List Of Target Genes Research Articles

Abstract 522: Limited and Gene-Specific Down-Regulation of PPAR Delta Target Genes by Dominant-Negative PPAR Gamma in Mesenteric Artery.

PPARγ is a ligand-activated transcription factor expressed in multiple tissues including the vasculature. Individuals with a mutation in the ligand-binding domain of PPARγ (P467L) are insulin resistant and have early onset hypertension. Targeted expression of P467L PPARγ to smooth muscle of transgenic mice (S-P467L) increased blood pressure, induced aortic dysfunction, and enhanced myogenic tone in the mesenteric artery. To examine the mechanisms underlying these changes, we used the Affymetrix exon array to perform gene expression profiling of mesenteric arteries from S-P467L and non-transgenic (NT) littermates (n =3 for S-P467L, n=4 for NT). Classic PPARγ target genes, as a group, were down-regulated (p<0.001, gene set analysis), consistent with reports that P467L PPARγ acts in a dominant negative manner. We also observed a down-regulation in pyruvate dehydrogenase kinase, isozyme 4 (PDK4, fold change = 0.4, p = 0.01), a known PPARδ target gene. We confirmed that PDK4 was a PPARδ target (fold change = 2.3±0.3, n = 5, Q-PCR) by 6-hr incubation of NT mesenteric arteries with PPARδ specific agonist GW0742 (1 μM). Based on this, we hypothesized that P467L PPARγ was interfering with and down-regulating PPARδ target genes in the mesenteric artery. To examine this, we integrated our expression dataset with a published study (PLoS One. 2011:e16344) which used expression microarrays and chromatin immunoprecipitation/next-generation sequencing (ChIP-Seq) to experimentally identify PPARδ target genes and PPARδ binding sites in a genome-wide manner. Using this target gene list and limiting our analysis to genes expressed in mesenteric arteries (94 genes), we identified 2 additional PPARδ targets that were modestly but significantly down-regulated (p<0.001) in mesenteric artery from S-P467L mice (TIMP4 -40%, CPT1A -22%). Both of these genes also have a PPARγ binding site according to ChIP-Seq (GEO accession, GSE13511 ); suggesting the possibility of interaction between PPARγ and PPARδ in regulation of target gene expression. Thus, our data shows that dominant-negative PPARγ down-regulates only a small subset of PPARδ target genes in mesenteric artery suggesting that P467L PPARγ exhibits a relative selectivity to PPARγ targets instead of global inhibition.

Gene expression of the lysophosphatidic acid receptor 1 is a target of transforming growth factor beta

The lysophosphatidic acid (LPA) receptor LPA1/Edg2 is the first identified LPA receptor. Although its wide tissue distribution and biological functions have been well studied, little is known about how LPA1 is transcriptionally regulated. In the current study, we showed that LPA1 is a physiological target of transforming growth factor beta (TGFβ)-mediated repression. In both normal and neoplastic cells, TGFβ inhibits LPA1 promoter activity, LPA1 mRNA expression, and LPA1-dependent chemotaxis and tumor cell invasion. Knockdown of the TGFβ intracellular effector Smad3 or Smad4 with lentivirally transduced shRNA relieved these inhibitory effects of TGFβ. Interestingly, the LPA1 promoter contains two potential TGFβ inhibitory elements (TIEs), each consisting of a Smad binding site and an adjacent E2F4/5 element, structurally similar to the TIE found on the promoter of the well-defined TGFβ target gene c-myc. Deletion and point mutation analyses indicate that the distal TIE located at 401 bp from the transcription initiation site, is required for TGFβ repression of the LPA1 promoter. A DNA pull-down assay showed that the -401 TIE was capable of binding Samd3 and E2F4 in TGFβ-treated cells. TGFβ-induced binding of the Smad complex to the native -401 TIE sequence of the LPA1 gene promoter was further verified by chromatin immunoprecipitation assays. We therefore identified a novel role of TGFβ in the control of LPA1 expression and LPA1-coupled biological functions, adding LPA1 to the list of TGFβ-repressed target genes.

