Multi-omics to characterize the functional relationships of R-loops with epigenetic modifications, RNAPII transcription and gene expression.

Abnormal accumulation of R-loops results in replication stress, genome instability, chromatin alterations and gene silencing. Little research has been done to characterize functional relationships among R-loops, histone marks, RNA polymerase II (RNAPII) transcription and gene regulation. We built extremely randomized trees (ETs) models to predict the genome-wide R-loops using RNAPII and multiple histone modifications chromatin immunoprecipitation (ChIP)-seq, DNase-seq, Global Run-On sequencing (GRO-seq) and R-loop profiling data. We compared the performance of ET models to multiple machine learning approaches, and the proposed ET models achieved the best and extremely robust performances. Epigenetic profiles are highly predictive of R-loops genome-widely and they are strongly associated with R-loop formation. In addition, the presence of R-loops is significantly correlated with RNAPII transcription activity, H3K4me3 and open chromatin around the transcription start site, and H3K9me1 and H3K9me3 around the transcription termination site. RNAPII pausing defects were correlated with 5'R-loops accumulation, and transcriptional termination defects and read-throughs were correlated with 3'R-loops accumulation. Furthermore, we found driver genes with 5'R-loops and RNAPII pausing defects express significantly higher and genes with 3'R-loops and read-through transcription express significantly lower than genes without R-loops. These driver genes are enriched with chromosomal instability, Hippo-Merlin signaling Dysregulation, DNA damage response and TGF-β pathways, indicating R-loops accumulating at the 5' end of genes play oncogenic roles, whereas at the 3' end of genes play tumor-suppressive roles in tumorigenesis.