The single-molecule accessibility landscape of newly replicated mammalian chromatin

We present replication-aware single-molecule accessibility mapping (RASAM), a method to nondestructively measure replication status and protein-DNA interactions on chromatin genome-wide. Using RASAM, we uncover a genome-wide state of single-molecule "hyperaccessibility" post-replication that resolves over several hours. Combining RASAM with cellular models for rapid protein degradation, we demonstrate that histone chaperone CAF-1 reduces nascent chromatin accessibility by filling single-molecular "gaps" and generating closely spaced dinucleosomes on replicated DNA. At cis-regulatory elements, we observe unique modes by which nascent chromatin hyperaccessibility resolves: at CCCTC-binding factor (CTCF)-binding sites, CTCF and nucleosomes compete, reducing CTCF occupancy and motif accessibility post-replication; at active transcription start sites, high chromatin accessibility is maintained, implying rapid re-establishment of nucleosome-free regions. Our study introduces a new paradigm for studying replicated chromatin fiber organization. More broadly, we uncover a unique organization of newly replicated chromatin that must be reset by active processes, providing a substrate for epigenetic reprogramming. Single-molecule methyltransferase footprinting of protein-DNA contacts from nascent chromatin fibers in BrdU-treated mESCs and K562 cells with third-generation PacBio sequencing.