Brittney Allyn (Mentor: Craig Bassing, PhD)

“Elucidating Topological Regulation of Antigen Receptor Gene Assembly”

Brittney Allyn, Katharina Hayer, Vincent Nganga, Eugene Oltz and Craig Bassing

The RAG-mediated assembly of antigen receptor genes is essential for adaptive immunity. Of great interest in the field is how V rearrangements occur across large genomic distances to a RAG-bound D/J recombination center (RC). The current model for the Igh locus is Cohesin and CTCF protein-mediated chromosome looping allows RAG to scan from the RC and capture V segments with a recombination signal sequence (RSS) of convergent orientation for synapsis, cleavage, and recombination via deletion of intervening sequences. However, this model cannot explain how V segments of divergent orientation recombine through inversion of intervening sequences as in Igk, Tcrb, and Tcrd loci. In addition to chromosome looping, diffusion-based interactions between chromatin regions of similar transcription activity (compartmentalization) can bring together distal genetic elements. In DN thymocytes, transcription of Dβ-Jβ segments permits formation of the RC and Dβ-to-Jβ recombination. Subsequent Tcrb folding then positions all Vβs in similar proximity to the RC. The most RC-distal Vβ (V1) is a powerful model to elucidate mechanisms governing long-range V recombination as it resides within a small region of active chromatin that harbors a single transcriptional element (V1 promoter) and one CTCF binding element (CBE). To elucidate how V1 rearrangement is regulated, I used CRISPR/Cas9 gene editing to establish mice with specific modifications of the V1 genomic region. Here I show that deletion of the V1 promoter abrogates germline V1 transcription, contact and rearrangement of V1 without disrupting CTCF binding of the V1 CBE. Deletion of the V1 CBE has no effect on germline V1 transcription but decreases contact and rearrangement of V1. Compartmentalization could promote Vβ rearrangements by diffusion-based synapsis with the RC, thus allowing Vβ-to-RC recombination via deletion or inversion depending on Vβ RSS orientation. To further test this model, I generated mice with an inverted V1 RSS and show, with preliminary analyses, inversional V1-RC rearrangements in total thymocytes. To further elucidate the mechanisms that govern V1 compartmentalization with the RC, I created mice with a transcriptional termination sequence inserted downstream of the V1 promoter. Here I show substantial loss of V1 recombination, consistent with lower contact of V1 with the RC, silent chromatin over the V1 RSS, or both. Together, my data support a working model in which compartmentalization is the principal force driving Vβ-RC interactions and rearrangements, while chromosome looping plays a contributory role in focusing these contacts to broaden Vβ usage.