53BP1 as a cofactor in the activation of the p53-family transcriptional program: A correlative multimodal microscopy approach

Understanding how molecular mechanisms regulate cell function in tissues requires approaches that link phenotypes to mechanisms across different spatial scales. Here, we present automated multimodal multiplexed microscopy for image cytometry (A.M 3 .I.CO), a correlative imaging platform that combines multiscale resolution, from millimeters to nanometers, with automated workflows for high-throughput phenotypic profiling and targeted single-molecule analysis in situ. Applying A.M 3 .I.CO, we observed that 53BP1 is recruited at transcribed p53 target genes independently of the activating stimulus, providing evidence for the formation of a putative complex between 53BP1, p53, and the basal transcription machinery in single cells, as also supported by in vivo observations in tumor models. Extending our measurements to p63 and p73, we found that 53BP1 localized at p63- and p73-targeted transcription sites, suggesting a broader role in participating in p53 family–dependent transcription. These findings indicate an additional genome-surveillance pathway in transcriptional control regulating diverse cellular functions while establishing A.M 3 .I.CO as a platform for spatially resolved functional analysis.