Genome-wide identification of new Wnt/β-catenin target genes in the human genome using CART method

<p>Abstract</p> <p>Background</p> <p>The importance of <it>in silico </it>predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of <it>cis </it>regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/β-catenin pathway target genes potentially involved in brain diseases.</p> <p>Results</p> <p>In this study, we propose 89 new Wnt/β-catenin pathway target genes predicted <it>in silico </it>by using a method based on multiple Classification and Regression Tree (CART) analysis. We used as decision variables the presence of transcription factor binding site motifs in the upstream region of each gene. This prediction was validated by RT-qPCR in a sample of 9 genes. As expected, LEF1, a member of the T-cell factor/lymphoid enhancer-binding factor family (TCF/LEF1), was relevant for the classification algorithm and, remarkably, other factors related directly or indirectly to the inflammatory response and amyloidogenic processes also appeared to be relevant for the classification. Among the 89 new Wnt/β-catenin pathway targets, we found a group expressed in brain tissue that could be involved in diverse responses to neurodegenerative diseases, like Alzheimer's disease (AD). These genes represent new candidates to protect cells against amyloid β toxicity, in agreement with the proposed neuroprotective role of the Wnt signaling pathway.</p> <p>Conclusions</p> <p>Our multiple CART strategy proved to be an effective tool to identify new Wnt/β-catenin pathway targets based on the study of their regulatory regions in the human genome. In particular, several of these genes represent a new group of transcriptional dependent targets of the canonical Wnt pathway. The functions of these genes indicate that they are involved in pathophysiology related to Alzheimer's disease or other brain disorders.</p

Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during <it>Drosophila melanogaster </it>embryogenesis

<p>Abstract</p> <p>Background</p> <p>Morphogenetic events that shape the <it>Drosophila melanogaster </it>embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of <it>D. melanogaster </it>embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.</p> <p>Results</p> <p>Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. <it>In situ </it>hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in <it>Drosophila </it>S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction.</p> <p>Conclusion</p> <p>Our data have yielded a list of developmentally regulated <it>D. melanogaster </it>genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling <it>D. melanogaster </it>embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.</p> <p>See related minireview by Vichas and Zallen: <url>http://www.jbiol.com/content/8/8/76</url></p