Abstract 2958: The novel protein kinase STK17A is a direct p53 target gene that mediates response to genotoxic and nutritional stress in a cancer cell context-dependent manner

Abstract STK17A (DRAK1) is a largely uncharacterized serine/threonine kinase that belongs to the death-associated protein kinase family. Prior microarray studies demonstrated that STK17A is induced with cisplatin in testicular cancer-derived human embryonal carcinoma (EC) cells. We now demonstrate the STK17A in a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner in a number of different cell contexts. A consensus and functional p53 response element was found upstream of the STK17A promoter and endogenous p53 was shown to bind to this site in a cisplatin-dependent manner. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with up-regulation of detoxifying and antioxidant genes and reduction in reactive oxygen species (ROS), whereas overexpression of STK17A increased ROS levels in EC cells. Interestingly, we found that STK17A is highly overexpressed in clinical glioblastoma (GBM) and cell lines compared to its expression in normal brain tissue and other cancers. High STK17A expression in GBM patients correlated with poor clinical outcome and decreased survival. STK17A knockdown in GBM cells decreased clonagenic cell growth, soft agar tumorigenicity, and survival in response to genotoxic and nutritional stress. Preliminary data will be presented implicating STK17A in the regulation of autophagy and the proximal autophagy component, ULK1. These findings indicate that STK17A may be added to a growing list of direct p53 target genes involved in autophagy that includes TIGAR, Sestrins, DAPK1 and DRAM and that by impacting autophagy STK17A may modulate response to genotoxic and nutritional stress in a cell context-depend manner. This study also suggests that STK17A is a potential novel prognostic maker and therapeutic target for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2958. doi:1538-7445.AM2012-2958

Abstract 2289: miR-497 targets cell cycle regulator WEE1 in neuroblastoma

Abstract Neuroblastoma (NB) is a pediatric cancer that originates from precursor cells of the sympathetic nervous system. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor, depending on patient age, histopathology and genetic subtype. Low expression of miR-497 was identified as being significantly (p<0.015) associated with poor event free and overall NB patient survival, indicating a potential tumor suppressor role (Bray et al., 2009). Furthermore, down-regulation of miR-497 has been demonstrated in a number of multi-drug resistant cancer cell lines. Here we present our findings on the functional effects of exogenous miR-497 expression in NB cell lines, and we identify WEE1 as a direct target of miR-497. Ectopic over-expression of miR-497 resulted in significantly decreased cell viability for MYCN amplified (MNA) Kelly cells (p<0.04) and NB1691 cells (p<0.03) (MTS assay), indicating that this miRNA must be targeting mRNA sequences from gene(s) with significant anti-proliferative effects. We examined the list of putative target genes generated by the TargetScan miRNA Target prediction algorithm and determined that, WEE1, a tyrosine kinase with a key role as an inhibitory regulator of the G2/M checkpoint, is a predicted target of miR-497. Expression analysis from our cohort of NB patients (n=47) identified that higher expression of WEE1 was significantly associated with poor overall survival and event free survival (p<0.03). In silico analysis (web based R2 microarray analysis platform from the Academic Medical Center (www.r2.humangenetics-amc.nl)) from a large independent data set (n=283) supports this association of WEE1 with less favourable outcome in NB (p<0.0001). miR-497 expression was found to be inversely correlated with WEE1 in the tumor cohort, consistent with direct targeting. Ectopic over-expression of miR-497 did not affect WEE1 mRNA levels but resulted in reduced WEE1 protein levels in both Kelly and NB1691 cells. Direct targeting of the 3′UTR of WEE1 by miR-497 was validated using luciferase reporter assays (p<0.002). In order to elucidate the mechanism of miR-497 down-regulation in poor prognosis tumors, we examined methylation DNA immunoprecipitation (MeDIP) data from 15 primary NB tumors and identified a hypermethylated site 166 bp up-stream of the miR-497. There was a significant inverse correlation between methylation at this site and levels of miR-497 expression. The miRNA methylation status was validated using bisulphite sequencing, and we also determined that miR-497 expression could be significantly increased following treatment of NB cell lines with the de-methylating agent 5-aza-cytidine. We conclude that miR-497 is down-regulated in neuroblastoma by hypermethylation and that functional studies indicate a tumor suppressive function for this miRNA, potentially through direct targeting of the G2/M checkpoint tyrosine kinase WEE1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2289. doi:1538-7445.AM2012-2289

