Detecting and visualizing gene fusions.

In recent years, gene fusions have gained significant recognition as biomarkers. They can assist treatment decisions, are seldom found in normal tissue and are detectable through Next-generation sequencing (NGS) of the transcriptome (RNA-seq). To transform the data provided by the sequencer into robust gene fusion detection several analysis steps are needed. Usually the first step is to map the sequenced transcript fragments (RNA-seq) to a reference genome. One standard application of this approach is to estimate expression and detect variants within known genes, e.g. SNPs and indels. In case of gene fusions, however, completely novel gene structures have to be detected. Here, we describe the detection of such gene fusion events based on our comprehensive transcript annotation (ElDorado). To demonstrate the utility of our approach, we extract gene fusion candidates from eight breast cancer cell lines, which we compare to experimentally verified gene fusions. We discuss several gene fusion events, like BCAS3-BCAS4 that was only detected in the breast cancer cell line MCF7. As supporting evidence we show that gene fusions occur more frequently in copy number enriched regions (CNV analysis). In addition, we present the Transcriptome Viewer (TViewer) a tool that allows to interactively visualize gene fusions. Finally, we support detected gene fusions through literature mining based annotations and network analyses. In conclusion, we present a platform that allows detecting gene fusions and supporting them through literature knowledge as well as rich visualization capabilities. This enables scientists to better understand molecular processes, biological functions and disease associations, which will ultimately lead to better biomedical knowledge for the development of biomarkers for diagnostics and therapies.

RNA-sequencing as useful screening tool in the combat against the misuse of anabolic agents.

The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the intended information from these regulated genes, biostatistical tools for pattern recognition were applied and resulted in a clear separation of the treatment groups. Those candidate genes could be verified in boars and in calves treated with anabolic substances. These results show the potential of RNA sequencing to screen for biomarker candidates to detect the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the assumption that gene expression biomarkers are independent of breed or even species and that biomarkers, identified in farm animals could also act as potential biomarker candidates to detect the abuse of anabolic substances in human sports.

SFTA2--a novel secretory peptide highly expressed in the lung--is modulated by lipopolysaccharide but not hyperoxia.

Tissue-specific transcripts are likely to be of importance for the corresponding organ. While attempting to define the specific transcriptome of the human lung, we identified the transcript of a yet uncharacterized protein, SFTA2. In silico analyses, biochemical methods, fluorescence imaging and animal challenge experiments were employed to characterize SFTA2. Human SFTA2 is located on Chr. 6p21.33, a disease-susceptibility locus for diffuse panbronchiolitis. RT-PCR verified the abundance of SFTA2-specific transcripts in human and mouse lung. SFTA2 is synthesized as a hydrophilic precursor releasing a 59 amino acid mature peptide after cleavage of an N-terminal secretory signal. SFTA2 has no recognizable homology to other proteins while orthologues are present in all mammals. SFTA2 is a glycosylated protein and specifically expressed in nonciliated bronchiolar epithelium and type II pneumocytes. In accordance with other hydrophilic surfactant proteins, SFTA2 did not colocalize with lamellar bodies but colocalized with golgin97 and clathrin-labelled vesicles, suggesting a classical secretory pathway for its expression and secretion. In the mouse lung, Sfta2 was significantly downregulated after induction of an inflammatory reaction by intratracheal lipopolysaccharides paralleling surfactant proteins B and C but not D. Hyperoxia, however, did not alter SFTA2 mRNA levels. We have characterized SFTA2 and present it as a novel unique secretory peptide highly expressed in the lung.

Estrogen induces repression of the breast cancer and salivary gland expression gene in an estrogen receptor alpha-dependent manner.

The focus of this study is on the expression and regulation of the estrogen-regulated breast cancer and salivary gland expression (BASE) gene that may function as a breast cancer marker. In MCF7 cells, BASE is repressed by estrogen in an estrogen receptor alpha (ER alpha)-dependent manner. Promoter analysis of the BASE gene led to the identification of a 2-kb upstream enhancer that harbors binding sites for ER alpha and FoxA1. The recruitment of both ER alpha and FoxA1 to this region was shown by chromatin immunoprecipitation analysis. Furthermore, mutation studies and knockdown experiments show a clear separation between gene expression mediated by FoxA1 and ER alpha-dependent gene regulation. Additionally, we provide information on BASE expression in human breast tumor samples.

E2-mediated cathepsin D (CTSD) activation involves looping of distal enhancer elements.

Estrogen receptor alpha (ERalpha) is a ligand dependent transcription factor that regulates the expression of target genes through interacting with cis-acting estrogen response elements (EREs). However, only a minority of ERalpha binding sites are located within the proximal promoter regions of responsive genes. Here we report the characterization of an ERE located 9kbp upstream of the TSS of the cathepsin D gene (CTSD) that up-regulates CTSD expression upon estrogen stimulation in MCF-7 cells. Using ChIP, we show recruitment of ERalpha and phosphorylated PolII at the CTSD distal enhancer region. Moreover, we determine the kinetics of transient CpG methylation on the promoter region of CTSD and for the first time, at a distal enhancer element. We show that ERalpha is crucial for long-distance regulation of CTSD expression involving a looping mechanism.

Genome-wide identification of high-affinity estrogen response elements in human and mouse.

Although estrogen receptors (ERs) recognize 15-bp palindromic estrogen response elements (EREs) with maximal affinity in vitro, few near-consensus sequences have been characterized in estrogen target genes. Here we report the design of a genome-wide screen for high-affinity EREs and the identification of approximately 70000 motifs in the human and mouse genomes. EREs are enriched in regions proximal to the transcriptional start sites, and approximately 1% of elements appear conserved in the flanking regions (-10 kb to +5 kb) of orthologous human and mouse genes. Conserved and nonconserved elements were also found, often in multiple occurrences, in more than 230 estrogen-stimulated human genes previously identified from expression studies. In genes containing known EREs, we also identified additional distal elements, sometimes with higher in vitro binding affinity and/or better conservation between the species considered. Chromatin immunoprecipitation experiments in breast cancer cell lines indicate that most novel elements present in responsive genes bind ERalpha in vivo, including some EREs located up to approximately 10 kb from transcriptional start sites. Our results demonstrate that near-consensus EREs occur frequently in both genomes and that whereas chromatin structure likely modulates access to binding sites, far upstream elements can be evolutionarily conserved and bind ERs in vivo.