ELUCIDATING GENOME TOPOLOGY REGULATION OF ANTIGEN RECEPTOR DIVERSIFICATION
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Genetics and Genomics
Biology
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The lymphocyte lineage- and developmental stage-specific folding of antigen receptor (AgR) loci is critical for assembling a broad repertoire of AgR genes through RAG-mediated rearrangement of distal V and (D)J gene segments. Two fundamental mechanisms fold chromosomes: cohesin-mediated chromosome loop extrusion and homotypic chromatin interactions. Cohesin-mediated loop extrusion anchored by CTCF has been implicated in AgR locus folding, but the precise roles of this mechanism outside of Igh are undefined. Moreover, roles of homotypic chromatin interactions in any AgR locus remain to be validated. To test the role of each of these mechanisms at the Tcrb locus, I used CRISPR/Cas9 in mice to inactivate CTCF binding elements (CBEs) or transcriptional elements and utilized high-throughput DNA sequencing methods to access locus conformation, transcription, protein binding, and chromatin state. Tcrb has numerous CBEs interspersed among Vβ segments in convergent orientation with three DJβ CBEs. My analyses indicate that, unlike Igh, homotypic chromatin interactions and bi-directional chromosome looping contribute to folding Tcrb for long-range recombination. Notably, I show that Vβ rearrangement can proceed independent of substrate orientation, ruling out scanning by DJβ-bound RAG as the sole mechanism of Vβ recombination, as is the case for Igh. Inactivation of the CBE adjacent to the most 5’Vβ segment (Trvb1) impaired loop extrusion anchored at this element extending to DJβ CBEs. Deletion of the DJβ CBEs revealed that they both anchor loops with Vβ CBEs and restrict Eβ activity to the DJβ clusters. However, unlike when Trbv1 CBE is inactivated, in the absence of DJβ CBEs, Vβ-DJβ loops were anchored at transcriptionally active DJβ chromatin. Inactivation of the Trvb1 promoter also eliminated loop extrusion anchored at Trvb1 by impairing cohesin loading. Similarly, deletion of the Tcrb enhancer (Eβ) eliminated cohesin loading at DJβ segments; however, did not disrupt DJβ CBE anchored loops with Vβs. Collectively, these data support that loop extrusion initiating within Vβ chromatin is a significant contributor to Tcrb folding. My data indicate that AgR loci employ different mechanisms of locus folding likely tailored to their genomic configuration and cellular context.