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 the CHD-family chromatin remodeler Hrp3 coordinates with the transcription elongation machinery, via the transcriptional regulator Prf1/RTF1, to position nucleosomes at sites of cryptic transcription initiation within gene bodies. In the absence of Hrp3, 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 define an elongation‑coupled chromatin pathway that preserves transcriptional fidelity and reveal how nucleosome remodeling shapes antisense transcription, cellular homeostasis, and adaptive potential.