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Etoposide induces nuclear re-localisation of AID.

Lambert LJ, Walker S, Feltham J, Lee HJ, Reik W, Houseley J - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.

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.

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Dynamics of AID re-localisation.A) Montage of frames from live-cell imaging of an NIH/3T3 cell expressing GFP-AID, treated for 2 hours with 200µM etoposide and then monitored after drug withdrawal. Each frame represents 40 minutes. This video can be seen in full in Movie S1. B) Montage of two cells undergoing cytoplasmic to nuclear re-localisation of AID after etoposide withdrawal. Frames are shown at 10 minute intervals, t=0 at the addition of etoposide, first frame shown here is at etoposide removal. This video can be seen in full in Movie S3.
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pone-0082110-g002: Dynamics of AID re-localisation.A) Montage of frames from live-cell imaging of an NIH/3T3 cell expressing GFP-AID, treated for 2 hours with 200µM etoposide and then monitored after drug withdrawal. Each frame represents 40 minutes. This video can be seen in full in Movie S1. B) Montage of two cells undergoing cytoplasmic to nuclear re-localisation of AID after etoposide withdrawal. Frames are shown at 10 minute intervals, t=0 at the addition of etoposide, first frame shown here is at etoposide removal. This video can be seen in full in Movie S3.

Mentions: Nuclear accumulation of ectopically-expressed AID has been reported in HEK293 cells treated with γ-rays, hydrogen peroxide or bleomycin [47]. These agents induce DSBs, but only amongst complex patterns of DNA damage, leaving the connection between DSB formation and AID re-localisation uncertain (see 48 and references therein). We therefore examined AID localisation after treatment with etoposide, a suicide inhibitor of topoisomerase II that induces precise DSBs (reviewed in 49). A FLAG-AID construct was transiently transfected into NIH/3T3 cells and treated with etoposide for 2 hours. In untreated cells, 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). We tested AID localisation after shorter etoposide exposures (24, 48, 72, 96 min), however, there was no increase in the number of cells showing nuclear AID despite the rapid appearance of H2AX phosphorylated at Ser139 (γH2AX) (Figure 1B). We then examined AID localisation during DSB repair by treating cells with etoposide for 2 hours, removing the drug and staining for AID after 2, 4, 6 and 24 hours. Across this time course, the proportion of cells showing detectable nuclear AID increased dramatically to a maximum of ~25% 4-6 hours after drug removal (Figure 1C). Greater re-localisation also occurred after drug removal, with many cells showing almost exclusively nuclear AID (Figure 1D). This phenotype was not caused by the presence of the N-terminal FLAG tag on the AID construct, as the same result was obtained with an N-terminal GFP tag (see later, Figure 2) and a C-terminal HA tag (Figure S1A). Therefore, AID re-localisation to the nucleus occurs in response to etoposide treatment, but is not a direct and immediate consequence of DSB formation.


Etoposide induces nuclear re-localisation of AID.

Lambert LJ, Walker S, Feltham J, Lee HJ, Reik W, Houseley J - PLoS ONE (2013)

Dynamics of AID re-localisation.A) Montage of frames from live-cell imaging of an NIH/3T3 cell expressing GFP-AID, treated for 2 hours with 200µM etoposide and then monitored after drug withdrawal. Each frame represents 40 minutes. This video can be seen in full in Movie S1. B) Montage of two cells undergoing cytoplasmic to nuclear re-localisation of AID after etoposide withdrawal. Frames are shown at 10 minute intervals, t=0 at the addition of etoposide, first frame shown here is at etoposide removal. This video can be seen in full in Movie S3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852760&req=5

pone-0082110-g002: Dynamics of AID re-localisation.A) Montage of frames from live-cell imaging of an NIH/3T3 cell expressing GFP-AID, treated for 2 hours with 200µM etoposide and then monitored after drug withdrawal. Each frame represents 40 minutes. This video can be seen in full in Movie S1. B) Montage of two cells undergoing cytoplasmic to nuclear re-localisation of AID after etoposide withdrawal. Frames are shown at 10 minute intervals, t=0 at the addition of etoposide, first frame shown here is at etoposide removal. This video can be seen in full in Movie S3.
Mentions: Nuclear accumulation of ectopically-expressed AID has been reported in HEK293 cells treated with γ-rays, hydrogen peroxide or bleomycin [47]. These agents induce DSBs, but only amongst complex patterns of DNA damage, leaving the connection between DSB formation and AID re-localisation uncertain (see 48 and references therein). We therefore examined AID localisation after treatment with etoposide, a suicide inhibitor of topoisomerase II that induces precise DSBs (reviewed in 49). A FLAG-AID construct was transiently transfected into NIH/3T3 cells and treated with etoposide for 2 hours. In untreated cells, 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). We tested AID localisation after shorter etoposide exposures (24, 48, 72, 96 min), however, there was no increase in the number of cells showing nuclear AID despite the rapid appearance of H2AX phosphorylated at Ser139 (γH2AX) (Figure 1B). We then examined AID localisation during DSB repair by treating cells with etoposide for 2 hours, removing the drug and staining for AID after 2, 4, 6 and 24 hours. Across this time course, the proportion of cells showing detectable nuclear AID increased dramatically to a maximum of ~25% 4-6 hours after drug removal (Figure 1C). Greater re-localisation also occurred after drug removal, with many cells showing almost exclusively nuclear AID (Figure 1D). This phenotype was not caused by the presence of the N-terminal FLAG tag on the AID construct, as the same result was obtained with an N-terminal GFP tag (see later, Figure 2) and a C-terminal HA tag (Figure S1A). Therefore, AID re-localisation to the nucleus occurs in response to etoposide treatment, but is not a direct and immediate consequence of DSB formation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus