Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently organized by long-range contacts. The conformation of chromatin at these three levels greatly influences DNA transcription. One class of chromatin regulatory proteins called insulator factors set up boundaries between heterochromatin and euchromatin and generate long-range loops. In Drosophila, three types of insulators (Su(Hw), dCTCF and BEAF) have been shown to regulate transcription and organize chromatin at the higher level by the formation of long-range interactions that were proposed to be mediated by the coalescence of several insulator proteins into clusters (insulator bodies). Our research aims to unravel the mechanism by which insulator bodies dynamically regulate chromatin structure and transcription by using single-molecule biophysics and quantitative modeling. On one hand, we will apply novel super-resolution fluorescence microscopy methods to investigate the structure and assembly dynamics of insulator bodies in single cells throughout the cell cycle and the role of their regulatory partners. On the other hand, we will reconstitute the looping activity of insulators in vitro and apply single-molecule manipulation methods to gain detailed insights into the molecular mechanisms involved in defining and regulating chromatin organization by insulators. This project has the potential to impact our understanding of several fundamental cellular processes: transcription regulation, cell-cycle dynamics, higher-order chromatin organization, and cell differentiation. The methods developed here will be directly applicable to the investigation of other nuclear super-structures, such as transcription and replication factories and Polycomb bodies, and thus will impact other research areas, such as DNA replication, transcription and cell division.