goCluster integrates statistical analysis and functional interpretation of microarray expression data

Motivation: Several tools that facilitate the interpretation of transcriptional profiles using gene annotation data are available but most of them combine a particular statistical analysis strategy with functional information. goCluster extends this concept by providing a modular framework that facilitates integration of statistical and functional microarray data analysis with data interpretation. Results: goCluster enables scientists to employ annotation information, clustering algorithms and visualization tools in their array data analysis and interpretation strategy. The package provides four clustering algorithms and GeneOntology terms as prototype annotation data. The functional analysis is based on the hypergeometric distribution whereby the Bonferroni correction or the false discovery rate can be used to correct for multiple testing. The approach implemented in goCluster was successfully applied to interpret the results of complex mammalian and yeast expression data obtained with high density oligonucleotide microarrays (GeneChips). Availability: goCluster is available via the BioConductor portal at www.bioconductor.org. The software package, detailed documentation, user- and developer guides as well as other background information are also accessible via a web portal at http://www.bioz.unibas.ch/gocluster. Contact: [email protected]

PromAn: an integrated knowledge-based web server dedicated to promoter analysis

PromAn is a modular web-based tool dedicated to promoter analysis that integrates distinct complementary databases, methods and programs. PromAn provides automatic analysis of a genomic region with minimal prior knowledge of the genomic sequence. Prediction programs and experimental databases are combined to locate the transcription start site (TSS) and the promoter region within a large genomic input sequence. Transcription factor binding sites (TFBSs) can be predicted using several public databases and user-defined motifs. Also, a phylogenetic footprinting strategy, combining multiple alignment of large genomic sequences and assignment of various scores reflecting the evolutionary selection pressure, allows for evaluation and ranking of TFBS predictions. PromAn results can be displayed in an interactive graphical user interface, PromAnGUI. It integrates all of this information to highlight active promoter regions, to identify among the huge number of TFBS predictions those which are the most likely to be potentially functional and to facilitate user refined analysis. Such an integrative approach is essential in the face of a growing number of tools dedicated to promoter analysis in order to propose hypotheses to direct further experimental validations. PromAn is publicly available at http://bips.u-strasbg.fr/PromA

Profiling spermatogenic failure in adult testes bearing Sox9-deficient Sertoli cells identifies genes involved in feminization, inflammation and stress

<p>Abstract</p> <p>Background</p> <p><it>Sox9 </it>(<it>Sry </it>box containing gene 9) is a DNA-binding transcription factor involved in chondrocyte development and sex determination. The protein's absence in testicular Sertoli nurse cells has been shown to disrupt testicular function in adults but little is known at the genome-wide level about molecular events concomitant with testicular break-down.</p> <p>Methods</p> <p>To determine the genome-wide effect on mRNA concentrations triggered by the absence of <it>Sox9 </it>in Sertoli cells we analysed adult testicular tissue from wild-type versus mutant mice with high-density oligonucleotide microarrays and integrated the output of this experiment with regulatory motif predictions and protein-protein network data.</p> <p>Results</p> <p>We report the genome-wide mRNA signature of adult testes lacking <it>Sox9 </it>in Sertoli cells before and after the onset of late spermatogenic failure as compared to fertile controls. The GeneChip data integrated with evolutionarily conserved <it>Sox9 </it>DNA binding motifs and regulatory network data identified genes involved in feminization, stress response and inflammation.</p> <p>Conclusions</p> <p>Our results extend previous observations that genes required for female gonadogenesis are up-regulated in the absence of <it>Sox9 </it>in fetal Sertoli cells to the adult stage. Importantly, we identify gene networks involved in immunological processes and stress response which is reminiscent of a phenomenon occurring in a sub-group of infertile men. This suggests mice lacking <it>Sox9 </it>in their Sertoli cells to be a potentially useful model for adult human testicular failure.</p

Fhl5/Act, a CREM-binding transcriptional activator required for normal sperm maturation and morphology, is not essential for testicular gene expression

<p>Abstract</p> <p>Background</p> <p>The LIM domain protein Fhl5 was previously found to interact with CREM, a DNA binding transcriptional regulator necessary for spermiogenesis in mammals. Co-transfection experiments using heterologous promoter constructs indicated a role for Fhl5 in transcriptional up-regulation of CREM-dependent testicular genes. Male mice lacking Fhl5 were reported to be fertile but displayed partially abnormal sperm maturation and morphology.</p> <p>Methods</p> <p>To identify Fhl5 testicular target genes we carried out two whole-genome expression profiling experiments using high-density oligonucleotide microarrays and total testis samples from Fhl5 wild-type versus homozygous mutant mice first in different and then in isogenic strain backgrounds.</p> <p>Results</p> <p>Weak signal differences were detected in non-isogenic samples but no statistically significant expression changes were observed when isogenic Fhl5 mutant and wild-type samples were compared.</p> <p>Conclusion</p> <p>The outcome of these experiments suggests that testicular expression profiling is extremely sensitive to the genetic background and that Fhl5 is not essential for testicular gene expression to a level detected by microarray-based measurements. This might be due to redundant function of the related and similarly expressed protein Fhl4.</p

