Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior [ChIP]
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 examination of 5 histone modifications and histone H3 in Drosophila fru P1-expressing and elav expressing neurons for males and females at three time-points (life stages): 48hr after puparium formation (48hAPF), 16-24hr adults (1-day adults), and 10-12 day adults. Libraries were prepared from 3-4 independent biological replicates per modification, time-point, and sex. Chromatin samples were obtained from whole pupae for 48hr APF or whole head tissue for adult stages. Sex and stage specific input chromatin libraries were generated for each stage and sex examined in our study.