Super-resolution imaging reveals disrupted higher-order heterochromatin organization at early-stage carcinogenesis on pathological tissue

Abnormal chromatin structure is one of the hallmarks in cancer and has long been used for cancer diagnosis, but the observed aberrant chromatin structures characteristic of cancer cells is limited to microscale features, largely due to due to the diffraction-limited resolution of conventional light microscopy. Recent advances in super-resolution microscopy overcome the technical limitations of conventional light microscopy and enable the in-situ visualization of the higher-order chromatin structure down to ~30 nm. Here we present PathSTORM, a variant of stochastic optical reconstruction microscopy (STORM) optimized for super-resolution imaging of densely packed higher-order chromatin organization at different epigenomic states on pathological tissue. We then apply this tool to characterize the structural alteration of higher-order heterochromatin, enriched with H3K9me3 and satellite repeats, at various stages of carcinogenesis in mouse models and human tissue specimens. PathSTORM reveals that a significant disruption of higher-order heterochromatin structure occurs in early carcinogenesis when tissue still appears histologically normal and becomes progressively severe during neoplastic progression. The disrupted higher-order heterochromatin structure also appears to be a common feature that reflects the evolution of carcinogenesis independent of molecular pathways in multiple tumor types. Taken together, PathSTORM can serve as a powerful tool to visualize higher-order chromatin structure in the spatial context of tissue architecture on pathological tissue. Our findings provide new insights on the role of higher-order heterochromatin structure in carcinogenesis, especially at the early stage, and have the potential for improving cancer diagnosis, risk stratification and facilitating the development and evaluation of new preventive strategies. To identify the specific genomic regions affected by the disrupted heterochromatin structure, using CUT&RUN, we profiled native (no crosslinking) chromatin structure of intestinal epithelial cells brushed from normal mouse intestine of wild-type and age-matched ApcMin/+mice at 5-6 weeks, which is in early carcinogenesis when tissue still appears histologically normal. CUT&RUN is a genome-scale protein localization technique that utilizes an enzyme-tethering strategy and serves as an alternative to chromatin immunoprecipitation (ChIP) to investigate the chromatin landscape and genome-wide occupancy of DNA-bound proteins. Briefly, a recombinant protein A-micrococcal nuclease (MNase) fusion bound to a primary antibody is tethered to the genomic regions occupied by the protein of interest, and followed by MNase-mediated cleavage of DNA and release of the targeted protein-DNA complexes to solution. The released DNA fragments are extracted and used for library construction and sequencing. We profiled the occupancy of H3K9me3 and H3K4me3, as well as a control lacking a primary antibody (but still including the proteinA-MNase to control for nonspecific fragment digestion and release) referred to as 'no antibody'.