Antibodies are generated by somatic recombination involving variable (V), diversity (D) and joining (J) gene segments. During the previous grant cycle, we initiated studies aimed to visualize VH-DHJH interactions in live cells. Using single color labeling we were able to track the trajectories adopted by VH and DHJH elements. We found that VH and DHJH elements were subjected to fractional Langevin motion, in which VH and DHJH elements bounce back and forth in a spring-like fashion. However, since only a single genomic region was marked, this approach did not permit us to directly monitor VH-DHJH interactions. To directly visualize VH- DHJH encounters we developed a novel approach. We generated BCR-ABL transformed pro- B cell lines that carried tandem arrays of wild-type TET-operator and mutant TET-operator binding sites in the VH and DHJH regions, respectively. To mark the VH and DHJH regions these cells were transduced with virus expressing WT-TET GFP and MUT-TET SNAP-TAG. The results were surprising. VH-DHJH motion was severely sub-diffusive. We found that VH regions were trapped in distinct chromatin configurations that were remarkably stable (<60 minutes). Only VH regions located nearby DHJH regions had a chance for a VH-DHJH encounter. Comparison of simulated and experimental data suggested that such severely sub-diffusive motion was imposed by geometric confinement (loop domains) and phase separation/gelation (reversible cross-links within VH-DHJH and DH-JH loop domains). A caveat of these studies is that they were performed using BCR-ABL transformed pro-B cells. Here we propose to perform and extend these studies using primary pre-pro-B and pro-B cells. Specifically, we would track VH-DHJH motion but now in primary B cell progenitors. We would describe VH-DHJH motion in physical terms, including diffusion coefficients, scaling exponents, velocities and spatial confinement. We would image across long-time scales to examine how long VH and DHJH regions are trapped in distinct configurations and determine when (timing) and how (speed) such configurations change. We would measure first-encounter times as well as pairing times. We would examine whether transcriptional regulators and chromatin remodelers modulate VH-DHJH motion and first-encounter times. We would identify critical residues in RAG1 that would dictate pairing times. We would visualize VDJ recombination in live cells. Data obtained from these experiments would reveal whether and how space and time intersect to modulate the motion of paired coding and regulatory elements.
It is now established that single nucleotide polymorphisms that are associated with disease predominantly map to regulatory regions. We suggest that a vast majority of these will affect the timing by which paired regulatory elements find each other in order to activate gene expression. These are very early studies but we expect that insights from these experiments will provide new insights and future directions relating to the role of space and time in gene regulation/somatic recombination and how this relates to health and the development of disease.
