Abstract
Most of the known Drosophila architectural proteins interact with an important cofactor, CP190, that contains three domains (BTB, M, and D) that are involved in protein–protein interactions. The highly conserved N-terminal CP190 BTB domain forms a stable homodimer that interacts with unstructured regions in the three best-characterized architectural proteins: dCTCF, Su(Hw), and Pita. Here, we identified two new CP190 partners, CG4730 and CG31365, that interact with the BTB domain. The CP190 BTB resembles the previously characterized human BCL6 BTB domain, which uses its hydrophobic groove to specifically associate with unstructured regions of several transcriptional repressors. Using GST pull-down and yeast two-hybrid assays, we demonstrated that mutations in the hydrophobic groove strongly affect the affinity of CP190 BTB for the architectural proteins. In the yeast two-hybrid assay, we found that architectural proteins use various mechanisms to improve the efficiency of interaction with CP190. Pita and Su(Hw) have two unstructured regions that appear to simultaneously interact with hydrophobic grooves in the BTB dimer. In dCTCF and CG31365, two adjacent regions interact simultaneously with the hydrophobic groove of the BTB and the M domain of CP190. Finally, CG4730 interacts with the BTB, M, and D domains of CP190 simultaneously. These results suggest that architectural proteins use different mechanisms to increase the efficiency of interaction with CP190.
Highlights
Introduction published maps and institutional affilA large family of transcription factors found in most higher eukaryotes comprises proteins containing a common highly conserved protein–protein interaction domain known as the Broad-complex, Tramtrack, and Bric-à-brac/poxvirus (BTB) domain [1,2]
The goal of this work was to identify novel potential architectural proteins with clusters of C2H2-type zinc-finger domains (C2H2) domains that interact with the BTB domain of CP190 and to understand the mechanisms of CP190 recruitment on the chromatin sites by the C2H2 proteins
To identify new potential architectural proteins that interact with the BTB domain of CP190, we used a library of 154 Drosophila C2H2 proteins attached to the activation domain of GAL4 (Supplementary Materials Table S1)
Summary
A large family of transcription factors found in most higher eukaryotes comprises proteins containing a common highly conserved protein–protein interaction domain known as the Broad-complex, Tramtrack, and Bric-à-brac/poxvirus (BTB) domain [1,2]. Transcription factors with the BTB domain (BTB TFs) have diverse functions in transcriptional regulation, chromatin remodeling, and protein degradation [3]. A considerable fraction of the BTB TFs, which are well characterized in Drosophila melanogaster, mice, and humans, contain C2H2-type zinc-finger domains (C2H2) [5]. The clusters of C2H2 domains in TFs are usually involved in high-affinity binding to specific sites on chromatin [6,7]. The human BCL6 protein contains the best-characterized BTB domain, which exists as a stable obligate homodimer [8].
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