Transcriptional regulation is crucial for a number of biological processes and involves the binding of sequence-specific transcription factors (TFs) which in turn affect the recruitment of RNA polymerase II and the rate of gene expression. This process is now known to involve many other layers of regulation such as the structure of chromatin. Genomic DNA can adopt many different structures apart from the usual B-form double stranded DNA (dsDNA), one such structure being an R-loop.
R-loops are formed when dsDNA is separated and a complementary ssRNA is able to bind one of the DNA strands, resulting in a RNA/DNA hybrid and a region of ssDNA. The development of recent techniques such as DNA-RNA immunoprecipitation (DRIP), a technique analogous to ChIP have shown that these structures are abundant throughout the mammalian genome. Furthermore, specific R-loops have bene shown to act as mediators of transcriptional regulation and cellular differentiation. These structures are particularly enriched in promoter regions, leading us to hypothesise that they play a role in transcriptional regulation and, more specifically, in modulation of TF binding.
To investigate this potential role, we will first map the location of R-loops genome wide in erythroid cell lines and combine this with publicly available data to identify regions of interest. We will then aim to target these structures to identify effects that R-loops have on TF binding, chromatin accessibility and/or gene expression.