Abstract 5310: Regulation of metastatic breast cancer by E2F transcription factors

Abstract Metastasis is the leading cause of mortality in breast cancer and the purpose of this study was to examine the role of the E2F transcription factors in mediating the dissemination of the primary cancer. E2F transcription factors have previously been demonstrated to play a critical role in regulation of cell cycle progression. However, in prior work we have demonstrated that gene expression signatures for the E2Fs have variable levels of activation in a heterogeneous tumor model. Using predictive bioinformatic methods to assess the probability of gene pathway activation, we have predicted variable roles for the E2Fs in key mouse models of breast cancer, including Myc, PyMT and Neu. We then genetically tested these computational predictions by interbreeding E2F knockout mice to the various transgenic tumor models. These crosses have revealed that the E2Fs have unique roles in tumor initiation and progression depending upon the oncogene driving the initial tumor. There are differential effects on tumor latency with the loss of individual E2F alleles, including decreased latency with E2F2 loss in the PyMT background but increased latency and reduced incidence in the Myc background. Moreover, we have observed key differences in tumor progression, with loss of E2Fs affecting the heterogeneity of the resulting tumors. In addition to tumor initiation, we have observed striking differences in tumor progression with loss of E2Fs increasing metastasis in the Myc model but reducing metastasis in the PyMT model. In order to determine the mechanism of action for these effects, we have undertaken a gene expression analysis from both primary tumors and the metastases. Comparison of tumor models in the various backgrounds has allowed us to generate a list of E2F target genes that have effects on metastasis. Comparison of these genes to human breast cancer has revealed that the key genes also have critical roles in metastatic human breast cancer survival. These E2F targets include genes that have previously been identified as metastasis suppressors and are being validated in this process. Taken together, this work suggests a critical role for the E2F transcription factors, not only as mediators of cell cycle but in regulating the metastatic progression of metastatic breast cancer. These data also illustrate the power of combining various genomic and gene expression analysis techniques with both mouse models and clinical data associated with gene expression analysis in human breast cancer. Using this approach we are able to predict and test for the role of critical pathways, such as the E2Fs, in the progression of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5310. doi:1538-7445.AM2012-5310

A Systems Biology Approach towards Deciphering the Unfolded Protein Response in Huntington’s Disease

Although the disease causing gene huntingtin has been known for some time, the exact cause of neuronal cell death during Huntington's disease (HD) remains unknown. One potential mechanism contributing to the massive loss of neurons in HD brains might be the Unfolded Protein Response (UPR) which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). As an adaptive response, UPR upregulates transcription of chaperones, temporarily attenuating new translation and activates protein degradation via the proteasome. However, at high levels of ER stress, UPR signalling can contribute to neuronal apoptosis.Our primary aims include (a) construction of the UPR signalling network, (b) curation and bioinformatical identification of UPR target genes and finally (c) examination of HD gene expression data sets for UPR transcriptional signatures and differential regulation of UPR pathways.The UPR signalling pathway is reconstructed based on literature review and using the "Unified Interactome database":http://www.unihi.org. Lists of UPR target genes detected by previous experiments or as predicted by computational analysis are compiled. This allows us to perform enrichment analysis for differential HD gene expression and to assess whether UPR expression signatures are prominent during HD pathogenesis. The canonical UPR pathway is complemented with additional protein interaction data allowing us to assess its embedding into the cellular context and to identify potential modifiers as well as novel drug targets. The in depth systems biology analysis can give us valuable insights about the involvement of the UPR in HD.

