Shared binding sites for the chromosomal architectural protein Su(Hw) mediate physical interactions between Drosophila TAD boundaries

Loop extrusion has been clearly shown to be insufficient as a mechanism to explain TAD formation, leaving a large gap in our understanding of how the specificity of TAD boundary interactions and inter-TAD chromosomal interactions are determined. Many TAD binding proteins have been implicated in boundary interactions, including the gypsy transposon boundary binding protein Su(Hw). How these proteins generate the often specific and orientation-dependent boundary interactions that underpin chromosomal architecture is largely unknown. Here, we investigate the role of the single Su(Hw) binding site located in each of the boundaries that flank the Drosophila eve locus, homie and nhomie. We show that Su(Hw), which binds hundreds of sites throughout the Drosophila genome, plays a large role in the highly selective and orientation-specific interactions of homie and nhomie. Despite its outsized role in the binding strength and stability of these interactions, other boundary binding proteins are implicated as the primary determinants of the specificity of the interactions. These studies provide an important example of the need to more fully investigate how strength and specificity of TAD boundary interactions are separately encoded in this important class of genome architectural elements.