Chimaeric Transcripts Research Articles

Abstract 2460: Characterizing PAX3-FOXO1 dependent cell death in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy

Abstract Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often by t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1. Expression of this chimaeric transcription factor is critical for tumorigenesis and cell survival. However, the exact mechanism of cell death after loss of PAX3-FOXO1 activity has not been determined so far. Our aim was thus to characterize the cell death pathways activated upon silencing of PAX3-FOXO1. In addition, we aimed at finding drugs further sensitizing aRMS cells to this mode of cell death. We used combined shRNA and CRISPR approaches, as well as a small molecule screen to demonstrate that after shRNA-mediated silencing of PAX3-FOXO1 expression, aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner. In accordance, we can show that the BH3-mimetic ABT-263 sensitizes aRMS cells to PAX3-FOXO1 silencing via this cell death pathway. Interestingly, ABT-199 was less effective, suggesting that Bcl-xl plays a major anti-apoptotic role in aRMS. Furthermore, induction of cell death is dependent on PI3K activity and antagonized by GSK3, suggesting that inhibition of PI3K in this sarcoma might have an anti-apoptotic impact. In contrast, combination of ABT-263 or GSK-3 inhibitors with small molecule drugs affecting PAX3-FOXO1 activity such as PLK1 and aurora kinase inhibitors can cooperate to enhance cell death in aRMS cells. These studies demonstrate the importance of elucidating biological mechanisms to guide development of rational drug combinations. Citation Format: Marco Wachtel, Johannes Ommer, Beat Schäfer. Characterizing PAX3-FOXO1 dependent cell death in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2460.

Abstract S4-02: A comprehensive analysis of fusion transcripts in breast cancer reveals associations between number of fusion transcripts, copy number events, gene expression profiles, and potentially clinical outcome

Abstract Fusion transcript 24analysis by five groups, each using "in house" analytical workflow to analyze RNA-seq data from 813 breast tumors from The Cancer Genome Atlas project detected 2514 high confidence RNA fusions (2 or more junction spanning reads detected by 2 or more groups). A subset of tumors with fusions were evaluated for chromosomal rearrangement using whole genome sequence data (WGS), and about half of the high confidence fusions could be validated, with the remainder likely subclonal and therefore below the limits of detection by WGS at the relatively low depth of coverage (30X) available. The majority of tumors contained one or more fusion transcript (range 0-24 fusions/tumor), but recurrence of specific chimaeric transcripts was low. Only ESR1->CCDC170, C20orf3->ACSS1, and USP22->MYH10 fusions were detected in 10 or more tumors. Additional ESR1 fusions were detected with AKAP12, BNC2, C6orf211, GNAS, POLH, USP25, and UTRN, within 19 tumors, primarily of the Luminal B subtype. RARA was the second most common 5' fusion partner with 17 tumors, predominately of the HER2-enriched subtype, expressing out of frame RARA fusions to 19 different 3' fusion partners. Other common 5' fusion partners included FBXL20, USP22, USP32, BCAS3, MED1, BPTF, ERBB2, MED24, and PPF1A1 (expressed in 10 or more tumors each). The most common 3' fusion partners were ERBB2, CCDC170, PITPNC1, ACSS1, MYH10, IKZF3, and RAB6A (10 or more tumors each). HER2-enriched tumors expressed significantly more fusions than other subtypes (4.9 fusions/tumor vs 3.3 for Basal-like, 1.3 for LumA, and 3.2 for LumB). Almost all HER2-enriched tumors expressed fusions (97%), whereas 85% of Basal-like tumors and 79% of Luminal B tumors expressed one or more fusions. Among Luminal A tumors, only 48% expressed one or more fusion transcript. Those Luminal A tumors with multiple fusion transcripts contained significantly more copy number aberrations and were enriched for expression of mitotic cell cycle genes (p=3.07E-18), characteristic of the recently described high risk CNH subclass of Luminal A tumors. We conclude that, consistent with other observations, breast tumors generally exhibit a high level of genomic instability, associated with multiple copy number alterations and multiple fusion transcripts. However, recurrence of specific fusion transcripts is low. About half of Luminal A tumors express fusion transcripts; and these tumors exhibit multiple copy number events, consistent with high level genomic instability, as well as enriched expression of mitotic cell cycle genes. These tumors appear to be related to the relatively high risk Luminal A CNH subclass, and express high levels of aurora kinases and polo-like kinases, which might be considered as therapeutic targets. Given the increasing emphasis on transcript profiling of clinical samples and the clinical significance of high risk Luminal A tumors, it may be timely to consider inclusion of fusion transcripts in such analyses as surrogate markers of genomic instability and potential therapeutic and/or prognostic indicators. Citation Format: Thompson EA, Asmann YW, Su X, Ellis MJ, Shao J, Hu Y, White KP, Cherniack AD, Hoadley KA, Serie DJ, Perez EA, Perou CM. A comprehensive analysis of fusion transcripts in breast cancer reveals associations between number of fusion transcripts, copy number events, gene expression profiles, and potentially clinical outcome. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-02.