A Systems Biology Approach towards Deciphering the Unfolded Protein Response in Huntington's Disease

AbstractAlthough the disease causing gene huntingtin has been known for some time, the exact cause of neuronal cell death during Huntington's disease (HD) remains unknown. One potential mechanism contributing to the massive loss of neurons in HD brains might be the Unfolded Protein Response (UPR) which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). As an adaptive response, UPR upregulates transcription of chaperones, temporarily attenuating new translation and activates protein degradation via the proteasome. However, at high levels of ER stress, UPR signalling can contribute to neuronal apoptosis.Our primary aims include (a) construction of the UPR signalling network, (b) curation and bioinformatical identification of UPR target genes and finally (c) examination of HD gene expression data sets for UPR transcriptional signatures and differential regulation of UPR pathways.The UPR signalling pathway is reconstructed based on literature review and using the "Unified Interactome database":http://www.unihi.org. Lists of UPR target genes detected by previous experiments or as predicted by computational analysis are compiled. This allows us to perform enrichment analysis for differential HD gene expression and to assess whether UPR expression signatures are prominent during HD pathogenesis. Results: The canonical UPR pathway is complemented with additional protein interaction data allowing us to assess its embedding into the cellular context and to identify potential modifiers as well as novel drug targets. Conclusions: The in depth systems biology analysis can give us valuable insights about the involvement of the UPR in HD.

An in silicoanalysis of dynamic changes in microRNA expression profiles in stepwise development of nasopharyngeal carcinoma

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA (mRNA) and protein synthesis. Recent studies have shown that some miRNAs are involved in the progression of nasopharyngeal carcinoma (NPC). However, the aberrant miRNAs implicated in different clinical stages of NPC remain unknown and their functions have not been systematically studied.MethodsIn this study, miRNA microarray assay was performed on biopsies from different clinical stages of NPC. TargetScan was used to predict the target genes of the miRNAs. The target gene list was narrowed down by searching the data from the UniGene database to identify the nasopharyngeal-specific genes. The data reduction strategy was used to overlay with nasopharyngeal-specifically expressed miRNA target genes and complementary DNA (cDNA) expression data. The selected target genes were analyzed in the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The microRNA-Gene-Network was build based on the interactions of miRNAs and target genes. miRNA promoters were analyzed for the transcription factor (TF) binding sites. UCSC Genome database was used to construct the TF-miRNAs interaction networks.ResultsForty-eight miRNAs with significant change were obtained by Multi-Class Dif. The most enriched GO terms in the predicted target genes of miRNA were cell proliferation, cell migration and cell matrix adhesion. KEGG analysis showed that target genes were significantly involved in adherens junction, cell adhesion molecules, p53 signalling pathway et al. Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-29a/c, miR-34b, miR-34c-3p, miR-34c-5p, miR-429, miR-203, miR-222, miR-1/206, miR-141, miR-18a/b, miR-544, miR-205 and miR-149 may play important roles on the development of NPC. We proposed an integrative strategy for identifying the miRNA-mRNA regulatory modules and TF-miRNA regulatory networks. TF including ETS2, MYB, Sp1, KLF6, NFE2, PCBP1 and TMEM54 exert regulatory functions on the miRNA expression.ConclusionsThis study provides perspective on the microRNA expression during the development of NPC. It revealed the global trends in miRNA interactome in NPC. It concluded that miRNAs might play important regulatory roles through the target genes and transcription factors in the stepwise development of NPC.

P5-04-03: Personalized Cancer Treatment Selection Using Computational Signaling Pathway Models.

