The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped (eve) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: λ DNA, nhomie forward (WT orientation), nhomie reverse (opposite of WT orientation), and homie forward (same orientation as WT homie). The nhomie forward replacement restores the WT physical and regulatory landscape: in MicroC experiments, the eve TAD is a ‘volcano’ triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The λ DNA replacement lacks boundary function: the endpoint of the ‘new’ eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j. While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve triangle peak is converted to a pair of ‘clouds’ of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted.

Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C₂H₂ zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino’s chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhino^G31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhino^G31D flies. Thus, Rhino’s chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.