Establishment of a recessive mutant small-eye rat with lens involution and retinal detachment associated with partial deletion and rearrangement of the Cryba1 gene.

From our stock of SDRs (Sprague-Dawley rats), we established a mutant strain having small opaque eyes and named it HiSER (Hirosaki small-eye rat). The HiSER phenotype is progressive and autosomal recessive. In HiSER eyes, disruption and involution of the lens, thickening of the inner nuclear layer, detachment and aggregation of the retina, rudimentary muscle in the ciliary body and cell infiltration in the vitreous humour were observed. Genetic linkage analysis using crossing with Brown Norway rat suggested that the causative gene(s) is located on chromosome 10. Microarray analysis showed that the expression level of the Cryba1 gene encoding βA3/A1-crystallin on chromosome 10 was markedly decreased in HiSER eyes. Genomic PCR revealed deletion of a 3.6-kb DNA region encompassing exons 4-6 of the gene in HiSERs. In HiSER eyes, a chimaeric transcript of the gene containing exons 1-3 and an approximately 250-bp sequence originating from the 3'-UTR of the Nufip2 gene, located downstream of the breakpoint in the opposite direction, was present. Whereas the chimaeric transcript was expressed in HiSER eyes, neither normal nor chimaeric βA3/A1-crystallin proteins were detected by Western blot analysis. Real-time RT (reverse transcription)-PCR analysis revealed that expression level of the Nufip2 gene in the HiSER eye was 40% of that in the SDR eye. These results suggest that the disappearance of the βA3/A1-crystallin protein and, in addition, down-regulation of the Nufip2 gene as a consequence of gene rearrangement causes the HiSER phenotype.

RNASeqBrowser: a genome browser for simultaneous visualization of raw strand specific RNAseq reads and UCSC genome browser custom tracks.

