Molecular mechanisms underlying synapsis of activation-induced deaminase (AID)-targeted S regions during class switch recombination (CSR) are poorly understood. By using chromosome conformation capture techniques, we found that in B cells, the Emicro and 3'Ealpha enhancers were in close spatial proximity, forming a unique chromosomal loop configuration. B cell activation led to recruitment of the germline transcript (GLT) promoters to the Emicro:3'Ealpha complex in a cytokine-dependent fashion. This structure facilitated S-S synapsis because Smicro was proximal to Emicro and a downstream S region was corecruited with the targeted GLT promoter to Emicro:3'Ealpha. We propose that GLT promoter association with the Emicro:3'Ealpha complex creates an architectural scaffolding that promotes S-S synapsis during CSR and that these interactions are stabilized by AID. Thus, the S-S synaptosome is formed as a result of the self-organizing transcription system that regulates GLT expression and may serve to guard against spurious chromosomal translocations.

Immunoglobulin class switch recombination is governed by long-range interactions between enhancers and germline transcript promoters to activate transcription and modulate chromatin accessibility to activation-induced cytidine deaminase (AID). However, mechanisms leading to the differential targeting of AID to switch (S) regions but not to constant (CH) regions remain unclear. We show that S and CH regions are dynamically modified with histone marks that are associated with active and repressed chromatin states, respectively. Chromatin accessibility is superimposable with the activating histone modifications, which extend throughout S regions irrespective of length. High density elongating RNA polymerase II (RNAP II) is detected in S regions, suggesting that the transcription machinery has paused and stalling is abolished by deletion of the S region. We propose that RNAP II enrichment facilitates recruitment of histone modifiers to generate accessibility. Thus, the histone methylation pattern produced by transcription localizes accessible chromatin to S regions, thereby focusing AID attack.

Class switch recombination (CSR) is regulated by the expression of activation-induced deaminase (AID) and germline transcripts (GLTs). AID-dependent double-strand breaks (DSBs) are introduced into switch (S) regions and stimulate CSR. Although histone acetylation (Ac) has been well documented in transcription regulation, its role in DNA damage repair remains largely unexplored. The 1B4.B6 B cell line and normal splenic B cells were activated to undergo CSR and analyzed for histone Ac by chromatin immunoprecipitation (ChIP). A detailed study of the Iγ3-Sγ3-Cγ3 locus demonstrated that acetylated histones are focused to the Iγ3 exon and the Sγ3 region but not to the intergenic areas. Histone H3 Ac is strongly correlated with GLT expression at four S regions, whereas H4 Ac was better associated with B cell activation and AID expression. To more directly examine the relationship between H4 Ac and AID, LPS-activated AID KO and WT B cells were analyzed and express comparable levels of GLTs. In AID-deficient B cells, both histones H3 and H4 are reduced where H4 is more severely affected as compared with WT cells. Our findings raise the intriguing possibility that histone H4 Ac at S regions is a marker for chromatin modifications associated with DSB repair during CSR.