RNA sequencing of Sleeping Beauty transposon-induced tumors detects transposon-RNA fusions in forward genetic cancer screens

Abstract

Forward genetic screens using Sleeping Beauty (SB)-mobilized T2/Onc transposons have been used to identify common insertion sites (CISs) associated with tumor formation. Recurrent sites of transposon insertion are commonly identified using ligation-mediated PCR (LM-PCR). Here, we use RNA sequencing (RNA-seq) data to directly identify transcriptional events mediated by T2/Onc. Surprisingly, the majority (∼80%) of LM-PCR identified junction fragments do not lead to observable changes in RNA transcripts. However, in CIS regions, direct transcriptional effects of transposon insertions are observed. We developed an automated method to systematically identify T2/Onc-genome RNA fusion sequences in RNA-seq data. RNA fusion-based CISs were identified corresponding to both DNA-based CISs (Cdkn2a, Mycl1, Nf2, Pten, Sema6d, and Rere) and additional regions strongly associated with cancer that were not observed by LM-PCR (Myc, Akt1, Pth, Csf1r, Fgfr2, Wisp1, Map3k5, and Map4k3). In addition to calculating recurrent CISs, we also present complementary methods to identify potential driver events via determination of strongly supported fusions and fusions with large transcript level changes in the absence of multitumor recurrence. These methods independently identify CIS regions and also point to cancer-associated genes like Braf. We anticipate RNA-seq analyses of tumors from forward genetic screens will become an efficient tool to identify causal events.

Figure 1.

Transcription near CIS insertions. Image showing FPKM values for (A) Eras and (B) Nf2 for each of the 14 SB osteosarcoma (OS) tumors. An exon map of the gene region is shown indicating the direction of transcription below the FPKM bar plots. For each tumor, a histogram of the raw number of reads observed within each 100-bp window is shown. The locations of LM-PCR-identified insertions are shown with triangles. Blue triangles represent positive orientation insertions that can activate transcription on the + strand, while green triangles represent negative orientation insertions that can activate transcription on the − strand. RNA fusions identified in the RNA-seq data are described in the figure legend (bottom right).

Figure 2.

Mapping to a modified mm9 genome. (A) IGV view of Eras genomic region from a tumor containing a transposon insertion. (B) Within the Eras transcript Exon 2, many unmapped read pairs were observed in the regions of fusions following mapping to the standard mm9 genome. (C) Remapping to the mm9 genome with added T2/Onc sequence reveals the presence of T2/Onc-genome fusion sequences. Many of the previously unmapped pairs now map to the T2/Onc sequence.

Figure 3.

Location of fusion within T2/Onc sequence indicates transposon mechanism of transcriptional alteration. (A) Map of a single copy of the T2/Onc transposon showing the locations of the various fusion products. The location of the fusion pair mapping to T2/Onc indicates how T2/Onc is modifying the transcript in order to generate the observed fusion event. (B) Transcriptional activation within the Capzb gene. (C) Transcriptional activation of the opposite strand of the Capzb gene. (D) Transcriptional activation on the opposite strand with apparent splicing in the Rere gene. (E) Premature truncation in Abca1. (F) Premature truncation and transcriptional activation happening at the same time in Abca1. Locations of LM-PCR junction fragments and fusion products are shown as described for Figure 1.

Figure 4.

Interplay between sequencing depth, LM-PCR depth, and RNA fusions. (A) Total number of fusions observed in each sample plotted against the sequencing depth. (B) The number of fragments in six tumors using the shLM-PCR technique, ordered in increasing number of fragments which is a semiquantitative indication of clonality and the number of fusions which support these shLM-PCR junction fragments. (C) Relationship between shLM-PCR number of supporting fragments and RNA fusion support. As more supporting shear fragments are observed, the percentage of insertions with observed fusions increases.

Figure 5.

Precise identification of CIS transposon targeting. Histograms, similar to Figure 1, allow precise identification of the molecular mechanism of insertion in CIS near (A) Ammecr1 and Rgag1, and (B) Cdkn2a. Locations of LM-PCR junction fragments and fusion products are shown as described for Figure 1.

Figure 6.

Insertion-modified transcripts (IMTs) in osteosarcoma tumors. Examples of increased IMTs via transcriptional activation for (A) Cngb3, (B) Poln, and (C) Braf, and for decreased IMTs with apparent activation of opposite strand transcript (D) Cdc42, (E) Xpo4, and (F) Max. FPKM, locations of reLM-PCR junction fragments and fusion products are shown as described for Figure 1.