BackgroundStrand specific RNAseq data is now more common in RNAseq projects. Visualizing RNAseq data has become an important matter in Analysis of sequencing data. The most widely used visualization tool is the UCSC genome browser that introduced the custom track concept that enabled researchers to simultaneously visualize gene expression at a particular locus from multiple experiments. Our objective of the software tool is to provide friendly interface for visualization of RNAseq datasets.ResultsThis paper introduces a visualization tool (RNASeqBrowser) that incorporates and extends the functionality of the UCSC genome browser. For example, RNASeqBrowser simultaneously displays read coverage, SNPs, InDels and raw read tracks with other BED and wiggle tracks -- all being dynamically built from the BAM file. Paired reads are also connected in the browser to enable easier identification of novel exon/intron borders and chimaeric transcripts. Strand specific RNAseq data is also supported by RNASeqBrowser that displays reads above (positive strand transcript) or below (negative strand transcripts) a central line. Finally, RNASeqBrowser was designed for ease of use for users with few bioinformatic skills, and incorporates the features of many genome browsers into one platform.ConclusionsThe features of RNASeqBrowser: (1) RNASeqBrowser integrates UCSC genome browser and NGS visualization tools such as IGV. It extends the functionality of the UCSC genome browser by adding several new types of tracks to show NGS data such as individual raw reads, SNPs and InDels. (2) RNASeqBrowser can dynamically generate RNA secondary structure. It is useful for identifying non-coding RNA such as miRNA. (3) Overlaying NGS wiggle data is helpful in displaying differential expression and is simple to implement in RNASeqBrowser. (4) NGS data accumulates a lot of raw reads. Thus, RNASeqBrowser collapses exact duplicate reads to reduce visualization space. Normal PC’s can show many windows of NGS individual raw reads without much delay. (5) Multiple popup windows of individual raw reads provide users with more viewing space. This avoids existing approaches (such as IGV) which squeeze all raw reads into one window. This will be helpful for visualizing multiple datasets simultaneously.RNASeqBrowser and its manual are freely available at http://www.australianprostatecentre.org/research/software/rnaseqbrowser or http://sourceforge.net/projects/rnaseqbrowser/Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1346-2) contains supplementary material, which is available to authorized users.

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells.

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

Abstract 757: Identification of the interactome of PAX3/FOXO1 in alveolar rhabdomyosarcoma.

Abstract About one third of the known cellular oncogenes are transcription factors (TFs). As many tumors are dependent on continuous activity of these proteins they represent highly interesting targets for therapeutic approaches e.g. by inhibition with small molecule inhibitors. Because of lack of enzymatic activity and accompanied difficulty of direct targeting, TF have been considered undruggable for a long time. This view recently changed with the demonstration that, instead of direct targeting, indirect targeting of protein partners required for TF activity might represent an appropriate alternative. Among transcription factors targeted by this approach are Notch, Bcl6 and EWS/FLI, the oncogenic fusion protein in Ewing sarcoma. In our studies we focus on alveolar Rhabdomyosarcoma (aRMS), an aggressive and highly metastatic pediatric sarcoma. ARMS is characterised by the expression of the fusion TF PAX3/FOXO1 which is essential for the survival of aRMS cells. Here, we aimed to identify interacting proteins that are involved in modulating the aberrant activity of PAX3/FOXO1 in aRMS. Towards this end, we purified the interactome of PAX3/FOXO1 from two different aRMS cell lines and identified the individual interactors by mass spectrometry. This led to the description of 150 candidate interactors which were reproducibly co-purified with PAX3/FOXO1 in four independent experiments. Most of the identified proteins are transcription factors or transcriptional regulators. To validate our findings, selected candidate interactors such as RUNX1 and TFAP2B were further shown to pull down PAX3/FOXO1 in co-immunoprecipitation studies. To analyse whether some of the candidate interactors have an influence on PAX3/FOXO1 activity we individually silenced the expression of a set of 60 candidates by siRNA and measured the expression of several PAX3/FOXO1 target genes including TFAP2B, FGFR4, and NMYC. These experiments revealed that interactors can have different effects on specific PAX3/FOXO1 target genes and suggest that specific transcriptional complexes are responsible for activating the transcription of different PAX3/FOXO1 target genes. This potentially reflects the diversity of mechanisms by which PAX3/FOXO1 regulates target gene expression including enhancer- and promoter-directed actions. Further characterization of selected interactors is necessary to understand the impact of these on the oncogenic function of PAX3/FOXO1 in more detail. The results of these experiments shed light on the regulatory pathway(s) and mechanisms involved in PAX3/FOXO1 mediated oncogenesis and thereby contribute to our understanding of the oncogenic function of tumor-specific chimaeric transcription factors. Citation Format: Maria Böhm, Marco Wachtel, Beat W. Schäfer. Identification of the interactome of PAX3/FOXO1 in alveolar rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2013-757

