A fluorescent assay for cryptic transcription inSaccharomyces cerevisiaereveals novel insights into factors that stabilize chromatin structure on newly replicated chromatin

Abstract

The disruption of chromatin structure can result in transcription initiating from cryptic promoters. A well-characterized, chromatin-destabilizing stress is the passage of RNA polymerase, and numerous factors function to stabilize chromatin on transcribed genes, suppressing cryptic transcription from sites within gene bodies. DNA replication is also inherently disruptive to chromatin, and multiple replication-coupled histone chaperones suppress cryptic transcription. However, these factors also have documented roles in transcription, and thus whether DNA replication per se can activate cryptic promoters has not been directly 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, Rtt106, Spt6, and Spt16, suppress transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Additionally, for the first time, we demonstrate modest cryptic transcription following the depletion of Rlf2/Cac1, a CAF-1 chromatin assembly complex component. Collectively, these results suggest that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.