Abstract
R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIP-seq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In intron-containing genes, R-loops are bounded between the transcription start site and the first exon-intron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation.
Highlights
R-loops are nucleic acid structures in which a strand of RNA is hybridized to a strand of DNA, while the other strand of DNA is looped out
To understand where R-loops are positioned in genomic promoter regions, we developed bisDRIP-seq. bisDRIP-seq is an approach to map R-loops at near-nucleotide resolution throughout the genome
Cytosines in the RNA-DNA hybrid portion of the R-loop are protected from bisulfite conversion
Summary
R-loops are nucleic acid structures in which a strand of RNA is hybridized to a strand of DNA, while the other strand of DNA is looped out. More recent studies provided evidence that R-loops in these critical regions can alter histone modifications and are associated with changes in gene transcription (Chen et al, 2015; Colak et al, 2014; Sun et al, 2013). The location of an R-loop in a gene is likely to dictate how that R-loop can impact promoter function. This is because eukaryotic promoter regions contain multiple functional domains that have distinct roles in transcription, including transcription start sites, transcription factor-binding sites, exon-intron junctions, CpG islands, and nucleosome-associated DNA (Lenhard et al, 2012). Understanding the precise location of R-loops can provide insight into how R-loops affect gene transcription
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