Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Abstract

The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Sμ locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Sμ R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Sμ repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Sμ repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Sμ repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.

SUPPLEMENTAL MATERIAL

We thank Michael S. Neuberger for providing detailed information about the precise locations of previously described mutations (34). We also thank members of the Lieber laboratory for comments on the paper.

This work was supported by NIH grants to M.R.L. and E.S. and by grants to A.A.K. from ARC (no. 3789), LNCC (comite Tarne-et-Garonne), and Canceropole GSO (ACI 2004-2007).