Higher-Order Chromatin Organisation by Insulator Proteins Revealed Using Super-Resolution Microscopy

Abstract

Insulators are regulatory DNA elements demarcating the boundaries between differentially expressed genomic regions. Five insulator binding proteins (IBPs), BEAF-32, dCTCF, Su(Hw), GAF and Zw5, and two co-factors, CP190 and Mod(mdg4)67.2, have been described in Drosophila melanogaster. These factors have been reported to maintain physical interactions between distant genomic sites. Such evidence predicts that insulator proteins should form multi-protein clusters that regroup specific sequences, by establishing long-range chromatin contacts, which ultimately help define gene expression programs.Despite the thorough mapping of protein binding sites and genome-wide interactions, the clustering of IBPs has been inferred from biochemical experiments, in which heterogeneity and dynamics are intrinsically averaged out. Direct monitoring of the spatial organization, dynamics and distribution of IBPs and their partners require single-cell microscopy approaches. However, these measurements have been so far hampered by the size of IBP clusters, the high local protein density, and the intrinsic resolution limit of conventional microscopies. Here, we implemented 3D Structured Illumination (SIM), multicolor and 3D localization-based (PALM/STORM) microscopies, with lateral resolutions of ∼100 and ∼20 nm respectively, to explore IBP clustering and its implication in gene expression regulation.We find that under normal growth conditions, BEAF-32, CP190, and dCTCF form 100-300 nano-clusters per nucleus, with a mean size of ∼40 nm, both in transfected and immuno-stained Drosophila S2 cells. The number and distribution of nano-clusters display cell-to-cell variability, consistent with IBP roles in maintaining chromatin organization throughout cell divisions. Two-color SIM and STORM showed different classes of IBPs and co-factors co-localize with characteristic patterns. Furthermore, different IBP clusters specifically associate with gene activity markers like RNA Pol2 and epigenetic histone modifications. Our study introduces a highly sensitive approach to functionally investigate the complex interplay between chromatin folding and gene regulation, with single-molecule resolution.