The Strength of an Ig Switch Region Is Determined by Its Ability to Drive R Loop Formation and Its Number of WGCW Sites

• Published in: Cell reports (Cambridge) Vol. 8; no. 2; pp. 557 - 569
• Main Authors: Zhang, Zheng Z., Pannunzio, Nicholas R., Han, Li, Hsieh, Chih-Lin, Yu, Kefei, Lieber, Michael R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.07.2014; Elsevier
• Subjects: Animals; B-Lymphocytes - metabolism; Cell Line, Tumor; GC Rich Sequence; Immunoglobulin Class Switching; Immunoglobulin Switch Region; Mice; Nucleic Acid Conformation
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2014.06.021

R loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R loop formation, and on the number of WGCW sites. [Display omitted] •Ig switch region strength is dependent on R loop formation•Ig switch region strength is dependent on the total number of WGCW sites•The R loop formation requires sequences rich in G clusters•G clusters can be at the upstream edge of or interspersed throughout the switch region Immunoglobulin heavy chain class switch recombination is the process by which IgM is converted to IgG, IgA, or IgE. Zhang et al. describe the DNA sequence requirements for the Ig switch regions at which DNA recombination occurs. Although the number of WGCW sites is important to provide AID cytosine deamination sites, the G clusters are needed to initiate R loop formation and provide the single-stranded DNA that is required for AID to act.