Visualizing Specific Genomic Loci using Fluorescently Labeled Transcription Activator-Like Effectors

Abstract

Understanding how gene transcription is spatially regulated by cis-regulatory elements (CREs) is both genetically and biomedically important. Cis-regulatory elements can be located thousands to millions of base pairs away from the promoter site, yet they somehow control and regulate transcription. Here we outline a new live cell, high resolution imaging methodology that allows for the visualization and quantification of these critical regulatory interactions. The method is based on the insertion of DNA sequences highly specific for a class of DNA binding proteins, known as Transcription Activator-Like Effectors (TALEs) into the human genome. TALEs are known to have high binding affinity and specificity to their target sites, and through conditional expression of fluorescently labeled TALEs, we are able to image specific chromosomal loci with improved signal-to-background and excellent localization precision. By using multicolor enhancer and promoter targeted TALEs, the technique can be coupled with super-resolution microscopy to detect and quantify subtle spatial changes occurring in the nucleus prior to or during transcriptional activation. This new methodology will help answer important questions on when, where and how genes are regulated during the process of cell differentiation or from external stresses and stimuli.