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HighlightsMicroscopy now allows the measurement of the polymeric structure of chromatin in intact, fixed nuclei, with a resolution of a few kilobases.Multiway contact interactions and physical separation of domains can be probed directly.Structural information can be combined with measurements of nascent transcription, nuclear position, cellular position, and cellular morphology.Single-cell imaging validates many of the features previously identified by bulk, proximity-based approaches, and challenges existing models of chromatin structure.In situ super-resolution imaging offers new potential for multiomic single-cell analyses.

Significance The relationship between long-range Polycomb-associated chromatin contacts and the linear propagation of histone H3 lysine 27 trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) is not well-characterized. Here, we nominate a role for developmental loci as genomic architectural elements that enable long-range spreading of H3K27me3. Polycomb-associated loops are disrupted upon loss of PRC2 binding and deletion of loop anchors results in alterations of H3K27me3 deposition and ectopic gene expression. These results suggest that Polycomb-mediated genome architecture is important for gene repression during embryonic development.

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SUMMARYPolycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction1-5. This process is essential for embryonic stem cell (ESCs) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spread of H3K27me3 on the linear genome6. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remains unclear7-10. Here, we apply H3K27me3 HiChIP11-13, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture14 to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells7-10. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes, such as Hmx1, Wnt6 and Hoxa. Genetic deletion of H3K27me3 loop anchors revealed a coupling of Polycomb-associated genome architecture and H3K27me3 deposition evidenced by disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. Further, we find that global alterations in PRC2 occupancy resulting from an EZH2 mutant15 selectively deficient in RNA binding is accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 acts as a “genomic wormhole”, using RNA binding to enhance long range chromosome folding and H3K27me3 spreading. Additionally, developmental gene loci have novel roles in Polycomb spreading, emerging as important architectural elements of the epigenome.