A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae [RNA-seq]

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which requires a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in S. cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA. Overall design: We generated a fluorescent reporter for transcription from a cryptic promoter within the S. cerevisiae FLO8 gene. Cells with a temperature sensitive mutation in SPT16 show increased fluorescence when grown at the non-permissive temperature. To confirm that increased fluorescence is due to transcription initiating at the FLO8 cryptic promoter, we performed strand-specific RNA-seq.