Using Chimaeric Expression Sequence Tag as the Reference to Identify Three-Dimensional Chromosome Contacts

Transcription-induced chimaeric transcripts, the potential post-transcriptional processing products, might reflect the spatial proximity of actively transcribed genes co-localized in transcription factories. A growing number of expression data deposited in databases provide us with the raw material for screening such chimaeric transcripts and using them as the probes to identify interactions between genes in cis or in trans. Based on the high-quality chimaeric transcripts gleaned from human expression sequence tag data with selection criteria, we identified the patterns of inter- and intrachromosomal gene–gene interactions. On top the contact pattern from interchromosomal interactions, we also observed an exponential behaviour of the intrachromosomal interactions within a certain length scale, which is consistent with the independent experimental results from Hi-C screening and with the Random Loop Model. A compatible result is found for mouse. Transcription-induced chimaeric transcripts, most of which might be accidental products with trivial functions, shed light on the spatial organization of chromosomes. These inter- and intrachromosomal interactions might contribute to the compaction of chromosomes, their segregation and formation of the chromosome territories, and their spatial distribution within the nucleus.

Abstract 5076: Integrative genomic and transcriptomic analysis of metaplastic carcinomas of the breast

Abstract Background Metaplastic breast carcinomas (MBCs) are characterised by neoplastic cells that display differentiation towards squamous epithelium or mesenchymal elements. These tumours are of triple-negative phenotype and have an aggressive clinical behaviour. The aims of this study were to characterise a series of MBCs at the genomic and transcriptomic level to identify potential drivers of this breast cancer special type. Design Twenty-five frozen MBCs and two MBC cell lines (HCC1569 and Hs578T) were subjected to genotyping (Affymetrix SNP6), 32K BAC microarray comparative genomic hybridisation (aCGH), microarray gene expression profiling (Illumina HT12) and massively parallel mRNA-sequencing (RNA-seq, Illumina GAII). Novel chimaeric transcripts were identified using Chimerascan v0.4.3. 10 cases with available germline DNA were subjected to targeted exomic sequencing (Illumina HiSeq). Somatic variants were called using the Genome Analysis Toolkit (GATK) using best practice guidelines. Fusion genes and mutations were validated using (RT)-PCR and Sanger sequencing, respectively. Results MBCs displayed a complex pattern of gene copy number aberrations, with multiple copy number gains and losses throughout the genome. Recurrent amplifications/ high-level gains at 3q25.2-q27.1, 8p11.21-q24.3 and 12p13.33 and recurrent homozygous deletions on 9p21.3 (CDKN2A/2B) and 10q23.31 (PTEN) were found. Unsupervised clustering of gene expression showed that MBCs associated with chondroid differentiation separated into a distinct cluster, whereas MBC's with spindle and epithelial morphology did not cluster apart. Integration of SNP6 copy number and gene expression data identified 198 genes overexpressed when amplified. RNA-seq revealed 1025 unique nominated fusion gene events (43-119 per tumour), including 254 fusions predicted to be in-frame. These included a promoter swap involving TBXLR1 fused to PIK3CA and a recurrent promoter swap involving NF1 that was also seen in the MBC cell line Hs578T. Targeted exome sequencing identified 402 somatic coding variants (12-96 per tumour), including recurrent mutations in TP53 (5/10) and genes involved in PI3K signaling. Conclusion Our results demonstrate that MBCs have complex genomes and are heterogeneous at both the genomic and transcriptomic levels. TP53, CDKN2A/2B and PTEN mutations and/ or homozygous deletions, and activation of the PI3K pathway are recurrent events in these tumours. 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 5076. doi:1538-7445.AM2012-5076

P3-06-02: Identification of Redundant, Tumor Subtype Specific Fusion Transcripts in Primary Breast Tumors.

