User-friendly solutions for microarray quality control and pre-processing on ArrayAnalysis.org.

Quality control (QC) is crucial for any scientific method producing data. Applying adequate QC introduces new challenges in the genomics field where large amounts of data are produced with complex technologies. For DNA microarrays, specific algorithms for QC and pre-processing including normalization have been developed by the scientific community, especially for expression chips of the Affymetrix platform. Many of these have been implemented in the statistical scripting language R and are available from the Bioconductor repository. However, application is hampered by lack of integrative tools that can be used by users of any experience level. To fill this gap, we developed a freely available tool for QC and pre-processing of Affymetrix gene expression results, extending, integrating and harmonizing functionality of Bioconductor packages. The tool can be easily accessed through a wizard-like web portal at http://www.arrayanalysis.org or downloaded for local use in R. The portal provides extensive documentation, including user guides, interpretation help with real output illustrations and detailed technical documentation. It assists newcomers to the field in performing state-of-the-art QC and pre-processing while offering data analysts an integral open-source package. Providing the scientific community with this easily accessible tool will allow improving data quality and reuse and adoption of standards.

Profiling of promoter occupancy by PPARalpha in human hepatoma cells via ChIP-chip analysis.

The transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is an important regulator of hepatic lipid metabolism. While PPARalpha is known to activate transcription of numerous genes, no comprehensive picture of PPARalpha binding to endogenous genes has yet been reported. To fill this gap, we performed Chromatin immunoprecipitation (ChIP)-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARalpha agonist GW7647. We found that GW7647 increased PPARalpha binding to 4220 binding regions. GW7647-induced binding regions showed a bias around the transcription start site and most contained a predicted PPAR binding motif. Several genes known to be regulated by PPARalpha, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARalpha binding to their promoter. A GW7647-induced PPARalpha-binding region was also assigned to SREBP-targets HMGCS1, HMGCR, FDFT1, SC4MOL, and LPIN1, expression of which was induced by GW7647, suggesting cross-talk between PPARalpha and SREBP signaling. Our data furthermore demonstrate interaction between PPARalpha and STAT transcription factors in PPARalpha-mediated transcriptional repression, and suggest interaction between PPARalpha and TBP, and PPARalpha and C/EBPalpha in PPARalpha-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARalpha in human liver and highlight the importance of cross-talk with other transcription factors.

Differential NF-kappaB pathways induction by Lactobacillus plantarum in the duodenum of healthy humans correlating with immune tolerance.

How do we acquire immune tolerance against food microorganisms and commensal bacteria that constitute the intestinal microbiota? We investigated this by stimulating the immune system of adults with commensal Lactobacillus plantarum bacteria. We studied the in vivo human responses to L. plantarum in a randomized double-blind placebo-controlled cross-over study. Healthy adults ingested preparations of living and heat-killed L. plantarum bacteria. Biopsies were taken from the intestinal duodenal mucosa and altered expression profiles were analyzed using whole-genome microarrays and by biological pathway reconstructions. Expression profiles of human mucosa displayed striking differences in modulation of NF-kappaB-dependent pathways, notably after consumption of living L. plantarum bacteria in different growth phases. Our in vivo study identified mucosal gene expression patterns and cellular pathways that correlated with the establishment of immune tolerance in healthy adults.

Tomato transcriptional responses to a foliar and a vascular fungal pathogen are distinct.

Plant activation of host defense against pathogenic microbes requires significant host transcriptional reprogramming. In this study, we compared transcriptional changes in tomato during compatible and incompatible interactions with the foliar fungal pathogen Cladosporium fulvum and the vascular fungal pathogen Verticillium dahliae. Although both pathogens colonize different host tissues, they display distinct commonalities in their infection strategy; both pathogens penetrate natural openings and grow strictly extracellular. Furthermore, resistance against both pathogens is conveyed by the same class of resistance proteins, the receptor-like proteins. For each individual pathogen, the expression profile of the compatible and incompatible interaction largely overlaps. However, when comparing between the two pathogens, the C. fulvum-induced transcriptional changes show little overlap with those induced by V. dahliae. Moreover, within the subset of genes that are regulated by both pathogens, many genes show inverse regulation. With pathway reconstruction, networks of tomato genes implicated in photorespiration, hypoxia, and glycoxylate metabolism were identified that are repressed upon infection with C. fulvum and induced by V. dahliae. Similarly, auxin signaling is differentially affected by the two pathogens. Thus, differentially regulated pathways were identified with novel strategies that allowed the use of state-of-the-art tools, even though tomato is not a genetic model organism.