Abstract Introduction: In clinical oncology, with breast cancer in the front line, - cancer genome sequencing is entering clinical diagnostics; - novel drugs target signaling pathways that drive tumor growth; - metastasized cancer is expected to change into a chronic disease, requiring therapy response monitoring and repeated personalized choice of targeted therapy. Hence, there is a need to clinically interpret available genomic data in order to diagnose which signaling pathway is driving tumor growth in a patient, including the causative mutation, such that appropriate targeted drugs can be chosen. Currently available pathway analysis tools and databases are not suitable for this. Companion diagnostics: To address the above need, we are developing computational models of signaling pathways, which can be used as companion diagnostic tools to translate genomic data into meaningful clinical information to enable selection of targeted drugs. The aim is to develop such models for the 10 to 20 most relevant oncogenic pathways, and use them to predict which one is most likely to drive tumor growth in an individual patient, including the probable underlying genomic defect. A model of the Wnt pathway: As a proof of concept, we have built a computational model of the Wnt pathway, which is active in colon adenoma and a main player in colon cancer, but also relevant in breast cancer. Our first generation model of the Wnt pathway covers its transcriptional program, for which we carefully selected a list of Wnt target genes. We have modeled this pathway by a Bayesian network, which interprets the expression levels of the target genes (from Affymetrix U133Plus2.0 arrays), and infers a probability that the Wnt pathway is active in a certain patient tumor sample. The parameters of the model are partly derived from literature and partly fitted on experimental data. Results: We first fitted a model on data from six pairs of Wnt knockdown experiments on colon cancer cell line LS174T, and tested it on a public set of 32 normal colon samples plus 32 colon adenomas from patients (GSE8671). This model perfectly predicted no Wnt activity in the normal samples and an active Wnt pathway in the adenomas. Next, we fitted the model on these 64 patient samples, and tested it on a set of 145 normal colon and colon cancer samples from patients (GSE20916). The model again predicted no Wnt activity in all 44 normal samples, and an active Wnt pathway in all cancer samples, except for four of the 36 samples from surgically removed colon carcinomas, which most likely are the most heterogeneous. Thirdly, we took the latter model, trained on colon samples, and tested it on three breast cancer data sets, one with 51 breast cancer cell lines (GSE12777) and two sets of breast cancer samples from patients (GSE12276, n = 204; GSE21653, n = 266). Also on this different tissue type, our model correctly predicted Wnt activity for the few breast cancer cell lines that are known to have an active Wnt pathway. For the two patient studies, a significantly higher number of samples of the basal subtype were predicted to have an active Wnt pathway compared to the other subtypes (Fisher's exact test: p = 0.021 for GSE12276; p = 2.7e-5 for GSE21653), in line with increasing evidence for Wnt activation in the basal subtype. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-04-03.

TIP: A probabilistic method for identifying transcription factor target genes from ChIP-seq binding profiles

ChIP-seq and ChIP-chip experiments have been widely used to identify transcription factor (TF) binding sites and target genes. Conventionally, a fairly 'simple' approach is employed for target gene identification e.g. finding genes with binding sites within 2 kb of a transcription start site (TSS). However, this does not take into account the number of sites upstream of the TSS, their exact positioning or the fact that different TFs appear to act at different characteristic distances from the TSS. Here we propose a probabilistic model called target identification from profiles (TIP) that quantitatively measures the regulatory relationships between TFs and target genes. For each TF, our model builds a characteristic, averaged profile of binding around the TSS and then uses this to weight the sites associated with a given gene, providing a continuous-valued 'regulatory' score relating each TF and potential target. Moreover, the score can readily be turned into a ranked list of target genes and an estimate of significance, which is useful for case-dependent downstream analysis. We show the advantages of TIP by comparing it to the 'simple' approach on several representative datasets, using motif occurrence and relationship to knock-out experiments as metrics of validation. Moreover, we show that the probabilistic model is not as sensitive to various experimental parameters (including sequencing depth and peak-calling method) as the simple approach; in fact, the lesser dependence on sequencing depth potentially utilizes the result of a ChIP-seq experiment in a more 'cost-effective' manner. mark.gerstein@yale.edu Supplementary data are available at Bioinformatics online.

Integrative nucleophosmin mutation-associated microRNA and gene expression pattern analysis identifies novel microRNA - target gene interactions in acute myeloid leukemia

