UC San Diego

The immunoglobulin heavy chain (Igh) locus is organized into distinct regions that encode multiple variable (Vh), diversity (Dh), joining (Jh) and constant (Ch) gene segments. DNA recombination takes place between the Vh, Dh and Jh segments at the Igh locus in developing B cells. The locus undergoes large-scale contraction to facilitate this recombination. However, its structural organization is unknown. It is likely that the structural organization plays a role in this process. By simultaneously visualizing three subregions of the Igh locus using 3D fluorescence in-situ hybridization, we show that looping of the distal Vh segments to the CH segments is observable in pro-B cells. This looping occurs at a significantly higher frequency in pro-B cells compared to CD8⁺ T cells. This indicated that there is a structural reorganization of the locus and not just a simple contraction of the chromatin fiber. To decipher the topology of the locus, 12 genomic markers were used that spanned the entire locus. Spatial distance distributions between different combinations of these markers were determined and compared to computer simulations of different models of chromatin structure. These comparisons revealed that the data agreed with a topology that predicted higher order organization of the chromatin fiber into multiple subcompartments connected by linkers (Multi-Loop Subcompartment model). Compartmentalization of the locus was visualized by labeling the entire locus with hybridization markers. Relative locations of the different Igh sub-regions in 3D space were determined using a trilateration technique. Striking conformational changes can be seen between pre- pro-B and pro-B cells, when the locus transitions from a de-contracted to a contracted state. The implications of the higher order organization of the locus on long-range genomic interactions are discussed. It is evident that the higher order organization is necessary for promoting long- range genomic interactions that would facilitate V(D)J recombination at the Igh locus. In absence of higher order organization, the expected frequency of interactions are much lesser