| Mature B lymphocytes undergo effector function diversification by means of class switch recombination (CSR), in which there is a cytokine- and/or antigen-dependent switch from the default IgM to downstream isotypes, IgG, IgE or IgA. This occurs in the Igh locus at the molecular level by an intrachromosomal deletion, resulting in gene rearrangement between switch regions. After CSR activation, germline transcripts (GLTs) of the targeted CH (isotype) locus are induced. CSR is dependent on B cell-specific long range interactions (LRIs), wherein Igh enhancers Eµ and 3'Eα are brought into close proximity, followed by recruitment of CH loci to this complex. We explored the extent to which these LRIs are mediated by GLT promoters in cis and/or by trans-acting factors that are crucial in regulating GLT production, specifically STAT6 and NF-κB. The enzyme AID is also required for CSR, where its activity leads to DNA double strand breaks (DSBs) and repair. In DNA break repair, 53BP1 has been shown to accumulate at damage sites and may facilitate LRIs. 53BP1-deficient B cells have significantly less CSR, to a much greater extent than all other DSB repair protein-deficient mice, indicating a key role for 53BP1 in CSR. Thus, we also explored if 53BP1 facilitates LRIs, and if AID-induced DSBs are required for this function.;To assess LRIs, we compared chromatin from resting (CD43– ) and LPS or LPS+IL4 activated B cells using the chromosome conformation capture (3C) technique. Our data indicate remarkable differences in factors mediating Igh LRIs. Interactions between a GLT promoter with 3'Eα appear to be facilitated by STAT6, which binds at the γ1 GLT promoter. Neither STAT6 nor the γ1 GLT promoter influenced Eµ-3'Eα interactions. In contrast, Eµ-3'Eα interactions appear facilitated by 53BP1, independent of and prior to AID-induced DSBs. Neither 53BP1 nor AID influenced 3'Eα-GLT promoter interactions. Finally, NF-κB appears to facilitate all Igh LRIs. The finding that 53BP1 facilitates LRIs is quite exciting, since this protein is not a transcription factor. Mediation of LRIs by a non-transcription factor protein to create an Eµ-3'Eα loop is a novel and unique finding not observed in other LRI-regulated genes. Together, this exquisitely regulated apparatus likely ensures that AID mutagenic activity and potentially damaging DNA DSBs are sequestered and properly repaired. |
