Abstract
The zinc finger-associated domain (ZAD), found in numerous Drosophila architectural proteins, such as Pita, enables homodimerization. Despite its prevalence in insects, only one human protein, ZFP276, possesses a ZAD-like domain. To date, the role of Pita has been studied in the formation of the boundaries of regulatory domains in the Bithorax complex, and the functional significance of its ZAD remains unclear. Using CRISPR/Cas9-mediated pita replacement with an attP site, we generated flies expressing modified Pita variants. Null pita mutants die in the late stages of embryogenesis. Flies expressing Pita lacking ZAD, PitaΔZ, exhibit reduced viability. Genome-wide chromatin immunoprecipitation revealed that PitaΔZ retains binding to housekeeping gene promoters and insulators, cooperating with other architectural C2H2 proteins and CP190. However, ZAD is essential for Pita binding to specific chromatin regions and its insulator function. Strikingly, the ZAD-like domain from human ZFP276 can functionally substitute for the ZAD in Pita. ZAD is critical for the insulator activity of Pita and its ability to efficiently bind to specific genomic regions. The human ZFP276 ZAD-like domain may function similarly to the ZAD of Pita, raising the question of why ZADs spread in insects but not in mammals.
