Chromatin Insulators: Master Regulators of the Eukaryotic Genome

Proper organization of the chromatin fiber within the three dimensional space of the eukaryotic nucleus relies on a number of DNA elements and their interacting proteins whose structural and functional consequences exert significant influence on genome behavior. Chromatin insulators are one such example, where it is thought that these elements assist in the formation of higher order chromatin loop structures by mediating long-range contacts between distant sites scattered throughout the genome. Such looping serves a dual role, helping to satisfy both the physical constraints needed to package the linear DNA polymer within the small volume of the nucleus while simultaneously orchestrating or excluding contacts between regulatory elements, such as enhancers and promoters, in order to direct the proper gene regulatory outputs needed to maintain cellular homeostasis. As a result of its central role in chromatin structure, insulators have been linked to a number of nuclear processes, although many aspects of their biology remain unanswered. The collection of work presented here addresses three of these concerns. Chapter I outlines the phylogenetic distribution of these elements, highlighting the lineage specificity of the Drosophila melanogaster insulator protein BEAF-32 and suggesting that insulator function poses a more significant agent for selection than conservation of the proteins themselves. Chapter II addresses a central debate in the insulator field regarding the function of insulator bodies, exposing an unexpected link between their formation, osmotic stress and cell death, while disproving the prevailing hypothesis set forth over a decade ago that essentially formed the foundation for how these elements function in vivo. Finally, in Chapter III, their contribution to inter-allelic complementation, or transvection, is addressed, where context- and dose-dependent effects on enhancer-promoter communication in trans were observed, suggesting that chromatin structure is the ultimate determinant of whether enhancer-promoter communication in trans leads to a sustained transcriptional output. Such findings provide a new perspective for a classic genetic phenomenon while highlighting a conserved feature of genome function. Taken collectively, this body of work reflects the broad nuclear functions attributed to these elements and suggests that chromatin insulators function as master regulators of the eukaryotic genome.