Project description:<p>High throughput RNA Sequencing has revealed that the human genome is widely transcribed. However, the extent of natural antisense transcription, the molecular mechanisms by which natural antisense transcripts (NATs) might affect their cognate sense genes, and the role of NATs in cancer are less well understood. Here, we use strand-specific paired-end RNA sequencing (ssRNASeq) on a cohort of 376 cancer patients covering 9 tissue types to comprehensively characterize the landscape of antisense expression. Our results reveal that greater than 60% of annotated transcripts have measureable antisense expression and the expression of sense and antisense transcript pairs is in general positively correlated. Furthermore, by studying the expression of sense/antisense pairs across tissues we identify lineage-specific, ubiquitous and cancer-specific antisense loci. Our results raise the possibility that NATs participate in the regulation of well-known tumor suppressors and oncogenes. Finally, this study provides a catalogue of cancer related genes with significant antisense transcription (oncoNAT). This resource will allow researchers to investigate the molecular mechanisms of sense/antisense regulation and further advance our understanding of their role in cancer.</p>
Project description:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and EM-NM-<, the start site of IM-NM-< germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is EM-NM-<-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-EM-NM-< interactions. ChIP-seq shows high level YY1 binding only at EM-NM-<, but low levels near some antisense promoters. PAIR-EM-NM-< interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to EM-NM-<. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement. ChIP Seq YY1 vs. input control
Project description:We characterized the expression patterns of sense-antisense transcripts, based on available cDNA sequences, in colon (colorectal) cancer tissues and in normal tissues surrounding the cancer tissues. Although expression balances (ratios) of most of sense and antisense transcript pairs did not change between patients or between normal and cancer tissues, we found 68 sense-antisense transcripts whose expression balances were altered specifically in colon cancer tissues.
Project description:RNA-sequencing (RNA-Seq) protocols and bioinformatic pipelines are designed to streamline downstream analyses on sequences believed to be the most important. Here, we have challenged this dogma by preserving ribosomal RNA (rRNA) in our samples and by lowering the minimal RNA size window of our small RNA-Seq analyses to 8 nt
Project description:Hight throughput techniques have revealed huge complexity in antisense RNAs in many organisms. We have explored the complexity of this class of transcripts and functional links to Polycomb silencing thought analysis of non-coding RNAs of Arabidopsis FLC. FLC is repressed and epigenetically silenced by prolonged cold, enabling plants to undergo the floral transition. Single nucleotide resolution tiling array revealed long non-coding transcripts covering the entire FLC locus. The most abundant of these are capped and polyadenylated, initiate over a 100 nucleotide window just downstream of the sense polyA site, are differentially spliced and terminate either within the sense gene or its promoter. Their levels correlate with FLC sense transcripts in all mutants and conditions tested except cold treatment. The antisense transcripts were strongly but transiently cold-induced, much earlier than other vernalization markers, and this coincided with reduction in sense FLC transcription but not sense FLC mRNA levels. Addition of the FLC antisense 5'/sense 3' region to a GFP transgene was sufficient to confer cold-induced silencing of thet fusion; however this silencing was not epigenetically manteined. These processes were all independent of the function of the Polycomb proteins required for maintenance of FLC silencing. Our data suggest that FLC antisense transcripts induce transient FLC transcriptional silencing, possibly through promoter interference, with the epigenetic silencing requiring subsequent recruitment of Polycomb machinery.
Project description:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and Eμ, the start site of Iμ germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to Eμ. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement.
Project description:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and Eμ, the start site of Iμ germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to Eμ. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement.
Project description:The histone variant H2A.Z, which has been reported to have both activating and repressive effects on gene expression, is known to occupy nucleosomes at the 5’ ends of protein-coding genes. We now find that H2A.Z is also significantly enriched in gene coding regions and at the 3’ ends of genes in budding yeast, where it co-localises with histone marks associated with active promoters. By comparing H2A.Z binding to global gene expression in budding yeast strains engineered so that normally unstable transcripts are abundant, we show that H2A.Z is required for normal levels of antisense transcripts as well as sense ones. High levels of H2A.Z at antisense promoters are associated with decreased antisense transcript levels when H2A.Z is deleted, indicating that H2A.Z has an activating effect on antisense transcripts. Decreases in antisense transcripts affected by H2A.Z are accompanied by increased levels of paired sense transcripts. Therefore, the effect of H2A.Z on protein coding gene expression is a reflection of its importance for normal levels of both sense and antisense transcripts. Htz1 ChIP-seq in wild-type (WT) and rrp6Δ yeast, along with negative control ChIP-seq in htz1Δ and input control. Strand-specific transcriptomic profiles of WT, htz1Δ, rrp6Δ and htz1Δrrp6Δ. Replicates are present for all samples except the negative and input control ChIP samples.