Etoposide Induces Nuclear Re-Localisation of AID

Laurens J Lambert,Jonathan Houseley,Wolf Reik,Jack Feltham,Heather J Lee,Simon Walker,Pierre Bobé

doi:10.1371/journal.pone.0082110

Laurens J Lambert, Jonathan Houseley + Show 5 more

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https://doi.org/10.1371/journal.pone.0082110

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Abstract

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.

Highlights

Genomes are protected from damage and mutation by a plethora of enzymes, certain cell types perform carefully orchestrated DNA rearrangements and mutational programs that create or enhance population diversity
activation-induced cytidine deaminase (AID) localisation was exclusively cytoplasmic as expected from previous studies [17,41,42], but after etoposide treatment occasional cells were observed in which AID was present in the nucleus and cytoplasm (Figure 1A)
AID re-localisation to the nucleus occurs in response to etoposide treatment, but is not a direct and immediate consequence of double strand breaks (DSBs) formation

Summary

Introduction

Genomes are protected from damage and mutation by a plethora of enzymes, certain cell types perform carefully orchestrated DNA rearrangements and mutational programs that create or enhance population diversity. Some daughters of activated B cells undergo class switch recombination (CSR), which changes the Ig constant region and alters downstream signalling in response to antigens [2]. The primary mutagen in both SHM and CSR is a single protein, Activation-induced cytidine deaminase (AID) [3,4], a member of the APOBEC family of RNA and DNA editing proteins that catalyse cytosine to uracil transitions Mutations at dA:dT base pairs occur in SHM these cannot be directly introduced by AID/UNG. DU:dG mispairs produced by AID are recognised by the Msh2/Msh heterodimer [15,16,17], instigating a non-classical mismatch repair pathway that results in the re-synthesis of surrounding DNA by the error prone polymerase η [18]

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