Systematic Analysis of Gene Expression Differences between Left and Right Atria in Different Mouse Strains and in Human Atrial Tissue

Background: Normal development of the atria requires left-right differentiation during embryonic development. Reduced expression of Pitx2c (paired-like homeodomain transcription factor 2, isoform c), a key regulator of left-right asymmetry, has recently been linked to atrial fibrillation. We therefore systematically studied the molecular composition of left and right atrial tissue in adult murine and human atria. Methods: We compared left and right atrial gene expression in healthy, adult mice of different strains and ages by employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in either atrium were validated by RT-qPCR and Western blot in further animals and in shock-frozen left and right atrial appendages of patients undergoing open heart surgery. Results: We identified 77 genes with preferential expression in one atrium that were common in all strains and age groups analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10, a member of the TGFb family, showed highest enrichment in right atrium. These differences were validated by RT-qPCR in murine and human tissue. Western blot showed a 2-fold left-right concentration gradient in PITX2 protein in adult human atria. Several of the genes and gene groups enriched in left atria have a known biological role for maintenance of healthy physiology, specifically the prevention of atrial pathologies involved in atrial fibrillation, including membrane electrophysiology, metabolic cellular function, and regulation of inflammatory processes. Comparison of the array datasets with published array analyses in heterozygous Pitx2c+/2 atria suggested that approximately half of the genes with left-sided enrichment are regulated by Pitx2c. Conclusions: Our study reveals systematic differences between left and right atrial gene expression and supports the hypothesis that Pitx2c has a functional role in maintaining ‘‘leftness’’ in the atrium in adult murine and human hearts

A statistical framework for testing functional categories in microarray data

Ready access to emerging databases of gene annotation and functional pathways has shifted assessments of differential expression in DNA microarray studies from single genes to groups of genes with shared biological function. This paper takes a critical look at existing methods for assessing the differential expression of a group of genes (functional category), and provides some suggestions for improved performance. We begin by presenting a general framework, in which the set of genes in a functional category is compared to the complementary set of genes on the array. The framework includes tests for overrepresentation of a category within a list of significant genes, and methods that consider continuous measures of differential expression. Existing tests are divided into two classes. Class 1 tests assume gene-specific measures of differential expression are independent, despite overwhelming evidence of positive correlation. Analytic and simulated results are presented that demonstrate Class 1 tests are strongly anti-conservative in practice. Class 2 tests account for gene correlation, typically through array permutation that by construction has proper Type I error control for the induced null. However, both Class 1 and Class 2 tests use a null hypothesis that all genes have the same degree of differential expression. We introduce a more sensible and general (Class 3) null under which the profile of differential expression is the same within the category and complement. Under this broader null, Class 2 tests are shown to be conservative. We propose standard bootstrap methods for testing against the Class 3 null and demonstrate they provide valid Type I error control and more power than array permutation in simulated datasets and real microarray experiments.Comment: Published in at http://dx.doi.org/10.1214/07-AOAS146 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Differential Gene Expression of Human Mast cell Activation Reveals Gene profiles of Innate and Adaptive Immunity.

High-density oligonucleotide microarray is a promising approach for high throughput analysis. It has been extensively used in many areas of biomedical research. Immunoglobulin E (IgE) mediated allergic response (type-1 hypersensitivity) is one of the most powerful reactions of the immune system. Tissue Mast Cells (MCs) and circulating basophils are the major effector cells in these reactions. By dissecting the regulatory circuitry of mast cells by analyzing the genome wide effects of antigen stimulation triggered by FcεRI, offers a potential for finding novel genes as ‘targets’ for therapeutic intervention. In this work, we tried to study the gene expression pattern in IgE sensitized and FcεRI cross linked cord blood derived MCs using one of the latest techniques, high density oligonucleotide expression probe array (HG-Focus array, Gene Chip, Affymetrix, Santa Clara, CA). Microarray hybridization of RNA from cord blood derived MCs revealed coordinated changes in gene expression in response to IgE stimulation and receptor cross linking at different time points. Among the most prominent findings, we observed 2 to 32-fold increased expression of different transcripts. Real-time PCR confirmed reliability of microarray data. This enabled us to classify and cluster genes by functional families as well as to understand known genes in signaling pathways. These results defined a list of primary candidates for finding novel genes as ‘targets’ for therapeutic intervention

Expression profile of genes involved in hydrogen sulphide liberation by _Saccharomyces cerevisiae_ grown under different nitrogen concentrations

The present work aims to elucidate molecular mechanisms underlying hydrogen sulphide production in _S. cerevisiae_ associated to nitrogen deficiency. To assess, at a genome-wide level, how the yeast strain adapted to the progressive nitrogen depletion and to nitrogen re-feeding, gene expression profiles were evaluated during fermentation at different nitrogen concentrations, using the DNA array technology. The results showed that most MET genes displayed higher expression values at the beginning of both control and N-limiting fermentation, just before the time at which the release of sulphide was observed. MET genes were downregulated when yeast stopped growing which could associate MET gene expression levels with cell growth. The over expression of MET genes after nitrogen addition was confirmed by a new release of H2S during the new set of fermentation experiments. In addition, to confirm gene expression profiles observed from macroarray results, real time RT-PCR was performed on 6 genes using additional sets of biological replicates. These genes were selected based on the assumption that differences in sulphide production observed among strains are due to genetic variations of the expression of genes involved in the Sulphate Reduction Pathway. An integration of expression data of genes involved in sulphur assimilation and sulphur amino acid biosynthesis with hydrogen sulphide production is presented

