Predicting single-molecule chromatin distribution from DNA sequence

Genome regulation unfolds through dynamic and stochastic single-molecule events. Deep learning models that read gene regulatory activities from DNA sequences have markedly advanced our ability to predict cell-population-level averages. However, they cannot capture the full spectrum of heterogeneity and dynamics at the single-molecule level, which directly reflect the underlying mechanisms of genome regulation. Here, we introduce a sequence-to-distribution (S2d) framework that directly models the entire probabilistic distribution of single-molecule chromatin states from DNA sequence alone. We used this framework to train Mola, a model of single-molecule chromatin accessibility. We harness Mola to investigate sequence-based regulatory code and mechanisms for multiple aspects of chromatin regulation at the single-molecule level. By quantifying sequence-driven nucleosome regulation, we reveal distinct modes of transcription factor engagement with nucleosomes, including a novel nucleosome-trapping factor, ZNF146. We characterized the sequence determinants governing the +1 nucleosome after transcription start sites and identified YY1 as the strongest +1 nucleosome-positioning factor. Leveraging long-context single-molecule modeling, we further discovered a shared sequence basis for the co-accessibility of regulatory elements and the co-footprinting of transcription factors. Joint prediction of DNA methylation and accessibility reveals a sequence-driven bimodal state mediated by methylation-sensitive transcription factors. Beyond static single-molecule snapshots, the S2d framework can generate pseudo-dynamic trajectories of chromatin states and propose a roadmap toward genome-wide chromatin dynamics. Moreover, we showed that Mola can predict and interpret the effects of genetic variants at the single-molecule level, revealing how sequence changes reshape distal regulatory coordination. Together, this sequence-to-distribution framework lays the foundation for understanding how genomic sequence encodes stochastic and dynamic genome regulation, and how genetic variation changes it, one molecule at a time.