Mechanisms of CP190 Interaction with Architectural Proteins in Drosophila Melanogaster

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.

Proximity labeling reveals a newin vivonetwork of interactors for the histone demethylase KDM5

“…Among these, we find proteins such as Boundary Element-Associated Factor of 32kDa (BEAF-32), Chromator (Chro), Putzig (Pzg), and Centrosomal protein 190kDa (Cp190) that function in regulating genomic architecture, suggesting a unstudied role for KDM5 in this process. (64)(65)(66)(67)(68)(69) In addition, we identified proteins critical for forming the transcriptional pre-initiation complex (TPIC), which is consistent with the promoter-proximal binding of KDM5 proteins across species. (25,44,(70)(71)(72) Using a recent cryo-EM structure of the human TPIC, we found that distinct surfaces interacted with KDM5, consistent with the specificity of biotinylation using TurboID-KDM5.…”

“…Among these, we find proteins such as BEAF-32, Chromator (Chro), Putzig (Pzg), and Cp190 that function in regulating genomic architecture, suggesting a unstudied role for KDM5 in this process. [66][67][68][69][70][71] To confirm our MS data, we performed western blots on input and streptavidin pulldowns and probed with antibodies specific for BEAF-32 and CP190. This confirmed enrichment of these two proteins in both NT-KDM5 and CT-KDM5 compared to dCas9:TurboID-expressing and control flies (Fig.…”

BTB domains: A structural view of evolution, multimerization, and protein–protein interactions

“…[62] The same mechanism was proposed for the interaction between the BTB of the Drosophila CP190 protein and unfolded peptides from architectural proteins. [63] Most of these peptides contain two short conserved hydrophobic regions that bind to the hydrophobic groove of the CP190 BTB domain according to mutagenesis data. [63] 2.…”

“…[63] Most of these peptides contain two short conserved hydrophobic regions that bind to the hydrophobic groove of the CP190 BTB domain according to mutagenesis data. [63] 2. Miz1-HUWE-1 interaction.…”

The Tramtrack group BTB protein domains are Arthropoda-specific multimerization modules

“…The most well-studied of these, CP190, has four C2H2 zinc finger domains that appear to be involved in protein-protein interactions rather than DNA binding [29, 30]. The N-terminal BTB/POZ domain of CP190 forms stable homodimers [21, 29, 31-33]. CP190 is required for the activity of housekeeping promoters and insulators [34-38].…”