Physical interaction between MSL2 and CLAMP assures direct co-operativity and prevents competition at composite binding sites

Abstract

AbstractMSL2, the DNA-binding subunit of theDrosophiladosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. We explore the nature of the cooperative binding to these GA-rich, composite se-quence elements in reconstituted naïve embryonic chromatin.We found that the cooperativity requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition.High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. We find that the cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs.The sites where MSL2/CLAMP bind stronglyin vitrolocate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2in vivobinding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sitesin vivo. This restriction must be due to components missing in our reconstitution, such asroX2lncRNA.