cancer treatment.

Core-clock genes Period 1 and 2 regulate visual cascade and cell cycle components during mouse eye development

The retinas from Period 1 (Per1) and Period 2 (Per2) double-mutant mice (Per1−/−Per2Brdm1) display abnormal blue-cone distribution associated with a reduction in cone opsin mRNA and protein levels, up to 1 year of age. To reveal the molecular mechanisms by which Per1 and Per2 control retina development, we analyzed genome-wide gene expression differences between wild-type (WT) and Per1−/−Per2Brdm1 mice across ocular developmental stages (E15, E18 and P3). All clock genes displayed changes in transcript levels along with normal eye development. RNA-Seq data show major gene expression changes between WT and mutant eyes, with the number of differentially expressed genes (DEG) increasing with developmental age. Functional annotation of the genes showed that the most significant changes in expression levels in mutant mice involve molecular pathways relating to circadian rhythm signaling at E15 and E18. At P3, the visual cascade and the cell cycle were respectively higher and lower expressed compared to WT eyes. Overall, our study provides new insights into signaling pathways -phototransduction and cell cycle- controlled by the circadian clock in the eye during development.

A Transcriptome Sequencing Study on Genome-Wide Gene Expression Differences of 3D Cultured Chondrocytes in Hydrogel Scaffolds with Different Gel Density.

Hydrogel is considered as a promising cell delivery vehicle in cartilage tissue engineering, whose tunable microenvironments may influence the function and fate of encapsulated chondrocytes. Here, the transcriptomes of chondrocytes that are encapsulated and cultured in hydrogel constructs respectively made of 0.8% and 4% alginate solution are investigated. Differences in chondrocyte transcriptome are detected via RNA-sequencing from these two cultural conditions. The differentially expressed genes (DEGs) are reflected in extracellular matrix (ECM) secretion, cell cycle, proliferation, cartilage development, and so on. Significantly, the expression of DEGs associated with cartilage ECM and cell proliferation are upregulated in 0.8% constructs; whilst the expressions of DEGs involved in cell cycle and matrix degradation are upregulated in 4% constructs. Moreover, interestingly, the expressions of chondrocyte hypertrophy markers are upregulated in 0.8% constructs; while 4% constructs seemingly favor the long-term maintenance of chondrocyte phenotype. Taken together, this study confirms on transcriptomic level that gel density affects gene expression and phenotype of the encapsulated chondrocytes; therefore, it may provide guidance for future design and fabrication of cartilage tissue engineering scaffolds.

Physiological and transcriptome changes induced by Pseudomonas putida acquisition of an integrative and conjugative element

Integrative and conjugative elements (ICEs) comprise ubiquitous large mobile regions in prokaryotic chromosomes that transmit vertically to daughter cells and transfer horizontally to distantly related lineages. Their evolutionary success originates in maximized combined ICE-host fitness trade-offs, but how the ICE impacts on the host metabolism and physiology is poorly understood. Here we investigate global changes in the host genetic network and physiology of Pseudomonas putida with or without an integrated ICEclc, a model ICE widely distributed in proteobacterial genomes. Genome-wide gene expression differences were analyzed by RNA-seq using exponentially growing or stationary phase-restimulated cultures on 3-chlorobenzoate, an aromatic compound metabolizable thanks to specific ICEclc-located genes. We found that the presence of ICEclc imposes a variety of changes in global pathways such as cell cycle and amino acid metabolism, which were more numerous in stationary-restimulated than exponential phase cells. Unexpectedly, ICEclc stimulates cellular motility and leads to more rapid growth on 3-chlorobenzoate than cells carrying only the integrated clc genes. ICEclc also concomitantly activates the P. putida Pspu28-prophage, but this in itself did not provoke measurable fitness effects. ICEclc thus interferes in a number of cellular pathways, inducing both direct benefits as well as indirect costs in P. putida.

Microarray analysis in pulmonary hypertension.

Microarrays are a powerful and effective tool that allows the detection of genome-wide gene expression differences between controls and disease conditions. They have been broadly applied to investigate the pathobiology of diverse forms of pulmonary hypertension, namely group 1, including patients with idiopathic pulmonary arterial hypertension, and group 3, including pulmonary hypertension associated with chronic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. To date, numerous human microarray studies have been conducted to analyse global (lung homogenate samples), compartment-specific (laser capture microdissection), cell type-specific (isolated primary cells) and circulating cell (peripheral blood) expression profiles. Combined, they provide important information on development, progression and the end-stage disease. In the future, system biology approaches, expression of noncoding RNAs that regulate coding RNAs, and direct comparison between animal models and human disease might be of importance.