PPARalpha-mediated effects of dietary lipids on intestinal barrier gene expression.

BACKGROUND: The selective absorption of nutrients and other food constituents in the small intestine is mediated by a group of transport proteins and metabolic enzymes, often collectively called 'intestinal barrier proteins'. An important receptor that mediates the effects of dietary lipids on gene expression is the peroxisome proliferator-activated receptor alpha (PPARalpha), which is abundantly expressed in enterocytes. In this study we examined the effects of acute nutritional activation of PPARalpha on expression of genes encoding intestinal barrier proteins. To this end we used triacylglycerols composed of identical fatty acids in combination with gene expression profiling in wild-type and PPARalpha-null mice. Treatment with the synthetic PPARalpha agonist WY14643 served as reference. RESULTS: We identified 74 barrier genes that were PPARalpha-dependently regulated 6 hours after activation with WY14643. For eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and oleic acid (OA) these numbers were 46, 41, and 19, respectively. The overlap between EPA-, DHA-, and WY14643-regulated genes was considerable, whereas OA treatment showed limited overlap. Functional implications inferred form our data suggested that nutrient-activated PPARalpha regulated transporters and phase I/II metabolic enzymes were involved in a) fatty acid oxidation, b) cholesterol, glucose, and amino acid transport and metabolism, c) intestinal motility, and d) oxidative stress defense. CONCLUSION: We identified intestinal barrier genes that were PPARalpha-dependently regulated after acute activation by fatty acids. This knowledge provides a better understanding of the impact dietary fat has on the barrier function of the gut, identifies PPARalpha as an important factor controlling this key function, and underscores the importance of PPARalpha for nutrient-mediated gene regulation in intestine.

Gene expression of transporters and phase I/II metabolic enzymes in murine small intestine during fasting.

BACKGROUND: Fasting has dramatic effects on small intestinal transport function. However, little is known on expression of intestinal transport and phase I/II metabolism genes during fasting and the role the fatty acid-activated transcription factor PPARalpha may play herein. We therefore investigated the effects of fasting on expression of these genes using Affymetrix GeneChip MOE430A arrays and quantitative RT-PCR. RESULTS: After 24 hours of fasting, expression levels of 33 of the 253 analyzed transporter and phase I/II metabolism genes were changed. Upregulated genes were involved in transport of energy-yielding molecules in processes such as glycogenolysis (G6pt1) and mitochondrial and peroxisomal oxidation of fatty acids (Cact, Mrs3/4, Fatp2, Cyp4a10, Cyp4b1). Other induced genes were responsible for the inactivation of the neurotransmitter serotonin (Sert, Sult1d1, Dtd, Papst2), formation of eicosanoids (Cyp2j6, Cyp4a10, Cyp4b1), or for secretion of cholesterol (Abca1 and Abcg8). Cyp3a11, typically known because of its drug metabolizing capacity, was also increased. Fasting had no pronounced effect on expression of phase II metabolic enzymes, except for glutathione S-transferases which were down-regulated. Time course studies revealed that some genes were acutely regulated, whereas expression of other genes was only affected after prolonged fasting. Finally, we identified 8 genes that were PPARalpha-dependently upregulated upon fasting. CONCLUSION: We have characterized the response to fasting on expression of transporters and phase I/II metabolic enzymes in murine small intestine. Differentially expressed genes are involved in a variety of processes, which functionally can be summarized as a) increased oxidation of fat and xenobiotics, b) increased cholesterol secretion, c) increased susceptibility to electrophilic stressors, and d) reduced intestinal motility. This knowledge increases our understanding of gut physiology, and may be of relevance for e.g. pre-surgery regimen of patients.

Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs.

Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.

Dose-dependent effects of dietary fat on development of obesity in relation to intestinal differential gene expression in C57BL/6J mice.