MicroRNAs are regulators of gene expression, which act mainly by decreasing mRNA levels of their multiple targets. Deregulated microRNA expression has been shown for acute myeloid leukemia, a disease also characterized by altered gene expression associated with distinct genomic aberrations such as nucleophosmin (NPM1) mutations. To shed further light on the role of deregulated microRNA and gene expression in cytogenetically normal acute myeloid leukemia with NPM1 mutation we performed an integrative analysis of microRNA and mRNA expression data sets. Both microRNA and gene expression profiles were investigated in samples from a cohort of adult cytogenetically normal acute myeloid leukemia patients (n=43; median age 46 years, range 23-60 years) with known NPM1 mutation status (n=23 mutated, n=20 wild-type) and the data were integratively analyzed. Putative microRNA-mRNA interactions were validated by quantitative reverse transcriptase polymerase chain reaction, western blotting and luciferase reporter assays. For selected microRNAs, sensitivity of microRNA-overexpressing cells to cytarabine treatment was tested by FACS viability and cell proliferation assays. Our integrative approach of analyzing both microRNA- and gene expression profiles in parallel resulted in a refined list of putative target genes affected by NPM1 mutation-associated microRNA deregulation. Of 177 putative microRNA - target mRNA interactions we identified and validated 77 novel candidates with known or potential involvement in leukemogenesis, such as IRF2-miR-20a, KIT-miR-20a and MN1-miR-15a. Furthermore, our data showed that deregulated expression of tumor suppressor microRNAs, such as miR-29a and miR-30c, might contribute to sensitivity to cytarabine, which is observed in NPM1 mutated acute myeloid leukemia. Overall, our observations highlight that integrative data analysis approaches can improve insights into leukemia biology, and lead to the identification of novel microRNA - target gene interactions of potential relevance for acute myeloid leukemia treatment.

Étude de la voie des rétinoïdes au sein des membranes fœtales humaines : mise en évidence de gènes cibles

Retinoids (active derivatives of vitamin A) were already demonstrated to be important morphogenes and their implication at the placental and fetal level was already established. A new field of research is now developed in order to show their role on fetal membranes constituted by amnion and chorion. To describe the role of retinoids on these membranes, our studies were focused on target gene research. Firstly, all metabolism enzymes needed to vitamin A pathways were demonstrated to be present and active in signal transduction. Secondly, a bioinformatic analysis was performed to assess a list of potential target genes that could be classified in different biological pathways (inflammation, retinoids, hormones, vascularization, extracellular matrix and water homeostasis). Then, it was demonstrated that the gene coding for PLAT, implied in the degradation of extracellular matrix during programmed or premature rupture of membranes, is regulated by retinoids in a two steps mechanism. Finally, preliminary data showed that some aquaporins, which control water transport across membranes, are expressed and regulated by retinoids in the fetal membranes. A disregulation in pathologies like oligo or poly-hydramnios can be anticipated. Improvement of our knowledge about the retinoid implications is a key point in order to obtain a precise and complete documented cartography of the vitamin A (regulating) in amniotic membranes (regulated) that will permit the development of new diagnostic and therapeutic strategies.

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

Abstract 928: Species and cell type independent core MYC target genes in cancer and stem cells

Abstract The c-Myc (hereafter Myc) transcription factor augments the induction of pluripotency of somatic cells and contributes to human tumorigenesis, but the mechanisms by which Myc mediates these phenotypes remain only partially understood. To decipher Myc's biological functions, it is essential to define its target genes. To date, numerous Myc target gene lists have been constructed in various cellular contexts from several species but no study has rigorously defined species and cell type independent core Myc targets. Using a model of human B lymphoma cells bearing inducible MYC, we identified direct Myc target genes via chromatin immunoprecipitation (ChIP), global nuclear run-on assay, and changes in mRNA levels. We also identified, for the first time, direct Myc targets in human embryonic stem cells (ESCs). Here we report that Myc regulates a core set of target genes that are shared in mouse and human ESCs as well as in four other human cancer cell types. The expression of this gene set is increased in Myc-expressing premalignant B lymphocytes and is highly elevated in lymphoma cells from Eμ-Myc transgenic mice. Remarkably, the expression of the core gene set significantly correlates with MYC expression across 8,129 microarray samples in Gene Expression Omnibus (GEO), which represent 312 cell and tissue types. Expression profiles of B cell lymphomas, acute leukemia, lung cancers and Ewing sarcomas are among those with the highest correlation. Annotation of this gene signature highlights Myc's primordial function in ribosomal biogenesis as one of its key roles in cancer and stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 928. doi:10.1158/1538-7445.AM2011-928

Microarray analysis of microRNA expression in LPS induced inflammation of human middle ear epithelial cells (HMEECs)

