Detection and characterization of R-loops at the murine immunoglobulin Sα region

IgA is the most abundant antibody in mammals. However, the mechanism of its class switching is still not clear. The formation of the R-loops, as the target for AID, has been proposed to play a crucial role during mammalian class switch recombination. Here, we provide a systematic evaluation of R-loops at Sα (IgA) in CH12F3-2A cells, which is a unique cell model system for class switch recombination because of its consistent switching to IgA upon stimulation. The results of R-loop analysis demonstrate distinct features specific to Sα. Some R-loops may initiate from the end of Iα, but all terminate exclusively within Sα. Time-course analysis also indicates that the percentage of R-loops peaks prior to the occurrence of class switch recombination. This is the first demonstration that R-loops form at Sα in vitro and in situ, despite variable G density and relatively few GGGG clusters in Sα. The short distance from the promoter to Sα may compensate for the less robust R-loop-forming factors at Sα relative to other switch regions. In conclusion, R-loops at the Sα region further support R-loop formation as a general feature of all stimulated switch regions.

► The systematic characterization of R-loops is performed in vitro and in situ at Sα in a murine model cell line of CSR. ► R-loop formation is detected within Sα and might be promoted by the short distance from the promoter. ► Time-course analysis results strongly support that R-loops are an intermediate of CSR. ► The theory that R-loops are a general feature of mammalian stimulated switch regions is supported by the identification of R-loops at Sα.