Short Title: 53bp1 supports p53-family transcriptional program

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.M3.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.M3.I.CO, we observed that 53BP1 is recruited at transcribed p53-target genes independently of the activating stimuli, with evidence for a putative complex formation involving 53BP1, p53, and the basal transcription machinery, as also demonstrated by in-vivo observations in tumor models. This localization extends to p63- and p73-targeted transcription sites, suggesting a broader role for 53BP1 in participating to p53 family–dependent transcription. These findings extend genome surveillance pathways to transcriptional control for the execution of extremely different cell functions and establish A.M3.I.CO as a platform for spatially resolved functional analysis.