Reproducible probe-level analysis of the Affymetrix Exon 1.0 ST array with R/Bioconductor

The presence of different transcripts of a gene across samples can be analysed by whole-transcriptome microarrays. Reproducing results from published microarray data represents a challenge due to the vast amounts of data and the large variety of pre-processing and filtering steps employed before the actual analysis is carried out. To guarantee a firm basis for methodological development where results with new methods are compared with previous results it is crucial to ensure that all analyses are completely reproducible for other researchers. We here give a detailed workflow on how to perform reproducible analysis of the GeneChip Human Exon 1.0 ST Array at probe and probeset level solely in R/Bioconductor, choosing packages based on their simplicity of use. To exemplify the use of the proposed workflow we analyse differential splicing and differential gene expression in a publicly available dataset using various statistical methods. We believe this study will provide other researchers with an easy way of accessing gene expression data at different annotation levels and with the sufficient details needed for developing their own tools for reproducible analysis of the GeneChip Human Exon 1.0 ST Array

Effects of Thyroxine Exposure on Osteogenesis in Mouse Calvarial Pre-Osteoblasts

The incidence of craniosynostosis is one in every 1,800-2500 births. The gene-environment model proposes that if a genetic predisposition is coupled with environmental exposures, the effects can be multiplicative resulting in severely abnormal phenotypes. At present, very little is known about the role of gene-environment interactions in modulating craniosynostosis phenotypes, but prior evidence suggests a role for endocrine factors. Here we provide a report of the effects of thyroid hormone exposure on murine calvaria cells. Murine derived calvaria cells were exposed to critical doses of pharmaceutical thyroxine and analyzed after 3 and 7 days of treatment. Endpoint assays were designed to determine the effects of the hormone exposure on markers of osteogenesis and included, proliferation assay, quantitative ALP activity assay, targeted qPCR for mRNA expression of Runx2, Alp, Ocn, and Twist1, genechip array for 28,853 targets, and targeted osteogenic microarray with qPCR confirmations. Exposure to thyroxine stimulated the cells to express ALP in a dose dependent manner. There were no patterns of difference observed for proliferation. Targeted RNA expression data confirmed expression increases for Alp and Ocn at 7 days in culture. The genechip array suggests substantive expression differences for 46 gene targets and the targeted osteogenesis microarray indicated 23 targets with substantive differences. 11 gene targets were chosen for qPCR confirmation because of their known association with bone or craniosynostosis (Col2a1, Dmp1, Fgf1, 2, Igf1, Mmp9, Phex, Tnf, Htra1, Por, and Dcn). We confirmed substantive increases in mRNA for Phex, FGF1, 2, Tnf, Dmp1, Htra1, Por, Igf1 and Mmp9, and substantive decreases for Dcn. It appears thyroid hormone may exert its effects through increasing osteogenesis. Targets isolated suggest a possible interaction for those gene products associated with calvarial suture growth and homeostasis as well as craniosynostosis. © 2013 Cray et al

Genomic and proteomic profiling of responses to toxic metals in human lung cells.

Examining global effects of toxic metals on gene expression can be useful for elucidating patterns of biological response, discovering underlying mechanisms of toxicity, and identifying candidate metal-specific genetic markers of exposure and response. Using a 1,200 gene nylon array, we examined changes in gene expression following low-dose, acute exposures of cadmium, chromium, arsenic, nickel, or mitomycin C (MMC) in BEAS-2B human bronchial epithelial cells. Total RNA was isolated from cells exposed to 3 M Cd(II) (as cadmium chloride), 10 M Cr(VI) (as sodium dichromate), 3 g/cm2 Ni(II) (as nickel subsulfide), 5 M or 50 M As(III) (as sodium arsenite), or 1 M MMC for 4 hr. Expression changes were verified at the protein level for several genes. Only a small subset of genes was differentially expressed in response to each agent: Cd, Cr, Ni, As (5 M), As (50 M), and MMC each differentially altered the expression of 25, 44, 31, 110, 65, and 16 individual genes, respectively. Few genes were commonly expressed among the various treatments. Only one gene was altered in response to all four metals (hsp90), and no gene overlapped among all five treatments. We also compared low-dose (5 M, noncytotoxic) and high-dose (50 M, cytotoxic) arsenic treatments, which surprisingly, affected expression of almost completely nonoverlapping subsets of genes, suggesting a threshold switch from a survival-based biological response at low doses to a death response at high doses

Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis

Background\ud DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. \ud \ud Results\ud A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. \ud \ud Conclusion\ud The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver. \ud \u