Tu1690 Genome-Wide Gene Expression Differences in Active and Quiescent Stages of Ulcerative Colitis From Endoscopic Pinch Biopsies: Insights Into Distinctive Pathogenesis

Tu1690 Genome-Wide Gene Expression Differences in Active and Quiescent Stages of Ulcerative Colitis From Endoscopic Pinch Biopsies: Insights Into Distinctive Pathogenesis

Expression2Kinases: mRNA profiling linked to multiple upstream regulatory layers

Genome-wide mRNA profiling provides a snapshot of the global state of cells under different conditions. However, mRNA levels do not provide direct understanding of upstream regulatory mechanisms. Here, we present a new approach called Expression2Kinases (X2K) to identify upstream regulators likely responsible for observed patterns in genome-wide gene expression. By integrating chromatin immuno-precipitation (ChIP)-seq/chip and position weight matrices (PWMs) data, protein-protein interactions and kinase-substrate phosphorylation reactions, we can better identify regulatory mechanisms upstream of genome-wide differences in gene expression. We validated X2K by applying it to recover drug targets of food and drug administration (FDA)-approved drugs from drug perturbations followed by mRNA expression profiling; to map the regulatory landscape of 44 stem cells and their differentiating progeny; to profile upstream regulatory mechanisms of 327 breast cancer tumors; and to detect pathways from profiled hepatic stellate cells and hippocampal neurons. The X2K approach can advance our understanding of cell signaling and unravel drugs mechanisms of action. The software and source code are freely available at: http://www.maayanlab.net/X2K. avi.maayan@mssm.edu Supplementary data are available at Bioinformatics online.

Complex traits analysis of chicken growth using targeted genetical genomics

Dissecting the genetic control of complex trait variation remains very challenging, despite many advances in technology. The aim of this study was to use a major growth quantitative trait locus (QTL) in chickens mapped to chromosome 4 as a model for a targeted approach to dissect the QTL. We applied a variant of the genetical genomics approach to investigate genome-wide gene expression differences between two contrasting genotypes of a marked QTL. This targeted approach allows the direct quantification of the link between the genotypes and the genetic responses, thus narrowing the QTL-phenotype gap using fewer samples (i.e. microarrays) compared with the genome-wide genetical genomics studies. Four differentially expressed genes were localized under the region of the QTL. One of these genes is a potential positional candidate gene (AADAT) that affects lysine and tryptophan metabolism and has alternative splicing variants between the two genotypes. In addition, the lysine and glycolysis metabolism pathways were significantly enriched for differentially expressed genes across the genome. The targeted approach provided a complementary route to fine mapping of QTL by characterizing the local and the global downstream effects of the QTL and thus generating further hypotheses about the action of that QTL.

Dietary unsaturated fatty acids affect the mammary gland integrity and health in lactating dairy cows

BackgroundInformation about the effects of unsaturated fatty acids (UFA) supplementation on the health and integrity of the mammary gland in lactating dairy cows is lacking. Therefore, the aim of this study was to determine the effects of unprotected dietary UFA on the global expression pattern of genes in the mammary gland tissue of grazing dairy cows, and to translate this information into relevant biological knowledge.MethodsTwenty-eight Holstein-Friesian dairy cows were randomly assigned to 4 different concentrated UFA-sources for 23 days after which all cows were switched to a non-UFA-supplemented concentrate for an additional 28 days. On the last day of both periods, mammary gland biopsies were taken to study genome-wide differences in gene expression on Bovine Genome Arrays.ResultsSupplementation with UFA reduced the concentration of short chain fatty acids (FA), C16 FA and saturated FA in the milk, whereas that of trans-FA increased. One major finding was that canonical pathways associated with remodelling and immune functions of the mammary gland were predominantly down-regulated during UFA supplementation and negatively correlated with the concentration of milk trans-FA.ConclusionsSupplementing grazing dairy cows with unprotected dietary UFA can affect the remodelling and immune functions of the mammary gland with potential consequences for its integrity and health, as well as milk quality.

