Independent Microarray Experiments Research Articles

1865PD - Specific patterns of long non-coding RNA expression in benign and malignant thyroid nodules

BackgroundAlthough there are increasing evidences of lncRNA role in cancer progression, and of its possible diagnostic and therapeutic significance, lncRNA in thyroid neoplasms are poorly investigated: studies are limited to papillary cancer, the differences in variants of papillary carcinoma are rarely considered, there are no investigations of lncRNA in benign nodules, follicular and anaplastic carcinomas. MethodsTo evaluate lncRNA expression in follicular adenoma (FA), follicular and classical variants of papillary carcinoma (fvPTC/clPTC), follicular (FTC) and anaplastic (ATC) carcinomas, a comprehensive dataset of 8 independent Microarray experiments on Affymetrix Human Genome U133 Plus 2.0 Array and RNA-Seq experiments (TCGA and PRJEB11591 projects) was analyzed. In silico validation of differential expression was performed. Putative functions of aberrantly expressed lncRNA were determined via co-expression and enrichment analysis of Gene Ontology, KEGG, Reactome terms. ResultsThe analysis revealed 8 lncRNA general for all investigated neoplasms, 22 - general for papillary carcinomas, 32 - specific for clPTC, 1 - for fvPTC, and 177 - specific for ATC. No lncRNA differentially expressed in FTC and FA was found. There are strong clustering of ATC, clustering of clPTC and fvPTC, no clustering within the FTC and FA. LncRNA found to be specific for ATC are probably associated with anaplastic features and cancer progression. Potential functions of lncRNA general for all studied neoplasms - L1CAM interactions; general for papillary carcinomas - tryptophan metabolism; specific for fvPTC - cell polarity and WNT signaling; specific for clPTC - extracellular matrix organization and endoderm formation; specific for ATC - cell cycle and mitosis. Known oncogenic and tumor suppressor lncRNA (NR2F1-AS1, LINC00511, SLC26A4-AS1, CRNDE, LINC01116, RMST) are found in thyroid carcinomas for the first time. ConclusionsCommon and specific patterns of lncRNA expression in main histological subtypes of thyroid nodules are determined. The findings enhance the understanding of lncRNA in thyroid cancer progression. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Identification of potential mechanism and hub genes for neuropathic pain by expression-based genome-wide association study.

Neuropathic pain (NP) is a common pathological pain state with limited effective treatments. This study was designed to identify potential mechanisms and candidate genes using gene expression-based genome-wide association study (eGWAS). All NP-related microarray experiments were obtained from Gene Expression Omnibus and ArrayExpress. Significantly dysregulated genes were identified between experimental and untreated groups, and the number of microarray experiments in which each gene was dysregulated was calculated. Significantly dysregulated genes were ranked according to P values of the chi-square test. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database, we performed functional and pathway enrichment analysis. Protein-protein interaction (PPI) network and module analysis was performed using Cytoscape software. A total of 115 candidate genes were identified from 19 independent microarray experiments by eGWAS based on the Bonferroni threshold ( P < 2.97 × 10 -6 ). Immune and inflammatory responses, and complement and coagulation cascades, were respectively the most enriched biological process and pathways for candidate genes. The hub genes with highest connectivity in PPI network and two modules Ccl2 and Jun, and Ctss application of the eGWAS methodology can identify mechanisms and candidate genes associated with NP. Our results support the validity and prevalence of inflammatory and immune mechanisms across different NP models, and Ccl2, Jun, and Ctss may be the hub genes for NP.

Identification and characterization of responsive genes in rice during compatible interactions with pathogenic pathovars of Xanthomonas oryzae

