S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.
R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast.
Department of Cell and Molecular Biology, University of California at Berkeley, Berkeley, California 94720, USA;
Lamia
L
Wahba
Department of Cell and Molecular Biology, University of California at Berkeley, Berkeley, California 94720, USA;
Lorenzo
L
Costantino
Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland 21218, USA.
Frederick J
FJ
Tan
Department of Cell and Molecular Biology, University of California at Berkeley, Berkeley, California 94720, USA;
Anjali
A
Zimmer
Department of Cell and Molecular Biology, University of California at Berkeley, Berkeley, California 94720, USA;
Douglas
D
Koshland
D002874:Chromosome Mapping; D004271:DNA, Fungal / Q000378:metabolism; D015870:Gene Expression; D016681:Genome, Fungal / Q000235:genetics*; D023281:Genomics; D006657:Histones / Q000378:metabolism; D011061:Poly A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D012331:RNA, Fungal / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004271:DNA, Fungal; D006657:Histones; D012331:RNA, Fungal; D011061:Poly A; D015719:Single-Strand Specific DNA and RNA Endonucleases
DNA:RNA hybrids; R loops; RNase H; polyA tracts; transcription