Investigations into the roles of R-loops in androgen signalling and prostate cancer

Background
Prostate cancer is the 2nd commonest cause of male cancer death in the UK. Growth of both normal and neoplastic prostate requires androgen signalling. However, androgen signalling mechanisms have not been fully elucidated. Recently, R-loops (three stranded, co-transcriptional DNA:RNA hybrids) have been implicated in DNA damage and regulation of gene expression. The aim of this project was to evaluate what role R-loops play in androgen regulated gene expression.

Methods
Prostate cancer cells were stimulated with androgens and the resulting changes in R-loop dynamics were assessed by 1) Predictive modelling based on nucleotide sequence contexts favouring R-loop formation and 2) DNA:RNA immunoprecipitation with next generation sequencing (DRIP-seq). The functional roles of R-loops were probed by overexpression of RNAse H1, a well characterised R-loop resolvase. R-loop interacting proteins were identified by DNA:RNA immunoprecipitation and Western blotting. The functions of these proteins were examined by siRNA knockdown experiments, DNA damage response assays, analysis of gene expression and chromatin immunoprecipitation.

Results
Predicted R-loop forming sequences were higher in genes downregulated in metastatic castration resistant prostate cancer. In LNCaP cells stimulated with androgens there was no correlation between gene expression and R-loop formation. R-loop dynamics did not change at androgen receptor bound enhancers. RNA-seq in cells overexpressing RNase H1 and treated with DHT showed unchanged canonical androgen signalling but did show potential off-target effects of RNase H1 overexpression. Two androgen responsive genes – NKX3.1 and KLK3 – accumulated R-loops. Only NKX3.1 accumulated R-loops with androgen treatment. Knock down of the DEAD box helicases DDX5 and DDX17 affected NKX3.1 and KLK3 gene expression and dysregulated global R-loop levels. DDX17 was recruited to the NKX3.1 R-loop but depletion of DDX5/DDX17 did not have locus-specific R-loop effects.

Conclusions
R-loops do not have general roles in androgen regulated gene expression but they may play a role in regulating specific loci.