Evidence for physical Interaction between the immunoglobulin heavy chain variable region and the 3' regulatory region

Abstract B cell-specific expression of immunoglobulin heavy chain (IgH) genes utilizes two cis regulatory regions, the intronic enhancer (Eμ), located in the JH-Cμ intron, and a complex regulatory region that lies 3′ to the IgH gene cluster, 3′ RR. We hypothesized that the 3′ RR is involved in IgH gene transcription in plasma cells via physical interaction between distal 3′ RR enhancers and target VH sequences, with loop formation by intervening DNA. In support of this hypothesis we report sequence data at DNA recombination breakpoints as evidence for loop formation preceding DNA inversion in a plasma cell line. In addition, using the chromosome conformation capture technique, physical interactions between VH and 3′ RR were analyzed directly and detected in MPC11 plasma cells and variants and normal splenic B cells but not detected in splenic T cells or in non-B cells. VH-3′ RR interactions were present in the absence of Eμ, but when the hs1,2 enhancer was replaced by a NeoR gene in a variant cell line lacking Eμ, H chain expression was lost, and interactions between VH and 3′ RR and among the 3′ RR regulators themselves were severely disrupted. In addition, the chromosome conformation capture technique detected interactions between the myc promoter and 3′ RR elements in MPC11, which like other plasmacytomas contains a reciprocal translocation between the c-myc and the IgH locus. In sum, our data support a hypothesis that cis VH-3′ RR and myc-3′ RR interactions involve physical interactions between these DNA elements.