Abstract
R-loops are three stranded nucleic acid structures involving an RNA:DNA hybrid and a displaced single stranded DNA (ssDNA). Though the majority of R-loop studies have investigated their pathological consequences in promoting genomic instability, R-loops also contribute to many physiological processes. In fact, from meta-analysis of R-loop datasets we know that R-loops cover about 3–5% of human genome, with their abundance tightly titrated by different enzymes or helicases; too many or too few R-loops impact normal cell functions. Aberrant R-loop accumulation has been implicated in cancer susceptibility and neurodegeneration, and increased R-loops levels throughout the genome observed in response to oncogenic signaling or mutations results in increased replication stress and DNA damage. Nonetheless, this also confers a vulnerability, and cancer cells harboring high levels of R-loops can be preferentially targeted by drugs that exacerbate R-loop-associated phenotypes. Here, we establish a protocol to detect RNA:DNA hybrids by immunohistochemistry (IHC) using the mouse and rabbit S9.6 antibodies. Using R-loop enhancing drugs, or by genetically manipulate DHX9 and SETX expression, helicases involved in R-loop metabolism, we provide evidence that our protocol is able to detect differences in R-loop levels. Finally, we show that S9.6 IHC is uniquely able to rapidly screen hundreds of cell and tumor samples demonstrating the heterogeneity in R-loop signal that can be observed. We also describe for the first time that R-loop expression determines sensitivity to the active vitamin D metabolite Calcitriol.