Chromatin immunoprecipitation (ChIP) is basically a fancy term for a technique that allows you to see where a protein of interest binds DNA. It achieves this by crosslinking DNA and protein, chopping up the DNA and then extracting the DNA fragments that are attached to the protein you’re interested in using antibodies. Chen et al. (1) have recently adapted this protocol to enable them to look at the location of R-loops within a cell. The aim of this approach was to increase our understanding that R-loops play and how they affect transcription. Interestingly, the team found that most of the R-loops captured were associated with gene promoters and were implicated with transcriptional pausing.
What are R-loops?
In the simplest sense, DNA in our cells is double stranded, whilst RNA is single stranded. An R-loop forms when the RNA strand can open up the DNA helix and bind to one of the strands creating a 3-stranded hybrid of DNA and RNA. So basically, the RNA displaces the complementary hydrogen bonding between the two DNA strands. The non-template single-stranded DNA is left to loop out.
R-loops form in a variety of contexts. One example is during transcription, when the nascent mRNA can interact with the DNA strand that was just used as a template to synthesise it. With one of the DNA strands looped out, this leaves highly actively transcribed genes vulnerable to DNA damage (Figure 1). Being able to locate R-loops in vivo will thus provide a better insight into gene transcription regulation and sights of DNA damage. But how do you locate R-loops?
The principle of ChIP involves probing for a protein of interest by using an antibody that recognises that protein. RNASEH1, was the protein chosen by Chen’s team to locate R-loops. For those of you who know you RNases this might seem concerning. This is because RNaseH preferentially binds RNA/DNA complexes and catalyses the cleavage of RNA. However, by using a mutated version of RNaseH making it ‘catalytically dead’, the team could exploit the preference of RNA/DNA binding to find R-loops without the disastrous consequence of the RNA being cleaved and degraded.
Once the R-loops were mapped it was found that the majority were found at gene promotors. Prevention of transcription elongation due to the addition of DRB (an inhibitor of the positive elongation factor P-TEFb), led to increased promoter-proximal pausing* and R-loop formation. The authors also showed that R-loop formation was promoted in G/C rich regions as the RNA/DNA hybrid would be more stable (3 hydrogen bonds compared with 2 in A/T, U/A binding) and a free RNA end which would enable efficient RNA invasion into DNA. Thus, R-loop presence was correlated both with increased promoter-proximal pausing and increased G/C content however whether the former is a cause or consequence is unclear. Nevertheless, R-ChIP is a great new method to map R-loops however it is currently limited by the requirement for mutant RNaseH to be expressed in the cells of interest.
*unsure of promoter proximal pausing.. read further reading (3), but in brief is the pausing of the polymerase soon after transcription has been initiated and may serve as an additional regulatory step
(A brief aside) Introns to the rescue
Introns – the non-coding regions of mRNA that are spliced out soon after transcription – were first characterised by Roberts and Sharp in 1976. Since mRNA lacks intron sequences that are still present in DNA, the DNA ‘intron’ sequences have to loop out whilst hybridised to mRNA (Figure 2). Interestingly, a recent study by Bonnet et al. (2) found that introns may serve to attenuate R-loop formation and consequently prevent the extent of DNA damage accumulation. Evidence for this came by inserting introns into intron-less genes that then showed decreased DNA damage. This additional role for introns may explain why they are so conserved throughout eukaryotes. Further work should examine how this instability varies in genetic variation and whether this serves as a increased risk for the development of diseases like cancer.
(1) Chen. R-ChIP using inactive RNase H reveals dynamic coupling of R-loops with transcriptional pausing at gene promoters Molecular Cell 68, 745-757 (2017)
(2) Bonnet. Introns protect eukaryotic genomes from transcription-associated genetic instability Molecular Cell 67, 608-621 (2017)
(3) Adelman. Promoter proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat Rev Genetics 13, 720-731 (2012)