Quantifying Transcription Factor Enrichment at Target Loci in Live Embryos

The mechanisms by which transcription factors efficiently search for and occupy their target gene loci within the crowded nuclear environment remain poorly understood. Recent discoveries made using high-resolution live imaging methods have revealed that transcription factors and other nuclear regulatory proteins form high-local concentration assemblies within nuclei. These assemblies, referred to as hubs, condensates, clusters, or foci, enhance how frequently factors bind to chromatin and can thus lead to robust occupancy or enrichment at target loci even in the face of low-affinity chromatin-protein interactions. However, quantifying the enrichment of transcription factors at target loci in live organisms is challenging. Here, we present a full workflow, from sample and microscope preparation to data analysis for calculating transcription factor enrichment at specific gene loci in live Drosophila embryos. In this method, the radial average fluorescence intensity of a tagged transcription factor centered at the transcription site is used to measure relative transcription factor enrichment. This chapter details how to acquire and analyze data using our pipeline while discussing common pitfalls, appropriate quality controls, and caveats. One key caveat we highlight is the confounding effect of using transcriptional sites too close to the nuclear periphery. For analysis, we present the Python package pyEnRICH, (PYthon framework for Enriched Radial Intensity Calculations for Hubs), and an accompanying tutorial complete with sample data to guide novices and experts through quantification of transcription factor enrichment at active gene loci.