Significance Glioblastoma Multiforme (GBM) is the most common and deadliest primary brain tumor in adults. As the median survival is approximately 14 mo there is an urgent need for novel therapies. Epigenetic modulators such as bromodomain and extraterminal (BET) proteins are important therapeutic targets in GBM. Bromodomain inhibitors (including I-BET151) suppress proliferation by repressing oncogenes and inducing tumor suppressor genes through unidentified pathways. Here we demonstrate that HOTAIR (HOX transcript antisense RNA) is overexpressed in GBM, where it is crucial to sustain tumor cell proliferation, and that inhibition of HOTAIR by I-BET151 is necessary to induce cell cycle arrest in GBM cells. Our study outlines the mechanism of action underlying the antiproliferative activity of I-BET151, showing for the first time, to our knowledge, that the oncogenic long noncoding RNA HOTAIR is a major target.
Abstract To elucidate the primary factors shaping mitochondrial DNA (mtDNA) mutagenesis, we derived a comprehensive 192-component mtDNA mutational spectrum using 86,149 polymorphic synonymous mutations reconstructed from the CytB gene of 967 chordate species. The mtDNA spectrum analysis provided numerous findings on repair and mutation processes, breaking it down into three main signatures: (i) symmetrical, evenly distributed across both strands, mutations, induced by gamma DNA polymerase (about 50% of all mutations); (ii) asymmetrical, heavy-strand-specific, C>T mutations (about 30%); and (iii) asymmetrical, heavy-strand-specific A>G mutations, influenced by metabolic and age-specific factors (about 20%). We propose that both asymmetrical signatures are driven by single-strand specific damage coupled with inefficient base excision repair on the lagging (heavy) strand of mtDNA. Understanding the detailed mechanisms of this damage is crucial for developing strategies to reduce somatic mtDNA mutational load, which is vital for combating age-related diseases.
Single-strand breaks (SSBs) represent one of the most common types of DNA damage, yet not much is known about the genome landscapes of this type of DNA lesions in mammalian cells. Here, we found that SSBs are more likely to occur in certain positions of the human genome-SSB hotspots-in different cells of the same cell type and in different cell types. We hypothesize that the hotspots are likely to represent biologically relevant breaks. Furthermore, we found that the hotspots had a prominent tendency to be enriched in the immediate vicinity of transcriptional start sites (TSSs). We show that these hotspots are not likely to represent technical artifacts or be caused by common mechanisms previously found to cause DNA cleavage at promoters, such as apoptotic DNA fragmentation or topoisomerase type II (TOP2) activity. Therefore, such TSS-associated hotspots could potentially be generated using a novel mechanism that could involve preferential cleavage at cytosines, and their existence is consistent with recent studies suggesting a complex relationship between DNA damage and regulation of gene expression.
Abstract Abstract Single strand breaks (SSBs) represent the major form of DNA damage, yet no technique exists to map these lesions genome-wide with nucleotide-level precision. Herein, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes of genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely contribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realm of cellular biology, not obtainable with current approaches.
While many genome sequences are complete, transcriptomes are less well characterized. We used both genome-scale tiling arrays and massively parallel sequencing to map the Caenorhabditis elegans transcriptome across development. We utilized this framework to identify transcriptome changes in animals lacking the nonsense-mediated decay (NMD) pathway. We find that while the majority of detectable transcripts map to known gene structures, >5% of transcribed regions fall outside current gene annotations. We show that >40% of these are novel exons. Using both technologies to assess isoform complexity, we estimate that >17% of genes change isoform across development. Next we examined how the transcriptome is perturbed in animals lacking NMD. NMD prevents expression of truncated proteins by degrading transcripts containing premature termination codons. We find that approximately 20% of genes produce transcripts that appear to be NMD targets. While most of these arise from splicing errors, NMD targets are enriched for transcripts containing open reading frames upstream of the predicted translational start (uORFs). We identify a relationship between the Kozak consensus surrounding the true start codon and the degree to which uORF-containing transcripts are targeted by NMD and speculate that translational efficiency may be coupled to transcript turnover via the NMD pathway for some transcripts. We generated a high-resolution transcriptome map for C. elegans and used it to identify endogenous targets of NMD. We find that these transcripts arise principally through splicing errors, strengthening the prevailing view that splicing and NMD are highly interlinked processes.
Abstract Background Fraction of functional sequence in the human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. Results In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many yet uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. Conclusions Altogether, these results argue that many unannotated and non-canonical human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions.
