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Antibodies are important immune molecules that are elicited by B cells to protect our bodies during viral infections or vaccinations. In humans, the antibody repertoire is diversified by programmed DNA lesion processes to ensure specific and high affinity binding to various antigens. Broadly neutralizing antibodies (bnAbs) are antibodies that have strong neutralizing activities against different variants of a virus. bnAbs such as anti-HIV bnAbs often have special characteristics including insertions and deletions, long complementarity determining region 3 (CDR3), and high frequencies of mutations, often at improbable sites of the variable regions. These unique features are rare mutational outcomes that are acquired during antibody diversification processes. In this review, we will discuss possible mechanisms that generate these rare antibody mutational outcomes. The understanding of the mechanisms that generate these rare mutational outcomes during antibody diversification will have implications in vaccine design strategies to elicit bnAbs.

Author(s): Mara Compagno [1]; Qi Wang [1]; Chiara Pighi [1]; Taek-Chin Cheong [1]; Fei-Long Meng [2]; Teresa Poggio [3]; Leng-Siew Yeap [2]; Elif Karaca [1]; Rafael B. Blasco [1]; Fernanda [...]

Abstract Background Pluripotency, the capacity for indefinite self-renewal and differentiation into diverse cell types is a unique state exhibited by embryonic stem (ES) cells. Transcriptional regulators, such as Oct4, are critical for pluripotency, but the role of epigenetic modifiers remains to be fully elucidated. Results Here, we show that ERG-associated protein with SET domain (ESET), a histone methyltransferase enzyme, maintains pluripotency through repression of Cdx2, a key trophectoderm determinant, by histone H3 lysine 9 trimethylation (H3K9me3) of the promoter region. Notably, this repression is mediated through the synergistic function of small ubiquitin-related modifier (SUMO)ylated ESET and Oct4. ESET localises to the promyelocytic leukaemia (PML) nuclear bodies and is SUMOylated in ES cells. Interaction of ESET with Oct4 depends on a SUMO-interacting motif (SIM) in Oct4, which is critical for the repression of Cdx2. Conclusion Loss of ESET or Oct4 results in strikingly similar phenotypes both in ES cells with their differentiation into trophectoderm cells, and in early embryos where there is a failure of development of the pluripotent inner cell mass (ICM) of blastocysts. We propose that SUMOylated ESET-Oct4 complex is critical for both the initiation and maintenance of pluripotency through repression of differentiation, particularly of the trophectoderm lineage by epigenetic silencing of Cdx2.

Insertions and deletions (indels) are low-frequency deleterious genomic DNA alterations. Despite their rarity, indels are common, and insertions leading to long complementarity-determining region 3 (CDR3) are vital for antigen-binding functions in broadly neutralizing and polyreactive antibodies targeting viruses. Because of challenges in detecting indels, the mechanism that generates indels during immunoglobulin diversification processes remains poorly understood. We carried out ultra-deep profiling of indels and systematically dissected the underlying mechanisms using passenger-immunoglobulin mouse models. We found that activation-induced cytidine deaminase-dependent ±1-base pair (bp) indels are the most prevalent indel events, biasing deleterious outcomes, whereas longer in-frame indels, especially insertions that can extend the CDR3 length, are rare outcomes. The ±1-bp indels are channeled by base excision repair, but longer indels require additional DNA-processing factors. Ectopic expression of a DNA exonuclease or perturbation of the balance of DNA polymerases can increase the frequency of longer indels, thus paving the way for models that can generate antibodies with long CDR3. Our study reveals the mechanisms that generate beneficial and deleterious indels during the process of antibody somatic hypermutation and has implications in understanding the detrimental genomic alterations in various conditions, including tumorigenesis.