Abstract Background: The role of fusion genes and associated fusion transcripts has long been recognized in hematopoietic malignancies. Until quite recently it has been difficult to detect such events on a genomic scale in solid tumors. Consequently, little is known about the potential role of fusion genes, transcripts, and proteins as driver mutations, biomarkers, or therapeutic targets in breast cancer. Methods: We have developed a novel analytical pipeline, Snowshoes-FTD, for detection of fusion transcripts in breast cancer cell lines and tumor samples (Asmann, et al. NAR 2011; May 27 ePub ahead of print). Preliminary analyses have been carried out with a panel of 8 each ER+, HER2+, and triple negative (TN) primary breast tumors, 8 primary human mammary epithelial cell (HMEC) lines from biopsy samples, plus 16 normal tissues from the Illumina Body Map dataset. Results: We have identified 120 redundant, tumor-specific fusion transcripts, expressed in two or more tumors and in no non-transformed samples. Sixteen of these represent intrachromosomal fusions and 104 arise from fusion of transcripts that map to two different chromosomes. Every breast tumor expressed one or more fusion transcripts. Twenty-nine fusion transcripts appeared to be tumor subtype specific. Among these, we have identified 2 HER2+, 10 ER+, and 17 triple negative specific redundant transcripts. In general, HER2+ tumors expressed fewer fusion transcripts (range 4 to 28/tumor) compared to TN (range11 to 44/tumor). Chromosomal distribution patterns were also markedly different among the tumor subtypes. For example, ER+ tumors expressed a preponderance of redundant fusion transcripts that involve chr1 and 2, whereas TN tumors had no fusion transcripts that map to either chromosome. Conversely, the predominant locus for TN fusion transcripts was chr19, which contains only one HER2+ fusion and no ER+ fusion transcripts. Conclusions: Primary breast tumors express many chimaeric transcripts, which we presume to arise primarily from genomic rearrangements. The majority of these transcripts are redundant, and a subset are tumor subtype specific. These transcripts may mark regions of chromosomal instability. HER2+ tumors, in general, appear to evidence less chromosomal instability, as inferred from fusion transcripts; although some HER2+ tumors appear to be quite unstable. TN tumors contain many more redundant fusion transcripts, implying increased genomic instability, particularly in chr19. We conclude that these fusion transcripts represent a class of heretofore unrecognized biomarkers that may be used for sub-classification of breast tumors. Some of these transcripts appear to encode proteins that may function as tumor-subtype-specific driver mutations and may have potential as therapeutic targets in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-06-02.

Abstract LB-276: Fusion transcripts in breast cancer cell lines and tumors

Abstract The role of fusion gene transcripts such as BRC-ABL1 has been long appreciated in hematopoietic malignancies. Recent evidence from next generation sequencing projects has suggested that gene fusion events and the resultant chimaeric transcripts may be expressed in solid tumors. We have developed a novel bioinformatics analytical pipeline to detect fusion gene transcripts from paired-end mRNA-seq data. The bioinformatics tool used for fusion transcript discovery employs multiple steps of false positive filtering and nominates the fusion transcript candidates with high confidence (approaching 100%). The screening and validation of the fusion candidates were quickly carried out using the recommended primer design regions as one of the outputs of the SnowShoes-FD pipeline. This pipeline was used to analyze the transcriptome of 22 breast cancer cell lines and 9 non-transformed breast cell populations. Fifty-four fusion candidates were nominated, all of which were validated using reverse transcription PCR and Sanger sequencing. Five fusion products were predicted to have a second fusion junction point between two fusion partners. These 54 fusion transcripts consist of 103 partner genes that are involved in cell cycle and/or nuclear receptor signaling. No fusion transcripts were identified from the non-transformed breast cell lines. We subsequently analyzed a panel of primary breast tumors, 8 each HER2+, triple negative, and ER+. Fifteen novel fusion transcript candidates have been nominated. Validation and functional analysis of these candidates is in progress. 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 LB-276. doi:10.1158/1538-7445.AM2011-LB-276

Escape from stress granule sequestration: another way to drug resistance?

