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R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells

Abstract

The mechanism responsible for immunoglobulin class switch recombination is unknown. Previous work has shown that class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the γ3 or the γ2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the class switch recombination mechanism

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Figure 1: In vitro transcription of class switch sequences.
Figure 2: Detection of single-stranded regions with bisulfite.
Figure 3: In vitro–transcribed Sγ3 sequences analyzed with bisulfite.
Figure 4: In vivo structure of Sγ3 after LPS stimulation.
Figure 5: Evidence of R-loops at Sγ2b in LPS-stimulated B cells.

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Acknowledgements

We thank F. Huang and M. Principale for assistance; and I. Haworth and J. Lee for discussions. These studies were supported by grants from the National Institutes of Health (M.R.L.). Manuscripts on in vitro class switch R-loops and in vivo R-loops49,50 were retracted very shortly after publication in 2000 because of data alteration by R. B. Tracy. There has been no reliance on the data or reagents of R. B. Tracy in this study or in any other studies by the Lieber laboratory.

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Correspondence to Michael R. Lieber.

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Yu, K., Chedin, F., Hsieh, CL. et al. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 4, 442–451 (2003). https://doi.org/10.1038/ni919

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