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Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination.

ABSTRACT: R-loops form at Sgamma3 and Sgamma2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine Sgamma3 and Sgamma2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the Sgamma3 and the Sgamma2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination.

SUBMITTER: Huang FT

PROVIDER: S-EPMC1458789 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Inflammation and tissue damage in systemic lupus erythematosus (SLE) are mediated by class-switched autoantibodies reactive with nucleic acids, nucleic acid-binding proteins, phospholipids and other self-antigens. While some healthy individuals produce IgM antibodies with specificities similar to those of lupus patients, immunoglobulin class switching to mature downstream isotypes appears to be required for the generation of pathogenic autoantibodies. To characterize the cellular and molecular basis of pathogenic autoantibody production in SLE, we studied the capacity of peripheral blood B cells of naive phenotype from patients with SLE, rheumatoid arthritis (RA) or healthy control subjects to spontaneously switch to IgG and IgA. In addition, we determined the DNA sequences of the upstream evolutionary conserved sequence (ECS)-Igamma promoter regulatory regions that control germline IH-CH transcription and class switch DNA recombination (CSR) to IgG1, IgG2 and IgG4. IgM+IgD+ B cells from patients with SLE, but not those from RA or healthy control subjects, underwent spontaneous CSR, as assessed by expression of germline Igamma1-Cgamma1, Igamma2-Cgamma2, Igamma3-Cgamma3, Igamma4-Cgamma4 and Ialpha1-Calpha1 transcripts, mature (switched) VHDJH-Cgamma1, VHDJH-Cgamma2, VHDJH-Cgamma3 and VHDJH-Calpha1 transcripts and secreted IgG and IgA. Although polymorphic DNA sequences were identified in the ECS-Igamma1, ECS-Igamma2 and ECS-Igamma4 promoter regions, the transcription factor-binding sites that mediate germline Igamma-Cgamma transcription were conserved in patients and controls. However, distinct patterns of nuclear protein binding to an ECS-Igamma promoter sequence that contains both positive and negative regulatory elements were observed in SLE patients and controls. These results support a role for exogenous signals, such as through CD40 ligation, rather than altered genomic sequence, in the increased production of class switched autoantibodies in SLE.

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Downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination.

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