The genome folds into Topologically Associating Domains (TADs) that define regions of preferential interactions between neighbor sites. Such 3D organization into TADs was thought to play a crucial role in spatial gene regulation notably by dictating the action of enhancers to activate their proper gene targets. In this model, it is expected that an enhancer would unlikely activate a gene if localized not within the same TAD unit. Since enhancers can act through long distances, the assembly of TADs, and the regulation of spatial folding, would define an essential mechanism in gene regulation. Insulators are DNA sequences thought to participate to delimit TADs, as TAD boundaries. They are capable of interacting with the other insulator at the opposite TAD boundaries, forming a loop. In addition, insulators can block enhancer activation of a gene when interposed between these two sequences. However, it should be noted that this model of TAD organization and insulator interaction has been validated for a few insulator sites. Research in this field is constantly evolving, and new studies are needed to better score insulators, their influences on genes, involving or not mechanisms underlying the formation and function of TADs, as well as the interactions between insulators, enhancers and genes. We have focused our research on the study of the main insulator in Drosophila, called Boundary Element Associated Factor of 32 Kda (BEAF-32) and analyzed the effect of BEAF-32 depletion on the spatial and functional structuring of Drosophila chromosomes. We will show how BEAF-32 influences long-distance enhancer-promoter interactions and how this may systematically impede on gene expression.