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Title: Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID}
Article:
Abstract:
AID( Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.
Introduction:
Activation-induced cytosine deaminase( AID) initiates immunoglobulin( Ig) gene diversification in activated B cells by deaminating C to U[1, 2]. Either UNG2 or MSH2/6 recognize and process this damage, and repair via error-prone pathways results in somatic hypermutation( SHM), class switch recombination( CSR) or gene conversion. AID-initiated damage can have pathological outcomes, evident as the chromosomal translocations associated B cell malignancies[3–7]. AID also participates in erasing CpG methylation to reprogram the genome in early development[8–12], promotes B cell tolerance[13, 14], and limits autoimmunity[15, 16]. AID is tightly regulated. AID localizes predominately to the cytoplasm but requires access to the nucleus to function. Posttranslational modifications and interactions with other proteins modulate cytoplasmic retention and nuclear import[17–19]. AID persistence in the nucleus is limited by proteosomal degradation[20, 21] and by CRM1-dependent nuclear export[22–24]. Catalytic activity of AID can be improved by active site point mutations, but while those mutations accelerate Ig gene diversification they also stimulate translocation and compromise cell viability[25]. Mutation or deletion of the C-terminal region that includes the nuclear export signal( NES) can diminish AID stability and the efficiency of CSR, but compromises cell fitness[26, 27]. AID abundance is constant during cell cycle[20, 26], but several kinds of observations have suggested that cell cycle may regulate AID activity. In DT40 chicken B cells, brief treatment with leptomycin B( LMB), an inhibitor of CRM1-dependent nuclear export, increases nuclear AID signal in G1 phase cells[28]; Polη, which copies donor DNA in AID-initiated gene conversion, co-localizes with the diversifying IgλR allele predominately in G1 phase[29]; UNG2 removes uracils produced upon deamination by AID predominately in G1 phase[30]; and RPA initially accumulates at Ig switch regions in G1 phase[31]. We have now asked if cell cycle regulates subcellular localization, stability or physiological activity of AID. We demonstrate that nuclear degradation occurs more slowly in G1 phase than in S-G2/M phase cells. We show that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags derived from cell cycle regulators CDT1 and Geminin[32] to destroy nuclear protein outside G1 or S-G2/M phase. We show that nuclear AID accelerates SHM and CSR, and is tolerated by cells in G1 phase, but compromises viability in S-G2/M phase. These results establish that cell cycle regulates abundance of nuclear AID and determines the ability of cells to respond to AID-initiated DNA damage. The AID derivatives that we have generated may be useful tools for engineering genes by iterative mutagenesis and selection, and cell cycle tags may be generally useful for studying DNA repair and recombination and RNA biogenesis, and for genome engineering.
Discussion:
G1 phase nuclear AID accelerates SHM and CSR without compromising cell viability in G1 phase, but is not well-tolerated in S-G2/M phases. The ability of G1 phase cells to tolerate nuclear AID may reflect the relative efficiency of repair of AID-initiated nicks in G1 phase compared to later phases of cell cycle, as well as the potential for nicks to be converted to potentially deleterious replicative DSBs in S phase( Fig 7). AID deaminates C to generate U, which is repaired by UNG2- or MSH2/6-dependent pathways which generate nicks( single-strand breaks) as critical intermediates( Fig 7A). The evidence that Ig gene diversification occurs in G1 phase[28–31] suggests that unrepaired nicks do not persist into S phase. DNA replication in S phase will convert a nick into a replicative DSB( Fig 7B), which can initiate a DNA damage response and activate checkpoints that prevent cell cycle progression[48]. S phase nuclear AID is therefore a potential source of replicative DSBs, and if these exceed the capacity for repair they can compromise cell proliferation. The resilience of G1 phase cells to AID-initiated damage was especially evident in the AIDF193A-mCherry-CDT1 transductants. The design of this construct completely restricted nuclear AID to G1 phase, by combining the effect of the CDT1 tag with the F193A mutation, which prevents exported AID from re-entering the nucleus in S-G2/M phase. In contrast, other AID derivatives designed to accelerate SHM have exhibited a negative impact on cell viability, including other NES mutants[26, 47], AID mutants selected for increased deamination activity[25, 26, 47] and the naturally occurring human AIDΔE5 dominant negative mutant[27]. Reduced viability was associated with the presence of nuclear AID in S phase, in AIDF193A-mCherry and AIDF193A-mCherry-GEM transductants( Fig 6D and 6E). We note that even though the GEM tag was predicted to restrict nuclear protein to S-G2/M phases, there was no nuclear AID-mCherry-GEM signal in any stage of cell cycle. In light of the evidence that nuclear AID is degraded more slowly in G1 than S or G2/M phase( Fig 2), it is likely that the AID-mCherry-GEM fusion protein was eliminated from the nucleus in G1 phase by degradation targeted to the GEM tag, and in S phase by degradation targeted to AID itself. AID has eight lysine targets for ubiquitination[20], and differential ubiquitination may be one source of temporal regulation. Cell cycle regulation of the error-prone pathways that promote SHM may enhance repair of AID-initiated damage in G1 phase, and also explain some apparent differences in the SHM spectra of the different transductants. An especially high fraction of transversion mutations from G to T was evident in AID-mCherry-GEM transductants( 11. 