Alteration of gene expression in mammary gland tissue of dairy cows in response to dietary unsaturated fatty acids

The aim of this study was to determine the effects of supplementing unprotected dietary unsaturated fatty acids (UFAs) from different plant oils on gene expression in the mammary gland of grazing dairy cows. A total of 28 Holstein–Friesian dairy cows in mid-lactation were blocked according to parity, days in milk, milk yield and fat percentage. The cows were then randomly assigned to four UFA sources based on rapeseed, soybean, linseed or a mixture of the three oils for 23 days, after which, all 28 cows were switched to a control diet for an additional 28 days. On the last day of both periods, mammary gland biopsies were taken to study genome-wide differences in gene expression on Affymetrix GeneChip® Bovine Genome Arrays (no. 900493) by ServiceXS (Leiden, The Netherlands). Supplementation with UFAs resulted in increased milk yield but decreased milk fat and protein percentages. Furthermore, the proportion of de novo fatty acids (FAs) in the milk was reduced, whereas that of long-chain FAs increased. Applying a statistical cut-off of false discovery rate of q-values <0.05 together with an absolute fold change of 1.3, a total of 972 genes were found to be significantly affected through UFA supplementation, indicating that large transcriptional adaptations occurred in the mammary gland when grazing dairy cows were supplemented with unprotected dietary UFA. Gene sets related to cell development and remodeling, apoptosis, nutrient metabolic process, as well as immune system response were predominantly downregulated during UFA supplementation. Such molecular knowledge on the physiology of the mammary gland might provide the basis for further functional research on dairy cows.

Meta-Analysis of Genome-Wide Gene Expression Differences in Onset and Maintenance Phases of Genetic Hypertension

Gene expression differences accompany both the onset and established phases of hypertension. By an integrated genome-transcriptome approach we performed a meta-analysis of data from 74 microarray experiments available on public databases to identify genes with altered expression in the kidney, adrenal, heart, and artery of spontaneously hypertensive and Lyon hypertensive rats. To identify genes responsible for the onset of hypertension we used a statistical approach that sought to eliminate expression differences that occur during maturation unrelated to hypertension. Based on this adjusted fold-difference statistic, we found 36 genes for which the expression differed between the prehypertensive phase and established hypertension. Genes having possible relevance to hypertension onset included Actn2, Ankrd1, ApoE, Cd36, Csrp3, Me1, Myl3, Nppa, Nppb, Pln, Postn, Spp1, Slc21a4, Slc22a2, Thbs4, and Tnni3. In established hypertension 102 genes exhibited altered expression after Bonferroni correction (P<0.05). These included Atp5o, Ech1, Fabp3, Gnb3, Ldhb, Myh6, Lpl, Pkkaca, Vegfb, Vcam1, and reduced nicotinamide-adenine dinucleotide dehydrogenases. Among the genes identified, there was an overrepresentation of gene ontology terms involved in energy production, fatty acid and lipid metabolism, oxidation, and transport. These could contribute to increases in reactive oxygen species. Our meta-analysis has revealed many new genes for which the expression is altered in hypertension, so pointing to novel potential causative, maintenance, and responsive mechanisms and pathways.

Abstract 5142: Detection of genomic changes in glioblastoma stem cell subpopulations by array comparative genomic hybridisation

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive tumors characterised by bad prognosis and limited treatment response. Stem-cell like GBM subpopulations are believed to cause tumor initiation and treatment resistance. Aim of this study was to clarify whether the GBM stem-cell like subpopulations (GSCLS) present in GBM cell cultures are characterized by specific genomic alterations. Fourteen GBM primary cultures and 6 cell lines were analyzed for the presence of GSCLS compartments by investigating their ability to grow in serum-free neurobasal medium. Growth in neurospheres was compared to the expression of the stem cell markers CD133, nestin and SOX2. Nine out of 14 GBM primary cultures and 3 out of 7 GBM cell lines were able to grow as stable non-adherent neurospheres in NBE-medium for 3 up to 14 months. From those GBM samples, able to grow as neurospheres in NBE-medium, 33% expressed all 3 stem cell markers, 42% two markers and 25% one stem cell marker. The samples who grew as monolayers or died in NBE-medium, expressed only one (30%) or no stem cell marker (70%). Genomic changes in selected primary cultures (N=8) and a GBM cell line grown in parallel for 6 to 60 weeks in spheres and monolayer cultures were comparably analyzed by (array)CGH. In all cases analyzed, GSCLS grown as neurospheres contained widely the identical chromosomal gains/amplifications and losses as the respective parental cell cultures. Differences concerned random low-level gains/losses of large chromosomal regions or whole chromosome arms. However, on closer inspection by aCGH small but distinct amplifications/deletions could be found in the GSCLS grown in neurospheres mainly affecting single gene loci. Further changes at the SOX2 gene locus and SOX2 gene expression were analyzed in GBM cell lines and primary culture. An increase of SOX2 gene expression was observed in GSCLS grown as neurospheres compared to the respective monolayer cultures as determined by qRT PCR. Preliminary data show that the SOX2 expression is higher in GBM tumor samples compared to the respective cell lines indicating loss of stem cell features during in vitro cell culture. Consequently, the association between the expression of stem cell markers, like SOX2, and the survival time of GBM patients is currently established. In ongoing experiments genome-wide gene expression differences between GSCLS neurospheres and the respective monolayer cultures are established and compared with the aCGH data. In conclusion, we demonstrate that the majority of human GBM primary cell cultures/cell lines contain stem cell-like subpopulations. These sphere-forming cell clones harbor small distinct gene amplifications/deletions as compared to the respective monolayer cell cultures and the genes residing in the affected regions are currently characterized. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5142.

