Elucidating Topological Regulation of Antigen Receptor Gene Assembly

Abstract

Abstract The compaction of antigen receptor (AgR) loci is thought to be important for efficient assembly of broad AgR gene repertoires through recombination of V gene segments with distal D and/or J gene segments of the recombination center (RC). Compaction of mammalian genomes occurs through CTCF protein-mediated chromosome looping, as well as by compartmentalization of chromatin based on transcriptional state. The large AgR loci generally have many CBEs strewn throughout their V segment regions positioned in convergent orientation with a few CBEs flanking their RC. The field hypothesizes that distal V CBEs promote recombination of nearby V gene segments by bringing them into proximity with D and J segments through looping to the convergent RC CBEs. To test this hypothesis, we have mutated CBEs within the mouse TCRβ locus. We find that deletion of the Trbv1 CBE, as well as deletion or inversion of the Trbv14 CBE, lowers, but does not ablate, contact and rearrangement of flanking Vβ gene segments with the distal RC. Unexpectedly, deletion of all convergent CBEs of the RC alters Vβ repertoire but has no obvious effect on overall levels of Vβ recombination, implying that compartmentalization might be the dominant mechanism for compaction and recombination of TCRβ loci. Consistent with this scenario, we show that deletion of the Trbv1 promoter inactivates Trbv1 chromatin and ablates interactions between Trbv1 and the RC. Our ongoing work seeks to determine relative roles of chromosome looping and compartmentalization in promoting and shaping AgR gene assembly.