Abstract Transcriptional output of human genome is far more complex than predicted by the current set of protein-coding annotations and most of the novel RNAs being produced appear to not encode proteins. This has transformed our understanding of genome complexity in recent years and suggested new paradigms of genome regulation. However, the fraction of the genome that is utilized to produce cellular RNA whose function we do not understand and even more so, their relative mass in a cell remains a controversial issue. RNA from normal human liver and brain, the K562 leukemia cell line and 6 paired Ewing primary and metastatic tumors was converted into cDNA using random hexamers and sequenced using single-molecule sequencing (SMS). No amplification, ligation, or size selection were used thus minimizing methodological biases. PolyA+ RNA, total RNA, and total RNA depleted of ribosomal RNA were studied. The SMS reads were aligned to the complete human genome and uniquely mapping reads from human tissue sources were further filtered to exclude sequences aligning to rDNA sequences, the mitochondrial genome, as well as to genomic repeats annotated by the RepeatMasker program as rRNA. After filtering, the remaining informative reads were used for subsequent analyses, including comparison to known annotations defined by the exons of UCSC Genes. This investigation makes the following key observations. 1. We show clearly that the so-called “dark matter RNAs”, which represent mostly non-coding RNA, not only exist in human cells but can comprise the majority of total non-ribosomal, non-mitochondrial RNA. In fact, we estimate that half to two-thirds of all such RNAs in a human cell is non-coding. 2. It shows a significant loss of this complexity if only polyA+ RNA is profiled. In this respect, most, if not all, contemporary RNA-seq papers continue to focus on this type of RNA and thus report significantly skewed results in terms of the true complexity of human RNA. 3. We show the presence of a large number of very long (100's of kbs), abundant intergenic transcribed regions located in areas of the genome that are devoid of protein-coding annotations. We show evidence that these very long and likely non-coding RNA transcripts are expressed during normal development, silenced in adult tissues and are then re-activated during cancer progression. Our understanding of the repertoire of human RNAs remains far from complete, and almost all RNA-Seq studies have missed this complexity due to the limited view obtained when using only the polyA+ RNA fraction. Moreover, many novel genomic regions give rise to RNAs differentially expressed in different tumor types and also in primary vs metastatic tumors derived from the same patient. This brings a tantalizing possibility that a great number of hitherto uncharacterized RNAs are involved in tumoregenesis and they could be used as both diagnostic and potentially therapeutic targets. 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 1177. doi:10.1158/1538-7445.AM2011-1177
Anti-inflammatory agents are used widely in treating numerous inflammatory conditions. The effect of Tr14, a multitargeted natural product, was compared to diclofenac, a non-selective cyclooxygenase inhibitor, on cutaneous wound repair in mice.
Objectives
To compare the effect of diclofenac with Tr14 on the transcriptome after cutaneous wounding in the mouse.
Methods
After abrasive wounding, the wounds were treated with topical Tr14 or diclofenac at clinically relevant doses. An additional group received subcutaneous Tr14 injections. The healing wounds were analyzed for RNA transcript profiling by RNAseq at specific times (12h, 24h, 36h, 72h, 96h, 120h, 196h) after injury. Differentially expressed genes (DEGs) were computed at each time point between diclofenac vs control or Tr14 vs control using EdgeR.
Results
Across time points, Tr14 treatment modulated a number of transcripts related to key wound repair pathways such as cellular differentiation, wound contraction, and cell mobility. Diclofenac, in contrast, changed gene expression mainly in two areas: Prominent effects were observed with regard to DNA chromatin regulation and ribosomal function, further effects were observed on the prostaglandin pathway and wound repair factors. In many of the key pathways modulated by Tr14, such as the defense response and cell motility, diclofenac tended to have an opposite effect on gene expression. At 12 hours post-injury, there were 521 transcripts significantly elevated and 1027 transcripts that were decreased by diclofenac treatment. By comparison, using a similar number of transcripts altered by Tr14 treatment, only 4 transcripts were increased in common, and 5 transcripts were decreased in common, suggesting that the therapies have different effects on the transcriptome.
Conclusions
The overall patterns of the Tr14 and diclofenac responses in the transcriptome during wound repair are very different. The Tr14 effect is most pronounced on the defense response, cell motility, and anti-apoptotic pathways. In contrast, diclofenac mainly affected histones and chromatin remodeling systems, as well as ribosomal systems that would be expected to alter the translational pattern of diclofenac-treated cells.
Disclosure of Interest
G. St. Laurent, III: None declared, B. Seilheimer: None declared, M. Tackett: None declared, J. Zhou: None declared, D. Shtokalo: None declared, Y. Vyatkin: None declared, P. Kapranov: None declared, I. Toma: None declared, T. Mccaffrey Speakers bureau: TM has received speaker9s honorarium from HEEL, GmbH
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