Gene regulatory landscape dissected by single-cell four-omics sequencing.

Cellular diversity is governed not only by the transcriptome but also by multiple layers of epigenomic regulation, including nucleosome occupancy, chromatin states and genome architecture1-3. Here, to comprehensively understand how these regulatory modalities converge to shape cellular identity, we developed a single-cell four-omics sequencing method that enables parallel profiling of genome conformation, histone modifications, chromatin accessibility and gene expression within the same cell (CHARM). Applying CHARM to mouse embryonic stem cells and cortical tissues, we reconstructed integrated epigenome profiles, uncovering distinct cell-cycle dynamics of chromatin accessibility and histone modification, and spatial clustering of regulatory elements in three-dimensional nuclear space. Leveraging an interpretable machine learning model, we further identified thousands of enhancer-promoter linkages with high accuracy that modulate gene expression in a cell-type- and subtype-specific manner. Together, CHARM enables integrative dissection of the three-dimensional epigenome at single-cell resolution, providing a versatile platform for decoding the regulatory landscape across diverse cells in complex tissues.