2025 Theses Doctoral

Surveillance and Co-option of LINE-1 Retrotransposons in Genome Evolution and Antibody Diversification

Lauring, Max C.

Activation-induced cytidine deaminase (AID) accomplishes somatic hypermutation (SHM) of V(D)J genes in germinal center B cells for antibody diversification and affinity maturation. How AID specifically targets V(D)J genes remains unclear. We report the discovery of LINE-1 (L1) retrotransposons upstream to many VH genes in the immunoglobulin heavy chain locus. These L1s are evolutionarily old, truncated, and retrotransposition-dead. Recombined VH promoters create long, strong antisense RNAs encoding nearby upstream L1s, triggering the recruitment of human silencing hub (HUSH) complex and AID, which we term L1-driven SHM. We show that L1-driven SHM occurs in vivo by utilizing HUSH knockout mice and engineered mice with VH genes devoid of upstream L1s. Insertion of transcriptionally-active L1s at non-immunoglobulin loci promotes off-target SHM. We demonstrate that old retrotransposons serve physiological roles and our findings reveal how B cells co-opted an anti-retrotransposon silencing mechanism to promote antibody diversity. In doing so, we established a new link between innate and adaptive immunity.

Furthermore, the regulation LINE-1 expression, and retrotransposon expression more broadly, is essential within the context of genome integrity and genome evolution. The structural and functional evolution of the human genome is influenced by de novo insertions of retrotransposons. The primary mechanism to restrict expression of retrotransposons – Long Interspersed Nuclear Elements (LINEs), Short Interspersed Nuclear Elements (SINEs), and Endogenous Retroviral Elements (ERVs) – is epigenetic regulation and transcriptional silencing. We demonstrate that transcription-associated catabolism of TEs by the RNA exosome complex serves as an additional layer of restriction against TE expression and mobilization in pluripotent and differentiated cells. We used long-read DNA sequencing to detect endogenous de novo TE insertions in a primary mouse embryonic stem cell that can conditionally deplete RNA exosome activity. Long-read RNA sequencing further confirmed that de novo TE insertions were exonized into resulting chimeric RNAs and germline TEs were aberrantly exonized. We mechanistically validated our long-read sequencing findings using an in vitro retrotransposition assay with exosome-specific auxin-inducible degron cell lines to show that RNA exosome directly suppresses TE mobilization. Our work reveals that RNA exosome controls TE expression co- and post-transcriptionally to mitigate aberrant TE exonization of nascent transcripts and de novo TE retrotransposition.