Abstract
BackgroundPita 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.ResultsUsing 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.ConclusionsThe 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.
Highlights
The development of modern approaches for the study of genome architecture, including chromosome conformation capture methods, coupled to high-throughput sequencing (Hi-C) and high-resolution microscopy techniques has revealed the hierarchical organization of genome [1, 2]
In Drosophila, we suggested the existence of a large family of architectural proteins, which typically contain N-terminal homodimerization domains and arrays of the zinc-finger Cys2-His2 (C2H2) domains [15,16,17,18,19,20,21,22]
Mapping regions within the Pita protein that interact with the BTB domain of CP190 To understand the interaction mechanism between the architectural protein Pita and the BTB domain of CP190, we attempted to precisely map the interaction regions in Pita
Summary
The development of modern approaches for the study of genome architecture, including chromosome conformation capture methods, coupled to high-throughput sequencing (Hi-C) and high-resolution microscopy techniques has revealed the hierarchical organization of genome [1, 2]. Regulatory elements, including enhancers, promoters, insulators, and silencers, actively interact with each other, which determines the correct and stable level of gene expression [6, 7]. The boundaries between TADs delineate specific genomic regions, and more effective interactions between regulatory elements occur within these regions than between different regions [8]. Because the LBD1 protein is the only one of these proteins to contain a well-described homodimerization domain [14], how specific interactions between enhancers and promoters occurs remains unclear. Pita is required for Drosophila development and binds to a long motif in active promoters and insulators. 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
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