Objectives The primary aim of this study was to reveal the relationship between inflammatory response of human middle ear epithelial cell (HMEEC) and microRNA (miRNA). Methods In experimental group, cells were treated with lipopolysaccharide (LPS) for 2 h. No LPS was treated in the control group. Total RNA was extractedand used for miRNA microarray analysis. The predicted targets of miRNA with significant change were obtained using miRBase. To assess the function of predicted target gene lists, we evaluated the frequency of specific gene ontology (GO) terms among the predicted target genes of the miRNA with significant change using DAVID (Database for Annotation, Visualization and Integrated Discovery). Results After normalization, the number of the differentially expressed genes was 15. Among them, 5 miRNAs were up-regulated and 10 miRNAs were down-regulated in LPS group compared with control group. The most enriched GO terms in the predicted target genes of miRNA with increased expression were developmental process, response to biotic stimulus, acute inflammatory response, and regulation of cell growth. The most enriched GO terms in the predicted target genes of miRNA with decreased expression were developmental process, cell differentiation, endocytosis, cell communication, IκB kinase/NFκB cascade, complement activation, innate immune response and cell adhesion. Conclusions In conclusion, we identified the differentially expressed miRNA in LPS induced acute inflammation of HMEECs whose expression profile may provide a useful clue for the understanding of pathophysiology of otitis media. Our work indicates that miRNA play important role in the pathogenesis of otitis media.

High responders to resistance exercise training demonstrate differential regulation of skeletal muscle microRNA expression

MicroRNAs (miRNA), small noncoding RNA molecules, may regulate protein synthesis, while resistance exercise training (RT) is an efficient strategy for stimulating muscle protein synthesis in vivo. However, RT increases muscle mass, with a very wide range of effectiveness in humans. We therefore determined the expression level of 21 abundant miRNAs to determine whether variation in these miRNAs was able to explain the variation in RT-induced gains in muscle mass. Vastus lateralis biopsies were obtained from the top and bottom ∼20% of responders from 56 young men who undertook a 5 day/wk RT program for 12 wk. Training-induced muscle mass gain was determined by dual-energy X-ray absorptiometry, and fiber size was evaluated by histochemistry. The expression level of each miRNA was quantified using TaqMan-based quantitative PCR, with the analysis carried out in a blinded manner. Gene ontology and target gene profiling were used to predict the potential biological implications. Of the 21 mature miRNAs examined, 17 were stable during RT in both groups. However, miR-378, miR-29a, miR-26a, and miR-451 were differentially expressed between low and high responders. miR-378, miR-29a, and miR-26a were downregulated in low responders and unchanged in high responders, while miR-451 was upregulated only in low responders. Interestingly, the training-induced change in miR-378 abundance was positively correlated with muscle mass gains in vivo. Gene ontology analysis of the target gene list of miR-378, miR-29a, miR-26a, and miR-451, from the weighted cumulative context ranking methodology, indicated that miRNA changes in the low responders may be compensatory, reflecting a failure to "activate" growth and remodeling genes. We report, for the first time, that RT-induced hypertrophy in human skeletal muscle is associated with selected changes in miRNA abundance. Our analysis indicates that miRNAs may play a role in the phenotypic change and pronounced intergroup variation in the RT response.

HnRNP A2/B1 Modulates Epithelial-Mesenchymal Transition in Lung Cancer Cell Lines

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been reported to be overexpressed in lung cancer and in other cancers such as breast, pancreas, and liver. However, a mechanism linking hnRNP A2/B1 overexpression and progression to cancer has not yet been definitively established. To elucidate this mechanism, we have silenced hnRNPA2/B1 mRNA in non-small-cell lung cancer cell lines A549, H1703, and H358. These cell lines present different levels of expression of epithelial-to-mesenchymal transition (EMT) markers such as E-cadherin, fibronectin, and vimentin. Microarray expression analysis was performed to evaluate the effect of silencing hnRNP A2/B1 in A549 cells. We identified a list of target genes, affected by silencing of hnRNP A2/B1, that are involved in regulation of migration, proliferation, survival, and apoptosis. Silencing hnRNP A2/B1 induced formation of cell clusters and increased proliferation. In the anchorage-independent assay, silencing hnRNP A2/B1 increased colony formation by 794% in A549 and 174% in H1703 compared with a 25% increase in proliferation, in both cell lines, in a two-dimensional proliferation assay. Silencing hnRNP A2/B1 decreased migration in intermediate cell line A549 and mesenchymal cell line H1703; however, no changes in proliferation were observed in epithelial cell line H358. Silencing hnRNP A2/B1 in A549 and H1703 cells correlated with an increase of E-cadherin expression and downregulation of the E-cadherin inhibitors Twist1 and Snai1. These data suggest that expression of hnRNP A2/B1 may play a role in EMT, in nonepithelial lung cancer cell lines A549 and H1703, through the regulation of E-cadherin expression.

