Inferring chromatin architecture at a single locus through probabilistic in situ DNA localization

Chromatin conformation is thought to be critical for enhancer function, but its dynamic, nanoscale organization is difficult to measure directly. Here we introduce PLOTTED (Probabilistic Localization of Oligopaint Tagged Target Element Distances), an integrated imaging and computational framework that infers chromatin architecture from targeted high-resolution imaging of cis-regulatory modules (CRMs). PLOTTED generates spatial distance distributions between DNA loci, enabling quantitative modeling of chromatin configurations across developmental time, spatial axes, and genotypes. Applying PLOTTED to the brinker locus in Drosophila embryos, we measured distances among three CRMs and used chromatin geometry as a proxy for regulatory activity. In wild type, CRM configurations shift dynamically at nuclear cycle 13, whereas these changes are delayed in mutants and vary along the dorsal-ventral and anterior-posterior axes. Importantly, these conformational changes correlate with altered gene expression. Together, our findings position PLOTTED as a probabilistic, single-locus framework for interpreting chromatin architecture in development and disease.