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance and chemotherapy failure in cancer. We have demonstrated that, in leukaemic cells, P-glycoprotein expression is regulated at the translational level. More recently, we have shown that in cells overexpressing P-glycoprotein, MDR1 mRNA does not aggregate into translationally silent stress granules. Importantly, this is not unique for MDR1, since other transcripts encoding transmembrane proteins, and which are thus translated at the endoplasmic reticulum, follow the same pattern. By using a series of chimaeric transcripts, we have demonstrated that transcript localization at the endoplasmic reticulum bypasses the signals dictating stress granule sequestration. Polysome profile analyses and protein synthesis experiments indicate that, upon stress withdrawal, endoplasmic-reticulum-bound transcripts resume translation faster than those at the cytosol, which have been sequestered into stress granules. This may represent a novel mechanism by which drug-resistant cells respond quickly to stress, helping them to survive the cytotoxic effect of chemotherapeutic drugs.

Tandem B1 SINE retro-elements may provide a basis for natural antisense transcription in the Magi1 locus of the mouse (Mus musculus)

Transposable elements, which are DNA sequences that can move between different sites in genomes, comprise approximately 40% of the genome of mammals and are emerging as important contributors to biological diversity. Here we report a transcription unit lying within intron 1 of the murine Magi1 (membrane associated guanylate kinase inverted 1) gene that codes for a cell-cell junction scaffolding protein. The transcription unit, termed Magi1OS (Magi1 Opposite Strand), originates from a region with tandem B1 short interspersed nuclear elements (SINEs) and is an antisense gene to Magi1. Mag1OS transcription initiates in a proximal B1 element that shows only 4% divergence from the consensus sequence, indicating that it has been recently inserted into the mouse genome and could be replication competent. Moreover, a chimaeric transcript may result from intra-chromosomal interaction and trans-splicing of the Magi1 antisense transcript (Magi1OS) and Ghrl, which codes for the multifunctional peptide hormone ghrelin. These two genes are 20 megabases apart on chromosome 6 and are transcribed in opposite directions. We propose that the Magi1OS locus may serve as a useful model system to study exaptation and retrotransposition of B1 SINEs, as well as to examine the mechanisms of intra-chromosomal trans-splicing.

Transcriptome sequencing to detect gene fusions in cancer

Recurrent gene fusions, typically associated with hematological malignancies and rare bone and soft tissue tumors1, have been recently described in common solid tumors2–9. Here we employ an integrative analysis of high-throughput long and short read transcriptome sequencing of cancer cells to discover novel gene fusions. As a proof of concept we successfully utilized integrative transcriptome sequencing to “re-discover” the BCR-ABL1 10 gene fusion in a chronic myelogenous leukemia cell line and the TMPRSS2-ERG 2,3 gene fusion in a prostate cancer cell line and tissues. Additionally, we nominated, and experimentally validated, novel gene fusions resulting in chimeric transcripts in cancer cell lines and tumors. Taken together, this study establishes a robust pipeline for the discovery of novel gene chimeras using high throughput sequencing, opening up an important class of cancer-related mutations for comprehensive characterization.

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.

Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

BackgroundThe peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS), which spans the promoter and untranslated regions of the ghrelin gene (GHRL). Here we further characterise GHRLOS.ResultsWe have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2). Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis), as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed.ConclusionGHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA) genes, including 5' capping, polyadenylation, extensive splicing and short open reading frames. The gene is also non-conserved, with differential and tissue-restricted expression. The overlapping genomic arrangement of GHRLOS with the ghrelin gene indicates that it is likely to have interesting regulatory and functional roles in the ghrelin axis.

RUNX1-RUNX1T1 Induces Over-Expression of γ-Catenin in Human CD34+ Cells, Increasing Self Renewal and Impairing Granulocytic Differentiation.

