Advances in Visualizing Cytosine Methylation and Allele‐Specific Epigenetic States in Tissue Context

Epigenetic regulation represents a heritable, sequence-independent layer of transcriptional control that critically shapes tissue morphology throughout development, homeostasis, and disease. Although immunohistochemical detection of histone modifications is now well established in diagnostic pathology, direct visualization of DNA methylation-the central mechanism underlying transcriptional silencing, genomic imprinting, and cellular identity-remains far more challenging. To address this limitation, a range of histochemical and cytochemical approaches has been developed to detect methylated cytosines within morphologically preserved tissues. Early strategies based on differential restriction enzyme sensitivity enabled global in situ assessment of CpG methylation, while bisulfite-dependent padlock-probe hybridization introduced sequence specificity through conversion-based discrimination of methylated and unmethylated cytosines. More recently, the ICON method has provided bisulfite-free, base-resolved detection of methylated CpG sites with single-cell precision, allowing direct visualization of allele-specific DNA methylation (ASM) within intact nuclei. These techniques reveal dynamic shifts between monoallelic and biallelic methylation during differentiation, exemplified by transitions at the Rankl promoter in osteogenic lineages. This review summarizes methodological advances in tissue-based DNA methylation detection and highlights how spatially resolved, sequence-specific approaches deepen our understanding of epigenetic regulation within histological architecture.