Rice is a staple food for one-half of the world’s population. However, pathogenic pathovars of Xanthomonas oryzae can reduce rice yield up to 60%. To explore the molecular features of rice response to two pathogenic pathovars of X. oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), the differentially expressed genes (DEGs) were studied in four independent microarray experiments on infected versus non-infected leaves in rice 24 h after inoculation. The gene ontology (GO) analysis on DEGs demonstrated that while stress related genes were upregulated, the genes encoding photosynthesis components were down regulated in bacterial-infected rice. Moreover, some discrepancy of the genes function was depicted between the two pathovars of X. oryzae, including apoptosis specific to Xoo. Further analysis on cis-regulatory elements (CREs) on the promoter of responsive transcription factors (TFs) revealed the critical role of some hormones such as methyl jasmonate (MeJA) and abscisic acid (ABA) as well as light in the control of genes expression against the bacterial infection. We used a systematic approach to build a network among the responsive TFs that led to discovery of the OsWRKY53 and ERF2 effects on interaction between these pathovars and rice. We also detected miRNA target sites in some of the downregulated genes. These results provided an insight into the genes regulation against bacterial infection and introduced important candidate genes for further investigations in rice.

AURKA induces EMT by regulating histone modification through Wnt/β-catenin and PI3K/Akt signaling pathway in gastric cancer.

Gastric cancer, a highly invasive and aggressive malignancy, is the third leading cause of death from cancer worldwide. Genetic association studies have successfully revealed several important genes consistently associated with gastric cancer to date. However, these robust gastric cancer-associated genes do not fully elucidate the mechanisms underlying the development and progression of the disease. In the present study, we performed an alternative approach, a gene expression-based genome-wide association study (eGWAS) across 13 independent microarray experiments (including 251 gastric cancer cases and 428 controls), to identify top candidates (p<0.00001). Additionally, we conducted gene ontology analysis, pathway analysis and network analysis and identified aurora kinase A (AURKA) as our candidate. We observed that MLN8237, which is a specific inhibitor of AURKA, decreased the β-catenin and the phosphorylation of Akt1 and GSK-3β, as well as blocked the Akt and Wnt signaling pathways. Furthermore, MLN8237 arrested the cells in the G2/M phase. The activity of Wnt and Akt signaling pathways affected the level of histone methylation significantly, and we supposed that MLN8237 affected the level of histone methylation through these two signaling pathways. Additionally, the treatment of MLN8237 influenced the level of H3K4 me1/2/3 and H3K27 me1/2/3. Chip data on cell lines suggested that MLN8237 increases the level of H3K27 me3 on the promoter of Twist and inhibits EMT (epithelial-mesenchymal transition). In summary, AURKA is a potential therapeutic target in gastric cancer and induces EMT through histone methylation.

A pharmacogenomic profile of human neural progenitors undergoing differentiation in the presence of the traditional Chinese medicine NeuroAiD.

NeuroAiD, a traditional Chinese medicine widely used to treat stroke patients in China, was recently demonstrated in rodent models and in clinical trials to possess neuroregenerative and neuroprotective properties. In order to understand the mechanisms employed by NeuroAiD to bring about its neuroproliferative and neuroprotective effects, we investigated the impact of MLC901, a reformulated version of MLC601, on human neural progenitors undergoing neural differentiation at the molecular level by performing three independent microarray experiments. Functional annotations of the genes regulated by MLC901 that were associated with neurogenesis were found to be enriched. We also identified potential targets (FGF19, GALR2, MMP10, FGF3 and TDO2) of MLC901 that could promote neurogenesis and neuroprotection in the human brain. This work highlighted some interesting targets and offered some insights into the possible mechanism of action of MLC901. The discovery could also provide a platform to the development of future therapeutic targets.

Identification of biomarker genes for resistance to a pathogen by a novel method for meta-analysis of single-channel microarray datasets.

The search for fast and reliable methods allowing for extraction of biomarker genes, e.g. responsible for a plant resistance to a certain pathogen, is one of the most important and highly exploited data mining problem in bioinformatics. Here we describe a simple and efficient method suitable for combining results from multiple single-channel microarray experiments for meta-analysis. A new technique presented here makes use of the fuzzy set logic for the initial gene selection and of the machine learning algorithm AdaBoost to retrieve a set of genes where expression profiles are the most different between the resistant and susceptible classes. As a proof of concept, our method has been applied to the analysis of a gene expression dataset composed of many independent microarray experiments on wheat head tissue, to identify genes that are biomarkers of resistance to the fungus Fusarium graminearum. We used microarray data from many experiments performed on wheat lines of various resistance level. The resulting set of genes was validated by qPCR experiments.