PromAn: an integrated knowledge-based web server dedicated to promoter analysis

PromAn is a modular web-based tool dedicated to promoter analysis that integrates distinct complementary databases, methods and programs. PromAn provides automatic analysis of a genomic region with minimal prior knowledge of the genomic sequence. Prediction programs and experimental databases are combined to locate the transcription start site (TSS) and the promoter region within a large genomic input sequence. Transcription factor binding sites (TFBSs) can be predicted using several public databases and user-defined motifs. Also, a phylogenetic footprinting strategy, combining multiple alignment of large genomic sequences and assignment of various scores reflecting the evolutionary selection pressure, allows for evaluation and ranking of TFBS predictions. PromAn results can be displayed in an interactive graphical user interface, PromAnGUI. It integrates all of this information to highlight active promoter regions, to identify among the huge number of TFBS predictions those which are the most likely to be potentially functional and to facilitate user refined analysis. Such an integrative approach is essential in the face of a growing number of tools dedicated to promoter analysis in order to propose hypotheses to direct further experimental validations. PromAn is publicly available at

Cross-platform gene expression signature of human spermatogenic failure reveals inflammatory-like response

BACKGROUND The molecular basis of human testicular dysfunction is largely unknown. Global gene expression profiling of testicular biopsies might reveal an expression signature of spermatogenic failure in azoospermic men. METHODS Sixty-nine individual testicular biopsy samples were analysed on two microarray platforms; selected genes were validated by quantitative real-time PCR and immunohistochemistry. RESULTS A minimum of 188 transcripts were significantly increased on both platforms. Their levels increased with the severity of spermatogenic damage and reached maximum levels in samples with Sertoli-cell-only appearance, pointing to genes expressed in somatic testicular cells. Over-represented functional annotation terms were steroid metabolism, innate defence and immune response, focal adhesion, antigen processing and presentation and mitogen-activated protein kinase K signalling pathway. For a considerable proportion of genes included in the expression signature, individual transcript levels were in keeping with the individual mast cell numbers of the biopsies. When tested on three disparate microarray data sets, the gene expression signature was able to clearly distinguish normal from defective spermatogenesis. More than 90% of biopsy samples clustered correctly into the corresponding category, emphasizing the robustness of our data. CONCLUSIONS A gene expression signature of human spermatogenic failure was revealed which comprised well-studied examples of inflammation-related genes also increased in other pathologies, including autoimmune disease

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

<p>Abstract</p> <p>Background</p> <p>The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton.</p> <p>Results</p> <p>Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not.</p> <p>Conclusion</p> <p>Our data demonstrate that Actichip is a powerful alternative to commercial high density microarrays for cytoskeleton gene profiling in normal or pathological samples. Actichip is available upon request.</p

Expression of HLA-G by mast cells is associated with hepatitis C virus-induced liver fibrosis.

International audienceBACKGROUND AND AIMS: Infection by hepatitis C virus is a worldwide health problem. An inadequate Th2 cytokine response promotes the fibrosis-cirrhosis fate. Immune-modulating molecules favoring a Th2 profile, such as HLA-G molecules of the HLA class Ib family, may play a role in chronic hepatitis. HLA-G contributes to the escape of tumors, and their involvement in viral infections has been increasingly described. The aim of this work was to study the expression of HLA-G in the liver, its cellular source and its regulation in cases of chronic C hepatitis. METHODS: HLA-G cells in blocks of liver derived from patients infected with HCV were labeled by immunohistochemistry and enumerated. Double immunofluorescence allowed the identification of the cellular source. HLA-G secretion by a human mast cell line was quantified by ELISA after various stimulations. After treatment with IFN-α real-time PCR was performed to determine the kinetics of cytokine expression profiles, followed by heat map clustering analysis. RESULTS: The number of HLA-G + cells was significantly associated with the area of fibrosis. For the first time, we identify the HLA-G+ cells as being mast cells. HLA-G secretion was significantly induced in human mast cells stimulated by IL-10 or interferons of class I. The transcriptome of the secretome of this cell line stimulated by IFN-α revealed that i) the HLA-G gene is upregulated late, ii) T lymphocytes and NK cells are recruited. CONCLUSIONS: These findings suggest an autocrine loop in the genesis of HCV liver fibrosis, based on mast cells expressing HLA-G

Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility

BACKGROUND Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapie

The ReproGenomics Viewer: an integrative cross-species toolbox for the reproductive science community.

International audienceWe report the development of the ReproGenomics Viewer (RGV), a multi-and cross-species working environment for the visualization, mining and comparison of published omics data sets for the reproductive science community. The system currently embeds 15 published data sets related to gametogenesis from nine model organisms. Data sets have been curated and conveniently organized into broad categories including biological topics, technologies, species and publications. RGV's modular design for both organisms and genomic tools enables users to upload and compare their data with that from the data sets embedded in the system in a cross-species manner. The RGV is freely available at http://rgv.genouest.org