Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster

Abstract

Background Pita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of three hox genes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins. Results Using in vitro pull-down analysis, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. Then we constructed transgenic lines expressing the Pita protein of the wild-type and mutant variants lacking CP190-interacting regions. We have demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, we found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions. Conclusions The results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster

“…Although solved with better resolution compared to the other known structures of the CP190 BTB domain (PDB codes 4U77 and 5EUP), this new structure demonstrates only minor differences (see Supplementary Materials Data for a detailed description). Previously, we mapped the 13-aa region (220-232 aa) in Pita and the 19-aa region (715-733 aa) in dCTCF that are critical for interaction with the BTB domain [45,48]. Pita also has a second minor region (114-164 aa) that interacts with the BTB domain with low affinity [45] (Figure 4a).…”

“…Previously, we mapped the 13-aa region (220-232 aa) in Pita and the 19-aa region (715-733 aa) in dCTCF that are critical for interaction with the BTB domain [45,48]. Pita also has a second minor region (114-164 aa) that interacts with the BTB domain with low affinity [45] (Figure 4a). Interestingly, Su(Hw) also has two adjacent regions, mapped to aa 88-150 and 150-238, that bind to the BTB domain [44] (Figure 4a).…”

“…The BTB domain of CP190 is required for interaction with the architectural proteins Su(Hw) [44], Pita [35,45], and dCTCF [46]. With another group of architectural proteins that includes ZIPIC and Opbp, the CP190 protein interacts via its centrosomal-targeting domain (M) [35,36].…”

“…The architectural proteins Su(Hw), Pita, and dCTCF in cooperation form boundaries/insulators in the Bithorax complex [47]. The CP190 protein is important for blocking cross-interaction between adjacent regulatory domains [45].…”

Structural basis for interaction between CLAMP and MSL2 proteins involved in the specific recruitment of the dosage compensation complex in Drosophila

“…Fly protein extracts were obtained as described (18). Immunostaining of polytene chromosomes was performed as described (12).…”