BCG-treated M2-macrophages enhance IFN-γ production of T cells (P3348)

Abstract M1- and M2-polarized Macrophage (MΦ) subtypes differentially regulate anti-mycobacterial defense. To examine their roles in human infection, we compared the gene expression profile of M1- vs. M2-MΦ to the expression profile of lesional skin from patients with restricted (tuberculoid, T-lep) vs. disseminated (lepromatous, L-lep) leprosy (a mycobacterial skin infection). Rank-Rank Hypergeometric Overlap (RRHO) is a novel algorithm for comparing full gene expression profiles from two independent microarray experiments that generates the most statistically significant overlapping gene list without requiring strict p-value/fold change cutoffs. RRHO analysis showed significant overlap of genes between M2-MΦ and L-lep lesions (n = 1559, max log hypergeometric p-value = 196), indicating a role for M2-MΦ in immune unresponsiveness. Bacillus Calmette-Guerin (BCG) is given to protect household contacts against leprosy. We hypothesized that BCG treatment may affect the function of M2-MΦ and investigated its effects in vitro. We found that BCG-treated M2-MΦ secreted less IL-10 (p &amp;lt; 0.05), with no effect on IL-12p40, compared to untreated M2-MΦ. In addition, BCG-treated M2-MΦ increased the frequency of IFN-γ producing cells in an antigen non-specific manner (p &amp;lt; 0.05), suggesting an adjuvant effect of BCG. Our data indicates M2-MΦ can be reprogrammed by BCG, resulting in increased IFN-γ production to enhance anti-mycobacterial immunity.

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.

Aryl Hydrocarbon Receptor-Induced Adrenomedullin Mediates Cigarette Smoke Carcinogenicity in Humans and Mice

Cigarette smoking (CS) is a leading cause of death worldwide. The aryl hydrocarbon receptor (AHR) is partially responsible for tobacco-induced carcinogenesis although the underlying mechanisms involving early effector genes have yet to be determined. Here, we report that adrenomedullin (ADM) significantly contributes to the carcinogenicity of tobacco-activated AHR. CS and AHR activating ligands induced ADM in vitro and in vivo but not in AHR-deficient fibroblasts and mice. Ectopic transfection of AHR rescued ADM expression in AHR(-/-) fibroblasts whereas AHR blockage with siRNA in wild type cells significantly decreased ADM expression. AHR regulates ADM expression through two intronic xenobiotic response elements located close to the start codon in the ADM gene. Using tissue microarrays we showed that ADM and AHR were coupregulated in lung tumor biopsies from smoker patients. Microarray meta-analysis of 304 independent microarray experiments showed that ADM is elevated in smokers and smokers with cancer. In addition, ADM coassociated with a subset of AHR responsive genes and efficiently differentiated patients with lung cancer from nonsmokers. In a novel preclinical model of CS-induced tumor progression, host exposure to CS extracts significantly elevated tumor ADM although systemic treatment with the ADM antagonist NSC16311 efficiently blocked tobacco-induced tumor growth. In conclusion, ADM significantly contributes the carcinogenic effect of AHR and tobacco combustion products. We suggest that therapeutics targeting the AHR/ADM axis may be of clinical relevance in the treatment of tobacco-induced pulmonary malignancies.

CD44 immune receptor linked to diabetes

Genome-wide association studies (GWAS) have identified numerous genes associated with type 2 diabetes, but selecting candidate genes for further verification can be problematic. This study used an expression-based GWAS in which data from 130 publicly available independent microarray experiments were collated to identify genes involved in the pathogenesis of type 2 diabetes. The gene encoding the immune cell receptor CD44 was the top-ranked gene. Follow-up studies showed that treatment of a mouse model with a CD44-targeted antibody decreased blood glucose levels, and that serum CD44 levels correlated with insulin resistance and glycaemic control in humans.

