upsFISH: An Occupancy-Reporting Fluorescence In Situ Hybridization Method for Single-Cell Detection of Chromatin Interactions

Understanding chromatin interactions is fundamental to gene regulation; however, existing approaches rely on spatial distance measurements or population-averaged contact frequencies, limiting their ability to directly report regulatory engagement at single-cell resolution. Here, we present ultraproximal specificity FISH (upsFISH), an occupancy-reporting fluorescence in situ hybridization method that detects chromatin interaction states through probe occupancy rather than spatial proximity. upsFISH employs a dual-arm primary probe targeting two genomic elements, together with secondary probes that bind unoccupied probe arms, generating combinatorial fluorescence signals that provide a binary, resolution-independent readout in single cells. Using the β-globin locus as a model, we show that upsFISH accurately detects enhancer-promoter interactions with high sequence specificity, including sensitivity to transcription factor binding motifs. Genetic and pharmacological perturbations demonstrate that upsFISH captures biologically meaningful changes in chromatin interactions and resolves allele-specific and cell-to-cell heterogeneity. Comparative analyses indicate that upsFISH outperforms conventional DNA FISH and micro-4C in detecting short-range regulatory interactions, although its advantage decreases with increasing genomic distance. Together, upsFISH establishes an occupancy-based framework for studying chromatin interactions in single cells.