Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior [TRAP I]
Chromatin modifications have essential roles in directing nervous system development and behavior. Drosophila courtship is an ideal model for understanding the relationships between chromatin modifications, gene expression, sexual dimorphisms in neuroanatomy, and reproductive behaviors. These behaviors are largely innate and are under the control of sex determination hierarchy genes doublesex (dsx) and fruitless (fru) that encode sex-specific transcription factors. Transcripts from the fru P1 promoter are sex-specifically spliced, resulting in male-specific proteins, FruM, which are required for male courtship behaviors. FruM has been shown to form a complex with Bonus/TIF1, a transcriptional cofactor, and interact with chromatin modifying proteins. This indicates that sex-differential modification of the chromatin landscape contributes to creating and maintaining the potential for sexually dimorphic behavior. To examine chromatin modifications genome-wide in fru P1-expressing neurons, we have developed a method to purify chromatin in a cell-type-specific manner using a tagged histone H2B under UAS control, “Chromatag”, paired with sequential ChIP-seq. Here, we evaluate five chromatin modifications: H3K27ac, H3K4me3, H3K36me3, H3K9me3, and H3K27me3 to investigate fru P1-expressing neurons at three developmental stages in both sexes. These modifications have been well characterized for their roles in transcriptional activation or repression. We observe greater changes in chromatin modification profiles across developmental stages than between sexes. In addition, we have generated cell-type specific RNA-seq data sets, using Translating Ribosome Affinity Purification (TRAP), to examine gene expression in fru P1-expressing neurons of both sexes. Informed by these data sets, we have identified several genes with roles in neuronal function and examined their expression in fru P1-expressing neurons, revealing sexually dimorphic expression patterns. Altogether, this work offers insights into stage and sex-differences in chromatin modifications and gene expression in fru P1-expressing neurons. Overall design: Cell-type-specific RNA-seq libraries for fru P1-expressing neurons were generated using Translating Ribosome Affinity Purification (TRAP). fru-P1 TRAP was performed at 48hr APF when fruM is at its peak expression in males and to coincide with the 48hr APF chromatin modification data in our study. TRAP (IP) and total mRNA (input) samples were prepared from whole 48hr APF pupae from both males and females with 4-5 biological replicates per sex.