Patterns of pain: Meta-analysis of microarray studies of pain

Existing microarray gene expression profiling studies of tonic/chronic pain were subjected to meta-analysis to identify genes found to be regulated by these pain states in multiple, independent experiments. Twenty studies published from 2002 to 2008 were identified, describing the statistically significant regulation of 2254 genes. Of those, a total of 79 genes were found to be statistically significant “hits” in 4 or more independent microarray experiments, corresponding to a conservative P<0.01 overall. Gene ontology-based functional annotation clustering analyses revealed strong evidence for regulation of immune-related genes in pain states. A multi-gene quantitative real-time polymerase chain reaction experiment was run on dorsal root ganglion (DRG) and spinal cord tissue from rats and mice given nerve (sciatic chronic constriction; CCI) or inflammatory (complete Freund’s adjuvant) injury. We independently confirmed the regulation of 43 of these genes in the rat-CCI-DRG condition; the genetic correlates in all other conditions were largely and, in some cases, strikingly, independent. However, a handful of genes were identified whose regulation bridged etiology, anatomical locus, and/or species. Most notable among these were Reg3b (regenerating islet-derived 3 beta; pancreatitis-associated protein) and Ccl2 (chemokine [C–C motif] ligand 2), which were significantly upregulated in every condition in the rat.Gene expression profiling (microarray) studies of chronic pain were subjected to meta-analysis. Two genes were identified that are consistently upregulated in chronic pain states.

On the Choice and Number of Microarrays for Transcriptional Regulatory Network Inference

BackgroundTranscriptional regulatory network inference (TRNI) from large compendia of DNA microarrays has become a fundamental approach for discovering transcription factor (TF)-gene interactions at the genome-wide level. In correlation-based TRNI, network edges can in principle be evaluated using standard statistical tests. However, while such tests nominally assume independent microarray experiments, we expect dependency between the experiments in microarray compendia, due to both project-specific factors (e.g., microarray preparation, environmental effects) in the multi-project compendium setting and effective dependency induced by gene-gene correlations. Herein, we characterize the nature of dependency in an Escherichia coli microarray compendium and explore its consequences on the problem of determining which and how many arrays to use in correlation-based TRNI.ResultsWe present evidence of substantial effective dependency among microarrays in this compendium, and characterize that dependency with respect to experimental condition factors. We then introduce a measure neff of the effective number of experiments in a compendium, and find that corresponding to the dependency observed in this particular compendium there is a huge reduction in effective sample size i.e., neff = 14.7 versus n = 376. Furthermore, we found that the neff of select subsets of experiments actually exceeded neff of the full compendium, suggesting that the adage 'less is more' applies here. Consistent with this latter result, we observed improved performance in TRNI using subsets of the data compared to results using the full compendium. We identified experimental condition factors that trend with changes in TRNI performance and neff , including growth phase and media type. Finally, using the set of known E. coli genetic regulatory interactions from RegulonDB, we demonstrated that false discovery rates (FDR) derived from neff -adjusted p-values were well-matched to FDR based on the RegulonDB truth set.ConclusionsThese results support utilization of neff as a potent descriptor of microarray compendia. In addition, they highlight a straightforward correlation-based method for TRNI with demonstrated meaningful statistical testing for significant edges, readily applicable to compendia from any species, even when a truth set is not available. This work facilitates a more refined approach to construction and utilization of mRNA expression compendia in TRNI.

Effect of continuous light on diurnal rhythms in Cyanothece sp. ATCC 51142

BackgroundLife on earth is strongly affected by alternating day and night cycles. Accordingly, many organisms have evolved an internal timekeeping system with a period of approximately 24 hours. Cyanobacteria are the only known prokaryotes with robust rhythms under control of a central clock. Numerous studies have been conducted to elucidate components of the circadian clock and to identify circadian-controlled genes. However, the complex interactions between endogenous circadian rhythms and external cues are currently not well understood, and a direct and mathematical based comparison between light-mediated and circadian-controlled gene expression is still outstanding. Therefore, we combined and analyzed data from two independent microarray experiments, previously performed under alternating light-dark and continuous light conditions in Cyanothece sp. ATCC 51142, and sought to classify light responsive and circadian controlled genes.ResultsFourier Score-based methods together with random permutations and False Discovery Rates were used to identify genes with oscillatory expression patterns, and an angular distance based criterion was applied to recognize transient behaviors in gene expression under constant light conditions. Compared to previously reported mathematical approaches, the combination of these methods also facilitated the detection of modified amplitudes and phase-shifts of gene expression. Our analysis showed that the majority of diurnally regulated genes, essentially those genes that are maximally expressed during the middle of the light and dark period, are in fact light responsive. In contrast, most of the circadian controlled genes are up-regulated during the beginning of the dark or subjective dark, and are greatly enriched for genes associated with energy metabolism. Many of the circadian controlled and light responsive genes are found in gene clusters within the Cyanothece sp. ATCC 51142 genome. Interestingly, in addition to cyclic expression patterns with a period of 24 hours, we also found several genes that oscillate with an ultradian period of 12 hours, a novel finding among cyanobacteria.ConclusionWe demonstrate that a combination of different analytical methods significantly improved the identification of cyclic and transient gene expression in Cyanothece sp. ATCC 51142. Our analyses provide an adaptable and novel analytical tool to study gene expression in a variety of organisms with diurnal, circadian and ultradian behavior.