Excessive intake of dietary fat is known to be a contributing factor in the development of obesity. In this study, we determined the dose-dependent effects of dietary fat on the development of this metabolic condition with a focus on changes in gene expression in the small intestine. C57BL/6J mice were fed diets with either 10, 20, 30 or 45 energy% (E%) derived from fat for four weeks (n = 10 mice/diet). We found a significant higher weight gain in mice fed the 30E% and 45E% fat diet compared to mice on the control diet. These data indicate that the main shift towards an obese phenotype lies between a 20E% and 30E% dietary fat intake. Analysis of differential gene expression in the small intestine showed a fat-dose dependent gradient in differentially expressed genes, with the highest numbers in mice fed the 45E% fat diet. The main shift in fat-induced differential gene expression was found between the 30E% and 45E% fat diet. Furthermore, approximately 70% of the differentially expressed genes were changed in a fat-dose dependent manner. Many of these genes were involved in lipid metabolism-related processes and were already differentially expressed on a 30E% fat diet. Taken together, we conclude that up to 20E% of dietary fat, the small intestine has an effective 'buffer capacity' for fat handling. From 30E% of dietary fat, a switch towards an obese phenotype is triggered. We further speculate that especially fat-dose dependently changed lipid metabolism-related genes are involved in development of obesity.

MADMAX - Management and analysis database for multiple ~omics experiments.

The rapid increase of ~omics datasets generated by microarray, mass spectrometry and next generation sequencing technologies requires an integrated platform that can combine results from different ~omics datasets to provide novel insights in the understanding of biological systems. MADMAX is designed to provide a solution for storage and analysis of complex ~omics datasets. In addition, analysis results (such as lists of genes) will be merged to reveal candidate genes supported by all datasets. The system constitutes an ISA-Tab compliant LIMS part which is independent of different analysis pipelines. A pilot study of different type of ~omics data in Brassica rapa demonstrates the possible use of MADMAX. The web-based user interface provides easy access to data and analysis tools on top of the database.

Genome-wide mRNA expression analysis of hepatic adaptation to high-fat diets reveals switch from an inflammatory to steatotic transcriptional program.

BACKGROUND: Excessive exposure to dietary fats is an important factor in the initiation of obesity and metabolic syndrome associated pathologies. The cellular processes associated with the onset and progression of diet-induced metabolic syndrome are insufficiently understood. PRINCIPAL FINDINGS: To identify the mechanisms underlying the pathological changes associated with short and long-term exposure to excess dietary fat, hepatic gene expression of ApoE3Leiden mice fed chow and two types of high-fat (HF) diets was monitored using microarrays during a 16-week period. A functional characterization of 1663 HF-responsive genes reveals perturbations in lipid, cholesterol and oxidative metabolism, immune and inflammatory responses and stress-related pathways. The major changes in gene expression take place during the early (day 3) and late (week 12) phases of HF feeding. This is also associated with characteristic opposite regulation of many HF-affected pathways between these two phases. The most prominent switch occurs in the expression of inflammatory/immune pathways (early activation, late repression) and lipogenic/adipogenic pathways (early repression, late activation). Transcriptional network analysis identifies NF-kappaB, NEMO, Akt, PPARgamma and SREBP1 as the key controllers of these processes and suggests that direct regulatory interactions between these factors may govern the transition from early (stressed, inflammatory) to late (pathological, steatotic) hepatic adaptation to HF feeding. This transition observed by hepatic gene expression analysis is confirmed by expression of inflammatory proteins in plasma and the late increase in hepatic triglyceride content. In addition, the genes most predictive of fat accumulation in liver during 16-week high-fat feeding period are uncovered by regression analysis of hepatic gene expression and triglyceride levels. CONCLUSIONS: The transition from an inflammatory to a steatotic transcriptional program, possibly driven by the reciprocal activation of NF-kappaB and PPARgamma regulators, emerges as the principal signature of the hepatic adaptation to excess dietary fat. These findings may be of essential interest for devising new strategies aiming to prevent the progression of high-fat diet induced pathologies.

Effect of synthetic dietary triglycerides: a novel research paradigm for nutrigenomics.