High resolution models of transcription factor-DNA affinities improve in vitro and in vivo binding predictions.

Accurately modeling the DNA sequence preferences of transcription factors (TFs), and using these models to predict in vivo genomic binding sites for TFs, are key pieces in deciphering the regulatory code. These efforts have been frustrated by the limited availability and accuracy of TF binding site motifs, usually represented as position-specific scoring matrices (PSSMs), which may match large numbers of sites and produce an unreliable list of target genes. Recently, protein binding microarray (PBM) experiments have emerged as a new source of high resolution data on in vitro TF binding specificities. PBM data has been analyzed either by estimating PSSMs or via rank statistics on probe intensities, so that individual sequence patterns are assigned enrichment scores (E-scores). This representation is informative but unwieldy because every TF is assigned a list of thousands of scored sequence patterns. Meanwhile, high-resolution in vivo TF occupancy data from ChIP-seq experiments is also increasingly available. We have developed a flexible discriminative framework for learning TF binding preferences from high resolution in vitro and in vivo data. We first trained support vector regression (SVR) models on PBM data to learn the mapping from probe sequences to binding intensities. We used a novel -mer based string kernel called the di-mismatch kernel to represent probe sequence similarities. The SVR models are more compact than E-scores, more expressive than PSSMs, and can be readily used to scan genomics regions to predict in vivo occupancy. Using a large data set of yeast and mouse TFs, we found that our SVR models can better predict probe intensity than the E-score method or PBM-derived PSSMs. Moreover, by using SVRs to score yeast, mouse, and human genomic regions, we were better able to predict genomic occupancy as measured by ChIP-chip and ChIP-seq experiments. Finally, we found that by training kernel-based models directly on ChIP-seq data, we greatly improved in vivo occupancy prediction, and by comparing a TF's in vitro and in vivo models, we could identify cofactors and disambiguate direct and indirect binding.

A32 A systems biology approach towards deciphering the unfolded protein response in Huntington's disease

BackgroundAlthough the disease causing gene huntingtin has been known for some time, the exact cause of neuronal cell death during Huntington's disease (HD) remains unknown. One potential mechanism contributing to...

Prediction of regulatory networks in mouse abdominal wall

Sequence specific transcription factors are essential for pattern formation and cell differentiation processes in mammals. The formation of the abdominal wall depends on a flawless merge of several developmental fields in time and space. The absence of Pitx2 leads to an open abdominal wall in mice, while mutations in humans result in umbilical defects, suggesting that a single homeobox transcription factor coordinates the formation and patterning of this anatomical structure. Gene expression analysis from abdominal tissue including the abdominal wall after removal of the major organs, of wild type, Pitx2 heterozygote and mutant mice, at embryonic day 10.5, identified 275 genes with altered expression levels. Pitx2 target genes were clustered using the “David Bioinformatics Functional Annotation Tool” web application, which bins genes according to gene ontology (GO) key word enrichment. This provided a way to both narrow the target gene list and to start identifying potential gene families regulated by Pitx2. Target genes in the most enriched bins were further analyzed for the presence and the evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the − 20 kb, intronic and coding gene sequences. Twenty Pitx2 target genes that passed all the above criteria were classified as genes involved in cell transport and growth. Data from these studies suggest that Pitx2 acts as an inhibitor of protein transport and cell apoptosis contributing to the open body wall phenotype. This work provides the framework to which the developmental network leading to abdominal wall syndromes can be built.