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

Abstract

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.

Fig 1. R-loop identification & feature enrichment in D. melanogaster adults.

Whole adult flies from strains DGRP379 and DGRP732 were separated by sex and subjected to DRIP-sequencing to detect R-loops. (A) DRIP-seq peak density across chromosomes for the full male and female datasets (i.e. without downsampling) shows an apparent depletion of X-linked R-loops in males. (B) Downsampling all female DRIP-seq reads and autosomal male DRIP-seq reads so that all chromosome arms have similar sequencing depths shows that X-linked R-loops are enriched in both males and females (Binomial test, * = p < 2.2e-16). (C) R-loop formation at chromatin states as described in [34]. (D) R-loop formation at various genetic features. (E) Metaprofiles of R-loop signal across protein-coding genes, autosomes versus X chromosome. For Panels (C) and (D): R-loop enrichment is shown as the observed number of DRIP-seq peaks overlapping each feature (or chromatin state) divided by the expected number of peaks (see Methods). P-values were calculated via a Permutation Test with Benjamini-Hochberg correction for multiple comparisons, * = corrected p < 0.05. For Panel (E): the solid lines represent the mean DRIP-seq signal within each metagene bin, and the shading represents the standard error of the mean.

Fig 2. Differential enrichment and motif analysis of R-loops.

(A) PCA analysis of DRIP conditions. (B) Venn diagram of non-differentially enriched (nonDE) and sex-biased (Female Enriched [FE] and Male Enriched [ME]) R-loops as identified by DiffBind. (C) STREME motif analysis by DE group; the top 5 motifs from each DE group are represented graphically (left), with z-score enrichment for each motif across DE groups plotted in the heatmap (right). (D) Motif enrichment on the X chromosome versus autosomes, plotted as log2 motifs per Mb. Binomial test, * = p < 0.001. (E) R-loop formation at chromatin states as described in [34]. (F) R-loop formation at various genetic features. (G) Metaprofiles of R-loop signal at genes within each DE group. For Panels (E) and (F): R-loop enrichment is shown as the observed number of DRIP-seq peaks overlapping each feature (or chromatin state) divided by the expected number of peaks (see Methods). P-values were calculated via a Permutation Test with Benjamini-Hochberg correction for multiple comparisons, * = corrected p < 0.05. For Panel (G): the solid lines represent the mean DRIP-seq signal within each metagene bin, and the shading represents the standard error of the mean.

Fig 3. X chromosome-specific R-loop enrichment.

(A) Differentially enriched R-loops by chromosome, plotted as R-loop peaks per Mb. Binomial test, * = p < 0.001. (B) Differentially enriched R-loop frequency on autosomes versus X chromosome, plotted as a fraction of total R-loops per DE group. (C) Gene expression analysis of R-loop-containing genes by DE group versus R-loop-absent (no R-loop) genes, on autosomes and the X chromosome, plotted as rlog-normalized expression. Wilcoxon test, *, **, *** = p < 0.05, 0.01, 0.001. (D) Distance to chromosomal entry site (CES) on the X chromosome across DE groups, plotted in log₂ base pairs (bp). Wilcoxon test, *** = p < 0.001. (E) MOF binding and (F) H4K16ac enrichment on the X chromosome in third-instar larva male salivary glands across DE groups. Wilcoxon test, **, *** = p < 0.01, 0.001. (G) MOF binding and (H) H4K16ac enrichment on autosomes and the X chromosome in third-instar larva female salivary glands across DE groups. Wilcoxon test, *,**,*** = p < 0.05, 0.01, 0.001.