Comparative analysis reveals genomic features of stress-induced transcriptional readthrough.

Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.

DoGFinder: a software for the discovery and quantification of readthrough transcripts from RNA-seq.

BACKGROUND: Recent studies have described a widespread induction of transcriptional readthrough as a consequence of various stress conditions in mammalian cells. This novel phenomenon, initially identified from analysis of RNA-seq data, suggests intriguing new levels of gene expression regulation. However, the mechanism underlying naturally occurring transcriptional readthrough, as well as its regulatory consequences, still remain elusive. Furthermore, the readthrough response to stress has thus far not been investigated outside of mammalian species, and the occurrence of readthrough in many physiological and disease conditions remains to be explored. RESULTS: To facilitate a wider investigation into transcriptional readthrough, we created the DoGFinder software package, for the streamlined identification and quantification of readthrough transcripts, also known as DoGs (Downstream of Gene-containing transcripts), from any RNA-seq dataset. Using DoGFinder, we explore the dependence of DoG discovery potential on RNA-seq library depth, and show that stress-induced readthrough induction discovery is robust to sequencing depth, and input parameter settings. We further demonstrate the use of the DoGFinder software package on a new publically available RNA-seq dataset, and discover DoG induction in human PME cells following hypoxia - a previously unknown readthrough inducing stress type. CONCLUSIONS: DoGFinder will enable users to explore, in a few simple steps, the readthrough phenomenon in any condition and organism. DoGFinder is freely available at https://github.com/shalgilab/DoGFinder .