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Detection and characterization of R-loops at the murine immunoglobulin Sα region
Yu-Pu Kao1, Wen-Chuan Hsieh, Shu-Ting Hung
1Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, Taiwan.
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 Sαin 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.
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 Sαin 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.