Identification of non-coding transcripts regulated by the transcription factor Rap1 by RNA-Seq analysis

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1 regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeller. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality towards productive transcription. Overall design: RNA-Seq analysis of Saccharomyces cerevisiae S288C strains transformed with OsTIR1 ligase and auxin-inducible degron (IAA7) tagged Rap1, or wild-type control strains. 3 biological replicates for each sample type and condition are analyzed. For each biological replicate, cells were grown overnight in YPD media, diluted, and treated with DMSO or 500 µM 3-indole-acetic acid (3-IAA, auxin). UNINDUCED samples were taken 30 minutes after treatment, and INDUCED samples were taken 30 minutes or 2 hours after treatment. For WT controls, samples were taken in mid-logarithmic growth. RNA was either subjected to rRNA depletion or polyA RNA purification to generate POLYA and TOTAL RNA-Seq libraries, respectively.

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Wu ACK, Patel H, Chia M, Moretto F, Frith D, Snijders AP, van Werven FJ