Newly identified tumor-associated role of human Sharpin

In order to discover previously unidentified cancer-associated genes, we analyzed genome-wide differences in gene expression between tumor biopsies and normal tissues. Among those differentially regulated genes, we identified Sharpin (Shank-associated RH domain-interacting protein) as a commonly up-regulated gene in multiple human cancer types. Although rat Sharpin is reported to interact with Shank1, a multidomain scaffold protein localized in postsynaptic densities, its exact roles are unknown. Whereas human Sharpin homologue was primarily localized in the cytosol of cultured cells, they were detected in both cytosol and nucleus of the cells from ovarian and liver cancer tissues using immunohistochemical staining. In addition, Chinese ovary hamster cells over-expressing Sharpin exhibited enhanced cancer-specific phenotypes in multiple in vitro tumor assays. Taken together, the results suggest that Sharpin is not an inert scaffold protein, but may play tumor-associated roles during cancer biogenesis.

To what extent is blood a reasonable surrogate for brain in gene expression studies: estimation from mouse hippocampus and spleen

Microarrays are designed to measure genome-wide differences in gene expression. In cases where a tissue is not accessible for analysis (e.g. human brain), it is of interest to determine whether a second, accessible tissue could be used as a surrogate for transcription profiling. Surrogacy has applications in the study of behavioural and neurodegenerative disorders. Comparison between hippocampus and spleen mRNA obtained from a mouse recombinant inbred panel indicates a high degree of correlation between the tissues for genes that display a high heritability of expression level. This correlation is not limited to apparent expression differences caused by sequence polymorphisms in the target sequences and includes both cis and trans genetic effects. A tissue such as blood could therefore give surrogate information on expression in brain for a subset of genes, in particular those co-expressed between the two tissues, which have heritably varying expression.

Genome‐wide expression analysis of recently processed formalin‐fixed paraffin embedded human prostate tissues

Formalin fixation and paraffin embedding (FFPE) is the standard practice to process surgical specimens in preparation for pathological evaluation. The FFPE procedure is known to introduce adverse effects on RNA quality and the ensuing RNA based expression analysis. However, the overall impact of the FFPE procedure alone on the reliability and accuracy of expression data, without influences from additional compromising factors introduced during long-term storage, has not been vigorously assessed. The quality of RNA extracted from recently processed FFPE prostate tissues was examined. FFPE and frozen blocks were prepared from matched surgical specimens and processed in parallel for RNA extraction, two rounds of linear RNA amplification, and genome-wide expression analysis. Expression ratios of prostate cancer versus benign prostatic hyperplasia (BPH) were compared between data derived from the paired FFPE tissues and frozen tissues. RNA extracted from recently processed FFPE prostate tissues was of high quality and suitable for RNA expression analysis. An unbiased analysis of expression data revealed nearly 80% concordance between FFPE tissues and frozen tissues, in genome-wide gene expression differences of cancer versus BPH, and across a wide spectrum of fold expression differences. FFPE procedure itself does not lead to adverse effects on genome-wide expression analysis. Prospective molecular archiving or modified long-term storage conditions should be implemented to expand the utility of FFPE tissues without compromising standard surgical pathology routines.

Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 ( CUG2)

We examined genome-wide differences in gene expression between tumor biopsies and normal tissues in order to identify differentially regulated genes in tumors. Cancer-upregulated gene 2 ( CUG2) was identified as an expressed sequence tag (EST) that exhibits significant differential expression in multiple human cancer types. CUG2 showed weak sequence homology with the down-regulator of transcription 1 ( DR1) gene, a human transcription repressor. We found that EGFP-CUG2 fusion proteins were predominantly localized in the nucleus, suggesting their putative role in gene regulation. In addition, CUG2-overexpressing mouse fibroblast cells exhibited distinct cancer-specific phenotypes in vitro and developed into tumors in nude mice. Taken together, these findings suggest that CUG2 is a novel tumor-associated gene that is commonly activated in various human cancers and exhibits high transforming activities; it possibly belongs to a transcription regulator family that is involved in tumor biogenesis.

Integrated Array-Comparative Genomic Hybridization and Expression Array Profiles Identify Clinically Relevant Molecular Subtypes of Glioblastoma

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.