Recent advancements in single-cell sequencing and spatial omics technologies have enhanced our understanding of diverse cellular identities, compositions, architectures, and functions. However, the single-cell three-dimensional (3D) organization of the epigenome is still not well understood, due to an absence of spatial single-cell methods that allow high-resolution, locus-specific detection of combinations of epigenetic marks while maintaining the 3D organization of the genome. Here, we develop Epigenetic Proximity Hybridization Reaction (Epi-PHR), a non-disruptive image-based single-cell spatial epigenetic profiling technology. Epi-PHR enables locus-specific and high-resolution in situ detection of combinations of epigenetic marks at hundreds of single gene targets within the same individual cells, while retaining the 3D organization of the genome. Dual-mark Epi-PHR in hippocampus tissue sections revealed region-specific epigenetic profiles. Phased Epi-PHR combined with chromatin tracing simultaneously detects allele-specific epigenetic states and chromatin conformations of a paternally imprinted Meg3 gene cluster in single cells, revealing associations between specific epigenetic mark enrichment and chromatin folding features for the distinct alleles from different parental origins. Surprisingly, we found that H3K9me3 abundance is positively associated with a chromatin domain boundary at the Meg3 locus among maternal copies. We expect Epi-PHR will be broadly applicable in research requiring single-cell spatial epigenetic information, and will help extend our understanding of the combinatorial epigenetic code and its relationship with chromatin organization.