The t(8;21) is one of the most frequent translocations in AML occurring at a frequency of 10–15% and at 30–40% incidence in cases of AML-FAB subtype M2. This translocation results in expression of the chimaeric transcription factor RUNX1-RUNX1T1 (aka AML1-ETO). Using a human cord blood model, we have previously reported that expression of this fusion gene inhibits granulocytic differentiation, a feature which is characteristic of 8;21 leukaemias. This model therefore provides a means to probe the mechanism by which RUNX1-RUNX1T1 participates in the inhibition of differentiation and the promotion of self-renewal. Data increasingly suggest that RUNX1-RUNX1T1 induces the dysregulation of target genes critical for haematopoietic differentiation. Here we report the use of Affymetrix HG-U133A microarrays (22,283 probe sets) to determine the effect of RUNX1-RUNX1T1 on gene expression during the development of human blood cell progenitors (CD34+ cells). These cells were isolated by MACS and RUNX1-RUNX1T1 expression was achieved by retroviral infection of these cells using a vector co-expressing GFP. Transduced cells were subsequently cultured in IL-3, SCF and G/GM-CSF supplemented medium. Using this strategy we generated matched RUNX1-RUNX1T1 and control populations representing progenitor cells (day 3) and individual developmental subsets isolated after 6 days of culture. The absolute Genechip data were imported into Genespring software and filtered to remove all probe sets that changed by <1.5 fold between control and RUNX1-RUNX1T1 transduced cells and were not statistically significant using ANOVA or paired t-test (P<0.05). We identified 26 genes that were differentially expressed between control and RUNX1-RUNX1T1 transduced myeloblasts (days 3–6). To focus on genes relevant to leukaemogenesis we also compared them to AML t(8;21) diagnostic (n=11) and normal donor (n=9) samples obtained from patients who were treated in the UK NCRI-AML Trial. In each of these data sets expression of RUNX1-RUNX1T1 was associated with over-expression of γ-catenin (>2.6 fold), an observation confirmed at the protein level. Furthermore, t(8;21) patients also over-expressed γ-catenin compared to FAB M2 patients without cytogenetic abnormality. γ-Catenin (like β-catenin) is believed to be intimately linked to the control of differentiation and self-renewal of stem cells. We therefore over-expressed this protein in CD34+ cells to determine whether this was sufficient to account for the dysregulation of development observed in RUNX1-RUNXT1 cells. We found that, as with cells expressing RUNX1-RUNX1T1, over-expressing γ-catenin promoted retention of myeloid colony-forming ability, such that by the second week of culture, myeloid colony forming efficiency exceeded that of control cultures by 3 fold. (P<0.05). We are currently studying the effect of γ-catenin over-expression in bulk liquid culture in the presence of IL-3, SCF and G/GM-CSF. Under these conditions over-expression of γ-catenin does not appear to increase the long-term proliferation of these cells compared to control, however, morphologically an inhibition of granulocytic differentiation was observed. In summary, RUNX1-RUNX1T1 promotes γ-catenin expression and this in turn appears to participate in leukaemogenesis associated with FAB M2.

An eleven nucleotide section of the 3′-untranslated region is required for perinuclear localization of rat metallothionein-1 mRNA

Localization of mRNAs provides a novel mechanism for synthesis of proteins close to their site of function. MT1 (metallothionein-1) is a small, metal-binding protein that is largely cytoplasmic but which can be found in the nucleus. The localization of rat MT1 requires the perinuclear localization of its mRNA by a mechanism dependent on the 3'-UTR (3'-untranslated region). The present study investigates the nature of this mRNA localization signal using Chinese-hamster ovary cells transfected with gene constructs in which either MT1 or the globin coding region is linked to different sequences from the MT1 3'-UTR. Deletion, mutagenesis and antisense oligonucleotide approaches indicate that nt 45-76 of the 3'-UTR, in particular nt 66-76, are required for the localization of either MT1 mRNA or chimaeric transcripts in which a beta-globin coding region is linked to sequences from the MT1 3'-UTR. This section of the 3'-UTR contains a CACC repeat. Two mutations that are predicted to alter the secondary structure of this region also impair localization. Our hypothesis is that the perinuclear localization signal in MT1 mRNA is formed by a combination of the CACC repeat and its structural context.

