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UNG shapes the specificity of AID-induced somatic hypermutation.

Pérez-Durán P, Belver L, de Yébenes VG, Delgado P, Pisano DG, Ramiro AR - J. Exp. Med. (2012)

Bottom Line: Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes.The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR).We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions.

View Article: PubMed Central - HTML - PubMed

Affiliation: B Cell Biology Laboratory, Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain.

ABSTRACT
Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.

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Fluorescence revertance assay to monitor AID activity. (A) Representation of the pMX-PIE-mOrangeSTOP and AID-ER-ΔhuCD4 or AIDE58Q-ER-ΔhuCD4 retroviral vectors used to transduce NIH-3T3 cells. (B) Detection of AID activity. NIH-3T3 cells were co-transduced with the retroviral vectors depicted in A and cultured with (bottom) or without (top) 1 µM OHT. mOrange+ cells were monitored by flow cytometry. Representative FACS analyses at day 9 are shown. (C) Time-course analysis of mOrange+ cell appearance in NIH-3T3 cells co-transduced as described in B. One representative experiment is shown.
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fig1: Fluorescence revertance assay to monitor AID activity. (A) Representation of the pMX-PIE-mOrangeSTOP and AID-ER-ΔhuCD4 or AIDE58Q-ER-ΔhuCD4 retroviral vectors used to transduce NIH-3T3 cells. (B) Detection of AID activity. NIH-3T3 cells were co-transduced with the retroviral vectors depicted in A and cultured with (bottom) or without (top) 1 µM OHT. mOrange+ cells were monitored by flow cytometry. Representative FACS analyses at day 9 are shown. (C) Time-course analysis of mOrange+ cell appearance in NIH-3T3 cells co-transduced as described in B. One representative experiment is shown.

Mentions: To monitor AID mutational activity we developed a sensitive fluorescence revertance assay. In brief, a stop codon overlapping with an AGCT AID mutational hotspot was introduced at positions 230–233 of the sequence encoding the mOrange fluorescent protein (mOrangeSTOP; Fig. 1 A and Fig. S1 A), a monomeric RFP1 variant which can be easily detected by flow cytometry (Shaner et al., 2004). This TAG stop codon generates a nonfluorescent truncated protein, but transversion mutations at its third nucleotide revert it to TAC or TAT tyrosine-encoding codons that reconstitute the full-length mOrange fluorescent protein. mOrangeSTOP was introduced into the GFP-containing retroviral vector pMX-PIE (Barreto et al., 2003) to allow the tracking of transduced cells (Fig. 1 A). Inducible AID activity was achieved by fusing AID to the estrogen-binding domain of estrogen receptor (ER; AID-ER), thus generating a protein that can be translocated into the nucleus—and therefore grant access to its DNA substrate—by tamoxifen (OHT) treatment (Doi et al., 2003). AID-ER, or the catalytically inactive mutant AIDE58Q-ER, was cloned into a second retroviral vector that contains a truncated, signaling-devoid form of the human CD4 molecule (ΔhuCD4) for tracking purposes (Fig. 1 A). To test the mOrangeSTOP revertance assay, we retrovirally transduced the mOrangeSTOP vector along with either AID-ER– or AIDE58Q-ER–containing vectors into NIH-3T3 mouse fibroblasts. After 3 d of puromycin selection, >95% of cells were GFP+ΔhuCD4+ (unpublished data). Cells were then cultured in the presence or absence of OHT for up to 11 d. We detected the appearance of mOrange+ cells in AID-ER transduced cultures as soon as 2 d after OHT treatment and their percentage increased with time (Fig. 1, B and C). In contrast, AIDE58Q-ER transduction failed to generate detectable mOrange+ cells and, in the absence of OHT AID-ER, only promoted marginal numbers of mOrange revertants (Fig. 1, B and C). These results show that AID mutational activity can be monitored by the generation of mOrange revertants in NIH-3T3 cells.


