Nucleosome Positioning Shapes Cryptic Antisense Transcription.

Maintaining transcriptional fidelity is essential for precise gene regulation and genome stability. Despite this, cryptic antisense transcription, occurring opposite to canonical coding sequences, is a pervasive feature across all domains of life. How such potentially harmful cryptic sites are regulated remains incompletely understood. Here, we show that nucleosome arrays within gene bodies play a key role in suppressing cryptic transcription. Using the fission yeast Schizosaccharomyces pombe as a model, we demonstrate that CHD1-family chromatin remodelers coordinate with the transcription elongation machinery, specifically the PAF complex, to position nucleosomes at sites of cryptic transcription initiation within gene bodies. In the absence of CHD1, AT-rich sequences within gene bodies lose nucleosome occupancy, exposing promoter-like sequences that drive cryptic initiation. While cryptic transcription is generally detrimental, we identify a subset of antisense transcripts that encode critical meiotic genes, suggesting that cryptic transcription can also serve as a source of regulatory innovation. These findings underscore the essential role of nucleosome remodelers in maintaining transcriptional fidelity and reveal their broader contributions to cellular homeostasis and evolutionary adaptability. Overall design: We aimed to characterize the diversity of antisense transcripts present in Schizosaccharomyces pombe (S. pombe). To achieve this, we employed Iso-Seq long-read sequencing to capture full-length transcripts. Our experimental design included two genetic backgrounds: wild-type (WT) S. pombe and an hrp1? hrp3? double mutant, which is known to exhibit increased levels of antisense transcription. Both genotypes were grown under standard laboratory conditions and subjected to two distinct stress treatments: caffeine and clotrimazole. This resulted in the following sample conditions for each genotype: untreated (control), caffeine-treated, and clotrimazole-treated. For sequencing, we multiplexed our samples in two separate runs. The first run included only the wild-type, untreated sample. The second run comprised all remaining conditions: both genotypes (WT and hrp1? hrp3?), each with and without the two stress treatments (caffeine and clotrimazole). This multiplexing strategy was chosen due to technical constraints.