Systems Biology of the Clock in Neurospora crassa

A model-driven discovery process, Computing Life, is used to identify an ensemble of genetic networks that describe the biological clock. A clock mechanism involving the genes white-collar-1 and white-collar-2 (wc-1 and wc-2) that encode a transcriptional activator (as well as a blue-light receptor) and an oscillator frequency (frq) that encodes a cyclin that deactivates the activator is used to guide this discovery process through three cycles of microarray experiments. Central to this discovery process is a new methodology for the rational design of a Maximally Informative Next Experiment (MINE), based on the genetic network ensemble. In each experimentation cycle, the MINE approach is used to select the most informative new experiment in order to mine for clock-controlled genes, the outputs of the clock. As much as 25% of the N. crassa transcriptome appears to be under clock-control. Clock outputs include genes with products in DNA metabolism, ribosome biogenesis in RNA metabolism, cell cycle, protein metabolism, transport, carbon metabolism, isoprenoid (including carotenoid) biosynthesis, development, and varied signaling processes. Genes under the transcription factor complex WCC ( = WC-1/WC-2) control were resolved into four classes, circadian only (612 genes), light-responsive only (396), both circadian and light-responsive (328), and neither circadian nor light-responsive (987). In each of three cycles of microarray experiments data support that wc-1 and wc-2 are auto-regulated by WCC. Among 11,000 N. crassa genes a total of 295 genes, including a large fraction of phosphatases/kinases, appear to be under the immediate control of the FRQ oscillator as validated by 4 independent microarray experiments. Ribosomal RNA processing and assembly rather than its transcription appears to be under clock control, suggesting a new mechanism for the post-transcriptional control of clock-controlled genes.

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.

Optimization of a microarray based approach for deriving representative gene expression profiles from human oocytes

The purpose of the present study was to optimize a protocol for deriving reproducible and representative gene expression profiles from very rare research samples of human oocytes using microarrays. Immature oocytes produced as a result of administration of gonadotrophins for the treatment of infertility were donated to research. Linear amplification (L-amp) and exponential amplification (E-amp) were both capable of generating sufficient product for hybridization to the microarrays even from the low amount of template mRNA present in a single human oocyte. Slightly higher numbers of transcripts were detected by microarray following linear rather than E-amp but both techniques generated a product with reliably reproducible sensitivity and fidelity providing oocytes were pooled in minimum numbers of three. The majority of the variance associated with amplification and hybridization to arrays comes from the molecular processing. Slightly greater additional variance is associated with biological differences in immature oocytes from the same or different donors. The findings suggest that representative gene expression profiles can be generated from human oocytes for comparative purposes following L-amp and hybridization to microarray. However, oocytes must be pooled for the starting template for each array and sufficient independent microarray experiments performed to minimize the variance associated with molecular processing.

Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients

BackgroundMore than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the wild type or mutant MECP2 in each of their cells. To test the hypothesis that MECP2 mutations result in genome-wide transcriptional deregulation and identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we performed gene expression profiling of pure populations of untransformed T-lymphocytes that express either a mutant or a wild-type allele.MethodsSingle T lymphocytes from a patient with a c.473C>T (p.T158M) mutation and one with a c.1308-1309delTC mutation were subcloned and subjected to short term culture. Gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis.ResultsExpression profiling yielded 44 upregulated genes and 77 downregulated genes. We compared this gene list with expression profiles of independent microarray experiments in cells and tissues of RTT patients and mouse models with Mecp2 mutations. These comparisons identified a candidate MeCP2 target gene, SPOCK1, downregulated in two independent microarray experiments, but its expression was not altered by quantitative RT-PCR analysis on brain tissues from a RTT mouse model.ConclusionInitial expression profiling from T-cell clones of RTT patients identified a list of potential MeCP2 target genes. Further detailed analysis and comparison to independent microarray experiments did not confirm significantly altered expression of most candidate genes. These results are consistent with other reported data.

Combinatorial Analysis of Transcription Factor Partners Reveals Recruitment of c-MYC to Estrogen Receptor-α Responsive Promoters

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

A Novel Role for Glucocorticoid-induced Leucine Zipper Protein in Epithelial Sodium Channel-mediated Sodium Transport

The steroid hormone aldosterone stimulates sodium (Na+) transport in tight epithelia by altering the expression of target genes that regulate the activity and trafficking of the epithelial sodium channel (ENaC). We performed microarray analysis to identify aldosterone-regulated transcripts in mammalian kidney epithelial cells (mpkC-CD(c14)). One target, glucocorticoid-induced leucine zipper protein (GILZ), was previously identified by serial analysis of gene expression (SAGE); however, its function in epithelial ion transport was unknown. Here we show that GILZ expression is rapidly stimulated by aldosterone in mpkCCD(c14) and that GILZ, in turn, strongly stimulates ENaC-mediated Na+ transport by inhibiting extracellular signal-regulated kinase (ERK) signaling. In Xenopus oocytes with activated ERK, heterologous GILZ expression consistently inhibited phospho-ERK expression and markedly stimulated ENaC-mediated Na+ current, in a manner similar to that of U0126 (a pharmacologic inhibitor of ERK signaling). In mpkCCD(c14) cells, GILZ transfection similarly consistently inhibited phospho-ERK expression and stimulated transepithelial Na+ transport. Furthermore, aldosterone treatment of mpkCCD(c14) cells suppressed phospho-ERK levels with a time course that paralleled their increase of Na+ transport. Finally, GILZ expression markedly increased cell surface ENaC expression in epidermal growth factor-treated mammalian kidney epithelial cells, HEK 293. These observations suggest a novel link between GILZ and regulation of epithelial sodium transport through modulation of ERK signaling and could represent an important pathway for mediating aldosterone actions in health and disease.

Genistein-induced changes in gene expression in Panc 1 cells at physiological concentrations of genistein.

To investigate the effect of genistein on gene expression in Panc 1 cells using microarray technology. Panc 1 cells were treated with 10 micromol/L genistein or DMSO (vehicle control) for 0, 1, 3, 6, or 12 hours. Total RNA from each sample was isolated, and biotin-labeled probes were hybridized to the human genome U133A chip, after which the chip was washed and scanned. Data were analyzed using DMT software (Affymetrix). For genes that showed large changes in expression due to genistein, these changes were confirmed using real-time PCR assays. Two independent microarray experiments showed that genistein significantly changed the expression of 47 genes: up-regulating of egr-1 and IL-8 and down-regulating of EGF-R AKT2, CYP1B1, NELL2, SCD, DNA ligase III, Rad as well as 18s and 28s rRNA and others. These alterations in expression were confirmed using real-time PCR, although the increase in change was not exactly the same in the 2 assays. Our data suggest the reported apparent ability of genistein to inhibit carcinogenesis may involve a number of pathways. The most obvious target is the EGF-R signaling pathway since the expression of 5 genes related to this pathway was reduced (EGFR, egr-1, AKT2, CYP1B1, and NELL2). Genistein may also act by disabling cancer cell self-protection by inhibiting expression of AKT2, CYP1B1, and DNA ligase III. Furthermore, genistein may inhibit car-cinogenesis by inhibiting expression of SCD. Finally, our data support findings indicating that genistein inhibits rRNA formation, which is an important mechanism by which genistein regulates tumor cell growth.