Multi-omic long-read sequencing reveals nucleosome control of transcriptional fidelity at the isoform level

Transcription and RNA processing are tightly coupled to chromatin organization, yet how nucleosome positioning is coordinated across the promoter and gene body to shape alternative isoform expression remains poorly understood. This question has been difficult to address because short-read chromatin profiling cannot simultaneously capture promoter and gene-body chromatin states on the same DNA molecule, and short-read RNA sequencing cannot resolve full-length transcripts without requiring computational assembly. To address this, we deleted two conserved chromatin remodelers, ISW1 and CHD1, in Saccharomyces cerevisiae and profiled chromatin fibers, nascent RNA, and full-length mature RNA using Oxford Nanopore long-read sequencing. We developed EpiFLAIR, a computational framework that assigns discrete long-range chromatin states across entire genes and integrates these states with isoform-level transcriptional outputs. Using EpiFLAIR, we show that single-molecule chromatin states together with sequence motifs are strong predictors of alternative TSS isoform expression. Loss of ISW1 and CHD1 produced divergent transcriptional changes by activating transcriptionally silenced promoters while repressing active ones. Single-molecule chromatin profiling suggested that this redistribution of transcriptional activity arose, in part, from an anticorrelation between gene-body and promoter chromatin accessibility along the same chromatin fiber, a finding uniquely enabled by long-read single-molecule resolution. Furthermore, disruption of regular nucleosome spacing impaired RNAPII processivity and co-transcriptional splicing, consequently altering mature RNA levels and splicing isoform composition. Together, our findings demonstrate that nucleosomes maintain transcriptional fidelity during initiation, elongation, and splicing to regulate isoform expression. More broadly, long-read single-molecule chromatin profiling represents a powerful approach for linking chromatin organization to isoform-level gene regulation.