ZAD mediates chromatin association and insulator activity of Drosophila Pita and is functionally interchangeable with the human ZFP276 ZAD-like domain

Drosophila Pita belongs to large group of architectural proteins that have C-terminal cluster of C2H2-type zinc-finger domains and N-terminal zinc finger-associated domain (ZAD) that can homodimerize. Despite widespread distribution of ZADs in insects, only one human protein, Zinc-finger protein 276 (ZFP276), has a ZAD-like domain. The functional role of Pita has been studied so far only in the formation of the boundaries of regulatory domains in Bithorax complex. Here we tested the role of ZAD in the functional activity of the Pita protein. Using CRISPR/Cas9, we replaced the promoter region of the pita gene with attP, allowing expression of modified protein variants. Pita null mutants die at a late embryonic stage, whereas flies expressing a Pita mutant lacking ZAD, Pita?Z, show reduced viability. ChIP-seq studies showed that Pita?Z effectively binds to promoters of housekeeping genes and insulators in cooperation with other known architectural C2H2 proteins and CP190. However, ZAD is required for the binding of Pita to a subset of chromatin regions and its function as an insulator protein. The ZAD-like domain from human ZFP276 can almost completely compensate for the ZAD functions in the Pita protein. The results directly demonstrate that ZAD is required for insulator activity of Pita and its ability to efficiently bind to specific genomic regions. The ZAD-like domain of human ZFP276 may function similarly to the ZAD of Pita, raising the question of why ZADs have not spread to mammalian C2H2 proteins as they have to most insects. Overall design: ChIP-seq signal of the Pita with FLAG and Pita antibodies in whole bodies of adult flies in Pitawt, Pita?Z, and PitaZ276 lines