摘要In eukaryotic organisms,the three-dimensional organization and epigenomic landscape of chromatin are fundamental to the regulation of gene expression.Previous studies have provided significant insights into CpG methylation,chromatin accessibility,and the dynamics of 3D architecture.However,a systematic delineation of how these epigenomic features regulate transcriptional activity remains limited.In this study,we develop nanoCAM-seq,a single-molecule sequencing technique designed to simultaneously profile higher-order chromatin interactions,chromatin accessibility,and endogenous CpG methylation.This approach provides an integrative view of chromatin features associated with cis-regulatory elements and reveals their coordinated dynamics during transitions of A/B compartments.Single-molecule analyses using nanoCAM-seq further reveal that promoters characterized by low CpG methylation and high chro-matin accessibility more frequently interact with multiple enhancers.Collectively,our findings establish nanoCAM-seq as a powerful approach for resolving the coordinated dynamics of chromatin architecture and epigenetic modifications,offering critical insights into the regulatory mechanisms underlying gene expression.