Chromatin architectures underlying plasmid-based assays for regulatory variant effects

Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, our understanding of how, or whether, chromatin structures form on plasmids transfected into mammalian cells remains limited. We developed plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) to accurately map chromatin architectures along individual, full-length transfected plasmid molecules at near-single-nucleotide resolution. Application of this method to diverse plasmids and cell lines demonstrates that plasmids are chromatinized in an organized, sequence-dependent manner and adopt a heterogeneous and incomplete chromatin architecture relative to nuclear-encoded chromatin fibers. Focal occupancy of nucleosomes and transcription factors along transfected plasmids is central to their transcriptional activity, and plasmids can recapitulate nuclear genome-encoded chromatin architectures with varying fidelity. Combining plasmid Fiber-seq with high-throughput reporter assays reveals the molecular mechanisms underlying pathogenic non-coding variants, which disentangles the effects of transcriptional activators and repressors with near-single-nucleotide resolution. Our findings reveal principles for accurate, fine-scale mapping of chromatin-dependent regulatory grammar.