AID-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Populations

Getahun

The Journal of Immunology

et al. 2015

Class switch recombination (CSR) generates isotype-switched antibodies with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA phosphoinositide 3-kinase (PI3K) catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. Here, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role of p110α in CSR. Furthermore, we identify a cooperative role of p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN reversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR.

Section: Resultsmentioning

confidence: 99%

“…Semi-quantitative RT-PCR was performed as described previously (42). Total RNA was purified with TriPure (Roche) and used for RT reaction according to manufacturer's instructions (Promega).…”

Section: Methodsmentioning

confidence: 99%

Imbalanced PTEN and PI3K Signaling Impairs Class Switch Recombination

Getahun

The Journal of Immunology

et al. 2015

“…While such defects of the potential 'CSR factory' components are absent in our model (where normal IgH CSR occurs), the KIKS locus might then simply demonstrate a defective recruitment of the knocked-in S regions into such CSR factories. Whether the Igk locus may intrinsically dictates differential outcome of AID-induced lesions, as it was recently demonstrated when S regions were knocked-in in other loci 20,32 , is another possibility. But the presence of ISDs, few S-S junctions, 53BP1 recruitment and gH2AX foci, proves that beside AIDinduced single-strand mutations, DSBs occur at the KIKS locus but are not appropriately handled for efficient synapsis and junction of distant breaks.…”

Section: Discussionmentioning

confidence: 99%

Efficient AID targeting of switch regions is not sufficient for optimal class switch recombination

et al. 2015

Antibody affinity maturation relies on activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) of immunoglobulin (Ig) loci. Class switch recombination (CSR) can in parallel occur between AID-targeted, transcribed, spliced and repetitive switch (S) regions. AID thus initiates not only mutations but also double-strand breaks (DSBs). What governs the choice between those two outcomes remains uncertain. Here we explore whether insertion of transcribed intronic S regions in a locus (Igk) strongly recruiting AID is sufficient for efficient CSR. Although strongly targeted by AID and carrying internal deletions, the knocked-in S regions only undergo rare CSR-like events. This model confirms S regions as exquisite SHM targets, extending AID activity far from transcription initiation sites, and shows that such spliced and repetitive AID targets are not sufficient by themselves for CSR. Beyond transcription and AID recruitment, additional IgH elements are thus needed for CSR, restricting this hazardous gene remodelling to IgH loci.

“…This cSµ region comprises a highly repetitive sequence and contains no open-reading frames. The gene targeting was performed as described previously (27). Correctly targeted clones were detected by Southern blot (EcoRI digest) with two probes that hybridized upstream of the 5’ homology arm (DQ52 probe) or downstream of the 3’ homology arm (J H4 probe) (26).…”

Section: Methodsmentioning

confidence: 99%

Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions

The Journal of Immunology

Eder

Elos

et al. 2016

Self Cite

Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of antibody gene diversification. However, precise mechanisms regulating AID-deamination frequency remain incompletely understood. Moreover, it is unknown whether different sequence contexts influence the preferential access of mismatch repair (MMR) or uracil glycosylase (UNG) to AID-initiated U:G mismatches. Here, we employed two knock-in models to directly compare the mutability of core Sµ and VDJ exon sequences and their ability to regulate AID-deamination and subsequent repair process. We find that S region is a much more efficient AID deamination target than V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by MMR deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID-deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating antibody gene diversity and AID targeting specificity.