UNG shapes the specificity of AID-induced somatic hypermutation.

Pérez-Durán P, Belver L, de Yébenes VG, Delgado P, Pisano DG, Ramiro AR - J. Exp. Med. (2012)

Fluorescence revertance assay to monitor AID activity. (A) Representation of the pMX-PIE-mOrangeSTOP and AID-ER-ΔhuCD4 or AIDE58Q-ER-ΔhuCD4 retroviral vectors used to transduce NIH-3T3 cells. (B) Detection of AID activity. NIH-3T3 cells were co-transduced with the retroviral vectors depicted in A and cultured with (bottom) or without (top) 1 µM OHT. mOrange+ cells were monitored by flow cytometry. Representative FACS analyses at day 9 are shown. (C) Time-course analysis of mOrange+ cell appearance in NIH-3T3 cells co-transduced as described in B. One representative experiment is shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3405504&req=5

fig1: Fluorescence revertance assay to monitor AID activity. (A) Representation of the pMX-PIE-mOrangeSTOP and AID-ER-ΔhuCD4 or AIDE58Q-ER-ΔhuCD4 retroviral vectors used to transduce NIH-3T3 cells. (B) Detection of AID activity. NIH-3T3 cells were co-transduced with the retroviral vectors depicted in A and cultured with (bottom) or without (top) 1 µM OHT. mOrange+ cells were monitored by flow cytometry. Representative FACS analyses at day 9 are shown. (C) Time-course analysis of mOrange+ cell appearance in NIH-3T3 cells co-transduced as described in B. One representative experiment is shown.
Mentions: To monitor AID mutational activity we developed a sensitive fluorescence revertance assay. In brief, a stop codon overlapping with an AGCT AID mutational hotspot was introduced at positions 230–233 of the sequence encoding the mOrange fluorescent protein (mOrangeSTOP; Fig. 1 A and Fig. S1 A), a monomeric RFP1 variant which can be easily detected by flow cytometry (Shaner et al., 2004). This TAG stop codon generates a nonfluorescent truncated protein, but transversion mutations at its third nucleotide revert it to TAC or TAT tyrosine-encoding codons that reconstitute the full-length mOrange fluorescent protein. mOrangeSTOP was introduced into the GFP-containing retroviral vector pMX-PIE (Barreto et al., 2003) to allow the tracking of transduced cells (Fig. 1 A). Inducible AID activity was achieved by fusing AID to the estrogen-binding domain of estrogen receptor (ER; AID-ER), thus generating a protein that can be translocated into the nucleus—and therefore grant access to its DNA substrate—by tamoxifen (OHT) treatment (Doi et al., 2003). AID-ER, or the catalytically inactive mutant AIDE58Q-ER, was cloned into a second retroviral vector that contains a truncated, signaling-devoid form of the human CD4 molecule (ΔhuCD4) for tracking purposes (Fig. 1 A). To test the mOrangeSTOP revertance assay, we retrovirally transduced the mOrangeSTOP vector along with either AID-ER– or AIDE58Q-ER–containing vectors into NIH-3T3 mouse fibroblasts. After 3 d of puromycin selection, >95% of cells were GFP+ΔhuCD4+ (unpublished data). Cells were then cultured in the presence or absence of OHT for up to 11 d. We detected the appearance of mOrange+ cells in AID-ER transduced cultures as soon as 2 d after OHT treatment and their percentage increased with time (Fig. 1, B and C). In contrast, AIDE58Q-ER transduction failed to generate detectable mOrange+ cells and, in the absence of OHT AID-ER, only promoted marginal numbers of mOrange revertants (Fig. 1, B and C). These results show that AID mutational activity can be monitored by the generation of mOrange revertants in NIH-3T3 cells.

Bottom Line: Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes.The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR).We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions.

View Article: PubMed Central - HTML - PubMed

Affiliation: B Cell Biology Laboratory, Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain.

ABSTRACT
Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.

Show MeSH
Related in: MedlinePlus