BACKGROUND: The effect of dietary fats on human health and disease are likely mediated by changes in gene expression. Several transcription factors have been shown to respond to fatty acids, including SREBP-1c, NF-kappaB, RXRs, LXRs, FXR, HNF4alpha, and PPARs. However, it is unclear to what extent these transcription factors play a role in gene regulation by dietary fatty acids in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the effects of various individual dietary fatty acids on hepatic gene expression in mice. We observed that the number of significantly changed genes and the fold-induction of genes increased with increasing fatty acid chain length and degree of unsaturation. Importantly, almost every single gene regulated by dietary unsaturated fatty acids remained unaltered in mice lacking PPARalpha. In addition, the majority of genes regulated by unsaturated fatty acids, especially docosahexaenoic acid, were also regulated by the specific PPARalpha agonist WY14643. Excellent agreement was found between the effects of unsaturated fatty acids on mouse liver versus cultured rat hepatoma cells. Interestingly, using Nuclear Receptor PamChip(R) Arrays, fatty acid- and WY14643-induced interactions between PPARalpha and coregulators were found to be highly similar, although several PPARalpha-coactivator interactions specific for WY14643 were identified. CONCLUSIONS/SIGNIFICANCE: We conclude that the effects of dietary unsaturated fatty acids on hepatic gene expression are almost entirely mediated by PPARalpha and mimic those of synthetic PPARalpha agonists in terms of regulation of target genes and molecular mechanism. Use of synthetic dietary triglycerides may provide a novel paradigm for nutrigenomics research.

The role of the small intestine in the development of dietary fat-induced obesity and insulin resistance in C57BL/6J mice.

BACKGROUND: Obesity and insulin resistance are two major risk factors underlying the metabolic syndrome. The development of these metabolic disorders is frequently studied, but mainly in liver, skeletal muscle, and adipose tissue. To gain more insight in the role of the small intestine in development of obesity and insulin resistance, dietary fat-induced differential gene expression was determined along the longitudinal axis of small intestines of C57BL/6J mice. METHODS: Male C57BL/6J mice were fed a low-fat or a high-fat diet that mimicked the fatty acid composition of a Western-style human diet. After 2, 4 and 8 weeks of diet intervention small intestines were isolated and divided in three equal parts. Differential gene expression was determined in mucosal scrapings using Mouse genome 430 2.0 arrays. RESULTS: The high-fat diet significantly increased body weight and decreased oral glucose tolerance, indicating insulin resistance. Microarray analysis showed that dietary fat had the most pronounced effect on differential gene expression in the middle part of the small intestine. By overrepresentation analysis we found that the most modulated biological processes on a high-fat diet were related to lipid metabolism, cell cycle and inflammation. Our results further indicated that the nuclear receptors Ppars, Lxrs and Fxr play an important regulatory role in the response of the small intestine to the high-fat diet. Next to these more local dietary fat effects, a secretome analysis revealed differential gene expression of secreted proteins, such as Il18, Fgf15, Mif, Igfbp3 and Angptl4. Finally, we linked the fat-induced molecular changes in the small intestine to development of obesity and insulin resistance. CONCLUSION: During dietary fat-induced development of obesity and insulin resistance, we found substantial changes in gene expression in the small intestine, indicating modulations of biological processes, especially related to lipid metabolism. Moreover, we found differential expression of potential signaling molecules that can provoke systemic effects in peripheral organs by influencing their metabolic homeostasis. Many of these fat-modulated genes could be linked to obesity and/or insulin resistance. Together, our data provided various leads for a causal role of the small intestine in the etiology of obesity and/or insulin resistance.

Comprehensive analysis of PPARalpha-dependent regulation of hepatic lipid metabolism by expression profiling.

PPARalpha is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARalpha in hepatic lipid metabolism, many PPARalpha-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARalpha-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARalpha target genes, livers from several animal studies in which PPARalpha was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARalpha-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARalpha-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein beta polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (HSL, Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Regulation of Pnpla2, Lipe, and Mgll, which are involved in triglyceride hydrolysis, was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARalpha agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARalpha. Our study illustrates the power of transcriptional profiling to uncover novel PPARalpha-regulated genes and pathways in liver.