Sex-specific variation in R-loop formation in Drosophila melanogaster

Author summaryR-loops are DNA:RNA hybrid structures that act as important regulators of gene expression and genomic stability and whose dysregulation can contribute to diseases such as neurological disorders and cancer. Here, we utilize DNA:RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq) to assess the sex-specific variability in R-loop formation in Drosophila melanogaster adults. Most R-loops are found at simple repeats in regions of high transcriptional activity such as active chromatin states, 5'UTRs, tRNAs, and topologically associating domain boundaries. In both sexes, we find that R-loops are more common on the X chromosome compared to autosomes, likely due to an increased density of X-linked simple repeats that favor R-loop formation. While R-loops are largely conserved between sexes, we uncover a small but significant subset of sex-biased R-loops. Female-enriched R-loops are associated with genes showing ovary-biased expression and form at unique genome features, compared to other R-loops. Male-enriched R-loops occur preferentially at dosage-compensated genes on the X chromosome, yet surprisingly, show reduced levels of the marker of dosage compensation, H4K16ac, raising the possibility that the H4K16ac histone modification may attenuate R-loop formation. Our identification of sex-biased R-loops suggests a specialized role for these structures in establishing and maintaining sex-specific epigenetic programs. R-loops are three-stranded nucleotide structures consisting of a DNA:RNA hybrid and a displaced ssDNA non-template strand. Previous work suggests that R-loop formation is primarily determined by the thermodynamics of DNA:RNA binding, which are governed by base composition (e.g., GC skew) and transcription-induced DNA superhelicity. However, R-loops have been described at genomic locations that lack these properties, suggesting that they may serve other context-specific roles. To better understand the genetic determinants of R-loop formation, we have characterized the Drosophila melanogaster R-loop landscape across strains and between sexes using DNA:RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq). We find that R-loops are associated with sequence motifs that are G-rich or exhibit G/C skew, as well as highly expressed genes, tRNAs, and small nuclear RNAs, consistent with a role for DNA sequence and torsion in R-loop specification. However, we also find motifs associated with R-loops that are A/T-rich and lack G/C skew as well as a subset of R-loops that are enriched in polycomb-repressed chromatin. Differential enrichment analysis reveals a small number of sex-biased R-loops: while non-differentially enriched and male-enriched R-loops form at similar genetic features and chromatin states and contain similar sequence motifs, female-enriched R-loops form at unique genetic features, chromatin states, and sequence motifs and are associated with genes that show ovary-biased expression. Male-enriched R-loops are most abundant on the dosage-compensated X chromosome, where R-loops appear stronger compared to autosomal R-loops. R-loop-containing genes on the X chromosome are dosage-compensated yet show lower MOF binding and reduced H4K16ac compared to R-loop-absent genes, suggesting that H4K16ac or MOF may attenuate R-loop formation. Collectively, these results suggest that R-loop formation in vivo is not fully explained by DNA sequence and topology and raise the possibility that a distinct subset of these hybrid structures plays an important role in the establishment and maintenance of epigenetic differences between sexes.