RNA-dependent DNA sequence specificity for X chromosome regulation

Chromatin regulatory complexes must selectively recognize specific loci within the vast genome to regulate gene expression. Beyond DNA motifs and histone marks, lncRNA scaffolding and biomolecular condensation are emerging as critical determinants of chromatin targeting across species. How contributions of proteins, DNA, and RNA components within epigenetic complexes are integrated to achieve precise genomic targeting remains unresolved. X chromosome dosage compensation in male Drosophila, mediated by the Male-Specific Lethal (MSL) complex, provides a powerful model for dissecting these mechanisms. Here, using time-resolved single-molecule optical tweezers assays, we show that MSL1, the central interaction platform of the MSL complex, can bind DNA sequence-specifically, but only within narrow concentration windows, outside of which binding becomes non-specific. In the presence of roX2 RNA, MSL1 recruits the RNA to DNA, where it promotes local accumulation of MSL1 at its target sites and restores sequence-specific binding across a broad concentration range. This highlights how an lncRNA can effectively fine-tune a protein’s DNA-binding specificity. In vivo, the MSL1 N-terminal disordered domain mediates interaction with roX2 lncRNA. Deletion of this region disrupts RNA binding, impairs X-chromosome localization, and leads to male lethality, underscoring its essential role in dosage compensation. Our findings reveal that MSL1 is not a passive scaffold but an active molecular integrator, simultaneously reading DNA sequence information and engaging lncRNA co-factors to organize a functional chromatin domain across the male X chromosome. We propose that local concentration of MSL1, mediated by roX2 and likely facilitated by MSL1-MSL1 interactions and biomolecular condensation, underpins MSL complex targeting in vivo. Our reconstituted three component DNA-RNA-protein system coupled with time resolved single-molecule measurements lays the groundwork for uncovering the fundamental principles that govern chromatin targeting mechanisms.