Genome-Wide Gene Expression Profiling of Fertilization Competent Mycelium in Opposite Mating Types in the Heterothallic Fungus Podospora anserina

are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation. strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species.This study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

<p>Abstract</p> <p>Background</p> <p>The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of <it>de novo </it>gene prediction programs, and annotation up-dating. We present a novel <it>in silico </it>procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the <it>in silico </it>outcome.</p> <p>Findings</p> <p>We used four criteria for <it>in silico </it>probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus <it>Podospora anserina </it>and the selection of a single 60-mer probe for each of the 10,556 <it>P. anserina </it>CDS.</p> <p>Conclusions</p> <p>A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.</p

A Functional and Regulatory Network Associated with PIP Expression in Human Breast Cancer

BACKGROUND: The PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes. PRINCIPAL FINDINGS: Microarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP-] cells were identified. Functional and regulatory network analyses based on a knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The network identified appears associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines, and contains many genes with a STAT5 regulatory motif in their promoters. CONCLUSIONS: Our global exploratory approach identified biological pathways modulated along with PIP expression, providing further support for its good prognostic value of disease-free survival in breast cancer. Moreover, our data pointed to the importance of a regulatory subnetwork associated with PIP expression in which STAT5 appears as a potential transcriptional regulator

Deciphering cellular states of innate tumor drug responses

BACKGROUND: The molecular mechanisms underlying innate tumor drug resistance, a major obstacle to successful cancer therapy, remain poorly understood. In colorectal cancer (CRC), molecular studies have focused on drug-selected tumor cell lines or individual candidate genes using samples derived from patients already treated with drugs, so that very little data are available prior to drug treatment. RESULTS: Transcriptional profiles of clinical samples collected from CRC patients prior to their exposure to a combined chemotherapy of folinic acid, 5-fluorouracil and irinotecan were established using microarrays. Vigilant experimental design, power simulations and robust statistics were used to restrain the rates of false negative and false positive hybridizations, allowing successful discrimination between drug resistance and sensitivity states with restricted sampling. A list of 679 genes was established that intrinsically differentiates, for the first time prior to drug exposure, subsequently diagnosed chemo-sensitive and resistant patients. Independent biological validation performed through quantitative PCR confirmed the expression pattern on two additional patients. Careful annotation of interconnected functional networks provided a unique representation of the cellular states underlying drug responses. CONCLUSION: Molecular interaction networks are described that provide a solid foundation on which to anchor working hypotheses about mechanisms underlying in vivo innate tumor drug responses. These broad-spectrum cellular signatures represent a starting point from which by-pass chemotherapy schemes, targeting simultaneously several of the molecular mechanisms involved, may be developed for critical therapeutic intervention in CRC patients. The demonstrated power of this research strategy makes it generally applicable to other physiological and pathological situations

The evolutionary rewiring of ubiquitination targets has reprogrammed the regulation of carbon assimilation in the pathogenic yeast Candida albicans

Date of Acceptance: 13/11/2012 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Correction for Sandai et al., The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans published 20-01-2015 DOI: 10.1128/mBio.02489-14Peer reviewedPublisher PD

RN181 suppresses hepatocellular carcinoma growth by inhibition of the ERK/MAPK pathway.

UNLABELLED: The activation of oncogenes and the inactivation of tumor suppressor genes by mutations or chronic hepatitis virus infections play key roles in the pathogenesis of hepatocellular carcinoma (HCC). Here we report that RN181, a really interesting new gene finger domain-containing protein, was down-regulated in highly malignant cell lines and in tumor cells of 139 HCC clinical samples in comparison with adjacent normal liver tissues. The expression of RN181 was strongly associated with the pathological grade of HCC. Alterations of the expression of RN181 by retrovirus-transduced up-regulation and short hairpin RNA-mediated down-regulation demonstrated the function of RN181 as a tumor suppressor because it decreased the proliferation and colony formation of HCC cells in vitro and inhibited tumor growth in vivo by suppressing cell proliferation and enhancing cell apoptosis in xenografted tumors. Proteomic analyses showed that RN181 regulates the expression of many proteins that are important in many cellular processes. Statistical analyses identified 33 proteins with consistent changes (≥2-fold) in RN181-transformed cells. Ten of these proteins were up-regulated by RN181, and 23 were down-regulated. Representative proteins were validated by western blotting. Interaction network investigations revealed that 20 RN181-regulated proteins could integrate several key biological processes such as survival, metabolism, and mitogen-activated protein kinase (MAPK) pathways. Remarkably, 11 of the 33 proteins are associated with MAPK signaling in one or more ways. RN181 suppressed the tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in cell lines and in tumor cells of xenografts and HCC clinical samples, and removing the suppression increased tumor growth. CONCLUSION: We have shown that RN181 suppresses the tumorigenesis of HCC through the inhibition of ERK/MAPK signaling in the liver. Our results provide new insights into the pathogenesis of HCC and may help with the development of novel therapeutic strategies

DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma

Epigenetic deregulation has emerged as a driver in human malignancies. There is no clear understanding of the epigenetic alterations in hepatocellular carcinoma (HCC) and of the potential role of DNA methylation markers as prognostic biomarkers. Analysis of tumor tissue from 304 patients with HCC treated with surgical resection allowed us to generate a methylation-based prognostic signature using a training-validation scheme. Methylome profiling was done with the Illumina HumanMethylation450 array (Illumina, Inc., San Diego, CA), which covers 96% of known cytosine-phosphate-guanine (CpG) islands and 485,000 CpG, and transcriptome profiling was performed with Affymetrix Human Genome U219 Plate (Affymetrix, Inc., Santa Clara, CA) and miRNA Chip 2.0. Random survival forests enabled us to generate a methylation signature based on 36 methylation probes. We computed a risk score of mortality for each individual that accurately discriminated patient survival both in the training (221 patients; 47% hepatitis C-related HCC) and validation sets (n = 83; 47% alcohol-related HCC). This signature correlated with known predictors of poor outcome and retained independent prognostic capacity of survival along with multinodularity and platelet count. The subset of patients identified by this signature was enriched in the molecular subclass of proliferation with progenitor cell features. The study confirmed a high prevalence of genes known to be deregulated by aberrant methylation in HCC (e.g., Ras association [RalGDS/AF-6] domain family member 1, insulin-like growth factor 2, and adenomatous polyposis coli) and other solid tumors (e.g., NOTCH3) and describes potential candidate epidrivers (e.g., septin 9 and ephrin B2). Conclusions: A validated signature of 36 DNA methylation markers accurately predicts poor survival in patients with HCC. Patients with this methylation profile harbor messenger RNA-based signatures indicating tumors with progenitor cell features