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Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

ABSTRACT: RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. Integration of copy number profiling with the RNA-sequencing results revealed that the majority of the gene fusions were promoter-donating events that occurred at copy number transition points or involved high-level DNA-amplifications. Sequencing of genomic fusion break points confirmed that DNA-level rearrangements underlie selected fusion transcripts. Furthermore, a significant portion (>60%) of the fusion genes were alternatively spliced. This illustrates the importance of reanalyzing sequencing data as gene definitions change and bioinformatic methods improve, and highlights the previously unforeseen isoform diversity among fusion transcripts.

SUBMITTER: Kangaspeska S

PROVIDER: S-EPMC3485361 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

Kangaspeska Sara S Hultsch Susanne S Edgren Henrik H Nicorici Daniel D Murumägi Astrid A Kallioniemi Olli O

PloS one 20121031 10

RNA-sequencing and tailored bioinformatic methodologies have paved the way for identification of expressed fusion genes from the chaotic genomes of solid tumors. We have recently successfully exploited RNA-sequencing for the discovery of 24 novel fusion genes in breast cancer. Here, we demonstrate the importance of continuous optimization of the bioinformatic methodology for this purpose, and report the discovery and experimental validation of 13 additional fusion genes from the same samples. In ...[more]

PMID: 23119097

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Project description:In the present study, the RNA sequencing (RNA-seq) data of uterine corpus endometrial carcinoma (UCEC) samples were collected and analyzed using bioinformatics tools to identify potential genes associated with the development of UCEC. UCEC RNA-seq data were downloaded from The Cancer Genome Atlas database. Differential analysis was performed using edgeR software. A false discovery rate <0.01 and |log2(fold change)|>1 were set as the cut-off criteria to screen for differentially expressed genes (DEGs). Differential gene co-expression analysis was performed using R/EBcoexpress package in R. DEGs in the gene co-expression network were subjected to Gene Ontology analysis using the Database for Annotation, Visualization and Integration Discovery. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was also performed on the DEGs using KOBAS 2.0 software. The ConnectivityMap database was used to identify novel drug candidates. A total of 3,742 DEGs were identified among the 552 UCEC samples and 35 normal controls, and comprised 2,580 upregulated and 1,162 downregulated genes. A gene co-expression network consisting of 129 DEGs and 368 edges was constructed. Genes were associated with the cell cycle and the tumor protein p53 signaling pathway. Three modules were identified, in which genes were associated with the mitotic cell cycle, nuclear division and the M phase of the mitotic cell cycle. Multiple key hub genes were identified, including cell division cycle 20, cyclin B2, non-SMC condensin I complex subunit H, BUB1 mitotic checkpoint serine/threonine kinase, cell division cycle associated 8, maternal embryonic leucine zipper kinase, MYB proto-oncogene like 2, TPX2, microtubule nucleation factor and non-SMC condensin I complex subunit G. In addition, the small molecule drug esculetin was implicated in the suppression of UCEC progression. Overall, the present study identified multiple key genes in UCEC and clinically relevant small molecule agents, thereby improving our understanding of UCEC and expanding perspectives on targeted therapy for this type of cancer.

Project description:BACKGROUND:The calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in Candida albicans, but it has not been experimentally demonstrated. METHODS:An inactivation mutant for CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2?M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively. RESULTS:RNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5'-GGAGGC(G/A)C(T/A)G-3'], which is different from the putative CaCrz1-binding motif [5'-G(C/T)GGT-3'] identified in the previous study, but similar to Saccharomyces cerevisiae ScCrz1-binding motif [5'-GNGGC(G/T)CA-3']. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression. CONCLUSIONS:CaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5'-TGAGGGACTG-3' site in its promoter. Video abstract.

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Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

Publications

Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms.

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