Structural and functional analysis of stage-specific Fab-7 boundary factor, Insensitive

Chromatin boundaries subdivide eukaryotic chromosomes into structurally and functionally autonomous domains. This is necessary to prevent promiscuous interactions between regulatory elements in one domain and genes or regulatory elements in an adjacent domain. An excellent model for understanding the regulatory functions of chromatin boundaries is the Fab-7 boundary in Drosophila melanogaster. While the Fab-7 boundary appears to be active irrespective of cell type or developmental stage, it is unusual amongst known boundaries in that proteins whose activity is developmentally restricted generate this constitutive activity. One factor, a BEN domain protein Insensitive, binds to the Fab-7 boundary during mid to late embryogenesis. Understanding how Insensitive confers boundary activity will help us understand the function of BEN domain proteins as well as how boundaries confer domain autonomy. Using electrophoretic mobility shift assays, we confirm the DNA binding activity of the BEN-domain and show it depends on a coiled-coil region within the C-terminus of Insensitive. Size exclusion chromatography and electron microscopy suggest that the Insensitive protein forms tetramer complexes capable of binding multiple DNA sites simultaneously to give DNA loops. These results support the idea that boundary proteins, in this case Insensitive, through various homo and hetero-interactions bind multiple DNA sites simultaneously. Our results also strongly support the model that DNA topological looping is how boundaries carry out their function.