Pack-MULE transposable elements mediate gene evolution in plants.

Mutator-like transposable elements (MULEs) are found in many eukaryotic genomes and are especially prevalent in higher plants. In maize, rice and Arabidopsis a few MULEs were shown to carry fragments of cellular genes. These chimaeric elements are called Pack-MULEs in this study. The abundance of MULEs in rice and the availability of most of the genome sequence permitted a systematic analysis of the prevalence and nature of Pack-MULEs in an entire genome. Here we report that there are over 3,000 Pack-MULEs in rice containing fragments derived from more than 1,000 cellular genes. Pack-MULEs frequently contain fragments from multiple chromosomal loci that are fused to form new open reading frames, some of which are expressed as chimaeric transcripts. About 5% of the Pack-MULEs are represented in collections of complementary DNA. Functional analysis of amino acid sequences and proteomic data indicate that some captured gene fragments might be functional. Comparison of the cellular genes and Pack-MULE counterparts indicates that fragments of genomic DNA have been captured, rearranged and amplified over millions of years. Given the abundance of Pack-MULEs in rice and the widespread occurrence of MULEs in all characterized plant genomes, gene fragment acquisition by Pack-MULEs might represent an important new mechanism for the evolution of genes in higher plants.

Variant MYST4-CBP gene fusion in a t(10;16) acute myeloid leukaemia.

We report a novel fusion of the MYST4 and CBP genes in an acute myeloid leukaemia (AML)-M4 patient exhibiting t(10;16)(q22;p13) and t(11;17)(q23;q21). The t(10;16)(q22;p13) resulted in a rearrangement, where MYST4-CBP and CBP-MYST4 chimaeric transcripts were products of in-frame fusions of MYST4 exon 17 to CBP exon 6 and CBP exon 4 to MYST4 exon 18 respectively. The potential resulting chimaeric proteins showed similarities with MYST3-CBP, MYST3-P300 and MYST3-NCOA2 putative fusion proteins found in other cases of AML.

Detection of AP12-MALT1 chimaeric gene in extranodal and nodal marginal zone B-cell lymphoma by reverse transcription polymerase chain reaction (PCR) and genomic long and accurate PCR analyses.

t(11;18)(q21;q21) has been recognized as a characteristic chromosomal translocation in mucosa-associated lymphoid tissue (MALT)-type lymphoma, and recent studies have demonstrated that this translocation results in the chimaeric transcript of API2 (apoptosis inhibitor 2)-MALT1 (mucosa-associated lymphoid tissue lymphoma translocation gene 1). In this study, we used reverse transcription polymerase chain reaction (RT-PCR) to analyse the incidence of this fusion product in a large series of MALT lymphoma, nodal marginal zone B-cell lymphoma (nMZBCL) and extranodal diffuse large B-cell lymphoma (DLBL) cases. RT-PCR analysis revealed that 17 of the 95 (17.9%) MALT lymphomas but none of the nine nMZBCLs or 16 DLBLs had API2-MALT1 fusion transcripts. The incidence of API2-MALT1 varied among MALT lymphomas arising from different sites and was highest for pulmonary MALT lymphomas (10 out of 16 cases, 62.5%). The presence of the API2-MALT1 fusion gene was also confirmed by long and accurate (LA)-PCR with genomic DNA, and the result correlated well with that obtained with the RT-PCR assay, thus demonstrating the usefulness of LA-PCR for the detection of the API2-MALT1 fusion gene.