Evolutionary Adaptation of an HP1-protein Chromodomain Integrates Chromatin and DNA Sequence Signals

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. Here, we elucidate the molecular basis of the interaction between the HP1 protein Rhino, a crucial factor of theDrosophilapiRNA pathway, and Kipferl, a DNA sequence-specific C₂H₂zinc finger protein and Rhino guidance factor. Through phylogenetic analyses, structure prediction, andin vivogenetics, 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 therhino^G31Dmutation phenocopykipferlmutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile ofrhino^G31Dflies. Thus, Rhino’s chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.