Metazoan transcription factors distinguish their response elements from a large excess of similar sequences. We explored underlying principles of DNA shape read-out and factor cooperativity in chromatin using a unique experimental system. We reconstituted chromatin on Drosophila genomes in extracts of preblastoderm embryos, mimicking the naïve state of the zygotic genome prior to developmental transcription activation. We then compared the intrinsic binding specificities of three recombinant transcription factors, alone and in combination, with GA-rich recognition sequences genome-wide. For MSL2, all functional elements reside on the X chromosome, allowing to distinguish physiological elements from non-functional ‘decoy’ sites. The physiological binding profile of MSL2 is approximated through interaction with other factors: cooperativity with CLAMP and competition with GAF, which sculpts the profile by occluding non-functional sites. An extended DNA shape signature is differentially read out in chromatin. Our results reveal novel aspects of target selection in a complex chromatin environment.
MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, coop-erates 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 sequence elements in reconstituted naive embryonic chromatin. We found that the cooperativity requires physical interaction between both proteins. Re-markably, 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 strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in our reconstitution, such as roX2 lncRNA.
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