Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA-minus RNA sequencing data.

BACKGROUND: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. RESULTS: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. CONCLUSION: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.

Cigarette smoke regulates the expression of TLR4 and IL-8 production by human macrophages.

BACKGROUND: Toll-like receptors (TLRs) are present on monocytes and alveolar macrophages that form the first line of defense against inhaled particles. The importance of those cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) has well been documented. Cigarette smoke contains high concentration of oxidants which can stimulate immune cells to produce reactive oxygen species, cytokines and chemokines. METHODS: In this study, we evaluated the effects of cigarette smoke medium (CSM) on TLR4 expression and interleukin (IL)-8 production by human macrophages investigating the involvement of ROS. RESULTS AND DISCUSSION: TLR4 surface expression was downregulated on short term exposure (1 h) of CSM. The downregulation could be explained by internalization of the TLR4 and the upregulation by an increase in TLR4 mRNA. IL-8 mRNA and protein were also increased by CSM. CSM stimulation increased intracellular ROS-production and decreased glutathione (GSH) levels. The modulation of TLR4 mRNA and surface receptors expression, IRAK activation, IkappaB-alpha degradation, IL-8 mRNA and protein, GSH depletion and ROS production were all prevented by antioxidants such as N-acetyl-L-cysteine (NAC). CONCLUSION: TLR4 may be involved in the pathogenesis of lung emphysema and oxidative stress and seems to be a crucial contributor in lung inflammation.

Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats.

During ischemic heart diseases and when heart failure progresses depletion of myocardial energy stores occurs. D-Ribose (R) has been shown to improve cardiac function and energy status after ischemia. Folic acid (FA) is an essential cofactor in the formation of adenine nucleotides. Therefore, we assessed whether chronic R-FA administration during the development of hypertrophy resulted in an improved cardiac function and energy status. In Wistar rats (n = 40) compensatory right ventricular (RV) hypertrophy was induced by monocrotaline (30 mg/kg; MCT), whereas saline served as control. Both groups received a daily oral dose of either 150 mg.kg(-1).day(-1) dextrose (placebo) or R-FA (150 and 40 mg.kg(-1).day(-1), respectively). In Langendorff-perfused hearts, RV and left ventricular (LV) pressure development and collagen content as well as total RV adenine nucleotides (TAN), creatine content, and RV and LV collagen content were determined. In the control group R-FA had no effect. In the MCT-placebo group, TAN and creatine content were reduced, RV and LV diastolic pressure-volume relations were steeper, RV systolic pressures were elevated, RV and LV collagen content was increased, and RV-LV diastolic interaction was altered compared with controls. In the MCT-R-FA group, TAN, RV and LV diastolic stiffness, RV and LV collagen content, and RV-LV diastolic interaction were normalized to the values in the control group while creatine content remained depressed and RV systolic function remained elevated. In conclusion, the depression of energy status in compensated hypertrophic myocardium observed was partly prevented by chronic R-FA administration and accompanied by a preservation of diastolic function and collagen deposition.

Mass-dependent decline of skeletal muscle function in cancer cachexia.

CD2F1 mice were inoculated with C26 adenocarcinoma cells, followed by assessment of ex vivo muscular function. Muscles from tumor-bearing mice had a significantly lower force output during a single maximal contraction and during repeated contractions than control muscles. The relative force output, however, did not differ when corrected for muscle mass. Thus, cachexia significantly reduces absolute skeletal muscle function, but muscle "quality" appears unaltered.

In vivo repression of an erythroid-specific gene by distinct corepressor complexes.

To assess the mechanisms of repression of the erythroid-specific carbonic anhydrase II (CAII) locus we used chromatin immunoprecipitation and show that an NCoR-histone deacetylase (HDAC)3 complex is recruited by the nuclear receptor v-ErbA to the intronic HS2 enhancer turning it into a potent silencer. Furthermore we demonstrate that efficient CAII silencing requires binding of a MeCP2-targeted HDAC-containing corepressor complex to the hypermethylated CpG-island at the promoter. Activation of transcription by either AZAdC or thyroid hormone results in loss of one of the two corepressor complexes. Thyroid hormone further replaces the enhancer-bound NCoR-corepressor complex by the TRAP220 coactivator. Treatment with the HDAC inhibitor trichostatin A (TSA) causes activation of CAII transcription and histone H3 and H4 hyperacetylation at the enhancer, apparently without affecting binding of the two corepressor complexes. Unexpectedly, histone H3 and H4 at the fully repressed promoter are already hyperacetylated despite the close apposition of the MeCP2-targeted HDAC complex. Acetylation of histone H4, but not H3, at the promoter is moderately increased following TSA treatment. Our data suggest that the hyperacetylated but repressed CAII promoter is (partially) remodeled and primed for activation in v-ErbA-transformed cells.