Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization.

In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (Fru^M) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of Fru^M in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of Fru^M. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru⁺ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, Fru^M appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find Fru^M motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that Fru^M acts cell-type-specifically to decommission regulatory elements in male fruitless neurons. Author summary: Courtship behavior in male Drosophila melanogaster is controlled by a well-defined neural circuit that is labeled by the male-specific transcription factor Fruitless (Fru^M). While Fru^M is known to change the number, anatomy and connectivity of neurons which comprise the circuit and has been suggested to repress the expression of a few gene targets, the mechanism of how Fru^M regulates genes across many different kinds of neurons is unknown. Using an approach to identify gene regulatory elements based on their chromatin accessibility states (ATAC-seq), we identified a large set of chromatin accessibility changes downstream of Fruitless. By examining the activity of these regulatory elements in-vivo, we found that their activity was 1) sexually dimorphic and 2) specific to a single class of Fru^M neurons, suggesting that Fru^M acts on different chromatin targets in different neuron classes comprising the courtship circuit. Further, we found a known Fru^M-regulated enhancer of the Fru^M-repressed gene Lgr3 to have closed chromatin specifically in Fru^M neurons. Combined with an enrichment of Fru^M motifs in regions which are closed in Fru^M neurons, we present a mechanism where Fru^M directs the decommissioning of sex-shared regulatory elements to masculinize neurons in a cell-type specific manner. [ABSTRACT FROM AUTHOR]