Revealing long-range heterogeneous organization of nucleoproteins with N⁶-methyladenine footprinting

A major challenge in epigenetics is uncovering the dynamic distribution of nucleosomes and other DNA-binding proteins, which plays a crucial role in regulating cellular functions. Established approaches such as ATAC-seq, ChIP-seq, and CUT&RUN provide valuable insights but are limited by the ensemble nature of their data, masking the cellular and molecular heterogeneity that is often functionally significant. Recently, long-read sequencing technologies, particularly Single Molecule, Real-Time (SMRT/PacBio) sequencing, have introduced transformative capabilities, such as N⁶-methyladenine (6mA) footprinting. This technique enables the detection of joint binding events and long-range chromatin organization on individual DNA molecules spanning multiple kilobases. Despite the potential of 6mA footprinting, existing analytical tools for 6mA detection in SMRT sequencing data suffer from significant limitations in both performance and applicability, especially with the latest sequencing platform. To address these gaps, we developed a novel 6mA-calling pipeline based on polymerase kinetics analysis. Our approach significantly outperforms current tools in terms of accuracy and computational efficiency, setting a new benchmark for 6mA detection. Utilizing our optimized experimental and computational framework, we extensively mapped nucleosome positioning and transcription factor occupancy at the single-molecule level, revealing critical features of the transcription-associated epigenetic landscape. Additionally, our work established high-resolution, long-range binding events in mitochondrial DNA, revealing simultaneous loading of two sets of replication machinery onto the displacement loop (D-loop). Our study highlights the potential of 6mA footprinting in capturing the coordinate binding of nucleoproteins and unraveling heterogeneous epigenetic states with unprecedented resolution.