The past decade has increased our understanding of how genome topology controls RAG endonuclease-mediated assembly of lymphocyte AgR genes. New technologies have illuminated how the large IgH, Igκ, TCRα/δ, and TCRβ loci fold into compact structures that place their numerous V gene segments in similar three-dimensional proximity to their distal recombination center composed of RAG-bound (D)J gene segments. Many studies have shown that CTCF and cohesin protein–mediated chromosome looping have fundamental roles in lymphocyte lineage- and developmental stage–specific locus compaction as well as broad usage of V segments. CTCF/cohesin–dependent loops have also been shown to direct and restrict RAG activity within chromosome domains. We summarize recent work in elucidating molecular mechanisms that govern three-dimensional chromosome organization and in investigating how these dynamic mechanisms control V(D)J recombination. We also introduce remaining questions for how CTCF/cohesin–dependent and –independent genome architectural mechanisms might regulate compaction and recombination of AgR loci.

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.

Antigen receptor (AgR) loci undergo distinct developmentally regulated changes that include a transcriptional burst and locus compaction to facilitate recombination between V and (D)J gene segments during the primary diversification process. In order to understand how this contributes to diverse AgR repertoires, we generated mice lacking T cell receptor beta (TCRβ) cis-regulatory and CTCF binding elements (CBEs) with potential to serve as locus loop anchors. We then systematically compared epigenetics, transcriptomes, and 3D genome topologies of wild-type and mutant thymocytes populations coupled with their TCRβ repertoires. We find that in DN thymocytes, contacts to the recombination center (RC) are limited to other transcriptionally active chromatin comprising of all Vβ segments, while excluding neighboring heterochromatic regions, supporting our prediction that compaction is driven by homotypic chromatin forces. We identify CBEs and cis regulatory elements that either a) serve as boundary elements, b) control select Vβ accessibility and interaction with the RC, or c) control proximal versus distal skewing of the repertoire. Collectively, our results reveal mechanisms by which CBEs cement TCRβ topological structures and allow efficient recombination of the distal Vβs during AgR diversification.