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 gamma3 or the gamma2b 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

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

“…R loops were analysed using bisulfite conversion of single-strand DNA 46 . R loop experiments were schematized in the Supplementary Fig.…”

DNA structures at chromosomal translocation sites

“…(32) RNA:DNA hybrids form during class switch recombination in vitro and in vivo; these have a classic R-loop conformation. (47,48) The RNA is generated by transcription, which is requisite for the R-loop formation. (47,48) The displaced DNA strand, which is G-rich, appears to remain as singlestranded DNA.…”

“…(47,48) The RNA is generated by transcription, which is requisite for the R-loop formation. (47,48) The displaced DNA strand, which is G-rich, appears to remain as singlestranded DNA. (47) However, EM data of these sequences in vitro have been interpreted as suggesting G-quartet formation.…”

“…(47,48) The displaced DNA strand, which is G-rich, appears to remain as singlestranded DNA. (47) However, EM data of these sequences in vitro have been interpreted as suggesting G-quartet formation. (49) Recent examples of well-studied non-B DNA structures, which are pathologically relevant are described below.…”

LncRNAs: Bridging environmental sensing and gene expression

“…Aberrant formation and/or stabilization of R-loops results in genomic instability as a result of prolonged exposure of a single-stranded DNA to the environment and/or by interfering with DNA replication machinery. 61,62 In contrast, R-loops play beneficial roles by mediating CRISPR interference, 63,64 promoting IgG class switching, 65,66 and by regulating transcription. 67,68 These structures can form in cis 60 or trans 69 in vivo, either by "threading back" of nascent RNAs during transcription by RNA Pol II, through the action of the homologous recombination machinery, or associated RNA-binding proteins as in the case of CRISPR.…”