1%) relative to AID-mCherry( 0%) or AID-mCherry-CDT1 transductants( 3. 4%; Fig 5D), a class of mutations that can be generated by activity of Rev1[49] or Polη[50]. In addition, a reduced frequency of mutations at A and T was evident in AID-mCherry-CDT1( 6. 8%) and AID-mCherry-GEM( 8. 4%) relative to AID-mCherry transductants( 17. 9%; Fig 5). The reduced frequency of mutations at A and T argues against a predominant role for Polη, which is especially active at mutating at A and T[51], and makes Rev1 a more likely candidate for the G to T transversions in AID-mCherry-GEM transductants. This suggests that Rev1 may function late in cell cycle. Consistent with this, Rev1 has been shown to repair UV damage at gaps that persist into G2 phase[52]. The CDT1 tag destabilizes nuclear protein outside G1 phase[32] and would not be predicted to increase nuclear abundance at any stage of cell cycle. Nonetheless, AID-mCherry-CDT1 nuclear signal exceeded that of AID-mCherry( Fig 4C). This somewhat paradoxical result could be explained if the CDT1 tag prevented cytoplasmic retention[19] or enabled more efficient nuclear import. Consistent with the latter possibility, treatment with LMB or LMB+MG132 did cause a slightly more rapid increase in nuclear signal in AID-mCherry-CDT1 than AID-mCherry transductants( S7 Fig). In addition, AID may be regulated by feedback loops that determine nuclear levels in G1 phase based on the level in another compartment or stage of cell cycle. In that case, low levels of AID in G2/M phase may lead to elevated nuclear levels in the next G1 phase, as was evident in the AID-mCherry-CDT1 transductants( Fig 4D), favoring a cell that has not carried out Ig gene diversification in one cell cycle to do so in the next. AID Ser3 is a site of negative regulation, and phosphorylation at this position was previously shown to downregulate SHM and CSR[34]. By analysis of the phosphomimetic mutant, AIDS3D, we showed that modification at this position regulates AID nuclear abundance and the rate of AID nuclear degradation in G1 phase( Fig 3). Phosphorylation at this site is modulated by the protein phosphatase, PP2A, a key signaling molecule, targets of which include cell cycle regulators. AID abundance also appears to be regulated by its catalytic activity. Both the nuclear and cytoplasmic signals of the AIDH56A-mCherry catalytic mutant were well below signals of wild type AID-mCherry( Fig 3). The Ramos AIDH56A-mCherry transductants express endogenous AID and hypermutate actively, so the reduced signal of the catalytic mutant must reflect a protein-intrinsic mechanism directed to the mutant protein. It is possible that sort of quality control mechanism may contribute to AID stability. Catalytically inactive AID may, for example, be a better target for ubiquitination. The possibility that AID might be regulated by a feedback mechanism that reported on AID-induced DSBs was previously raised by Chaudhuri and collaborators, who claimed that a catalytically inactive AID double mutant, AIDH56R E58Q, was a poor substrate for Ser38 phosphorylation in B cells carrying out CSR[53]. Central to that claim was evidence of apparently reduced recovery of the Ser38-phosphorylated derivative of AIDH56R E58Q. AIDH56R E58Q bears amino acid substitutions at two residues at the catalytic active site. Reduced abundance of the AIDH56R E58Q was evident in data included in supplementary data accompanying that report but not commented on by the authors[53]. Our evidence for reduced abundance of the AIDH56A-mCherry catalytic mutant( Fig 3) provides an alternative explanation for poor recovery of Ser38-phosphorylated AIDH56R E58Q. It would be appropriate to revisit the claim that an ATM-dependent signal necessary for AID Ser38 phosphorylation requires DNA damage for induction[53] in experiments in which results are subjected to more rigorous quantification. Several groups have reported that treatments that cause DSBs( ionizing radiation, etoposide) can induce nuclear localization of AID[24, 53, 54]. A DSB-dependent signal may be relevant to CSR, but unlikely to be relevant to SHM. The DNA intermediates in CSR carry DSBs, which initiate a DNA damage response mediated by γ-H2AX, which is essential to CSR[41, 55]. In contrast, SHM intermediates contain DNA nicks, not DSBs( Fig 7); and γ-H2AX is not essential for SHM[56] or induced in hypermutating B cells. The CDT1 and GEM tags can be readily adapted to study repair in other contexts, and they also have potential practical utility. It has long been apparent that AID-initiated mutagenesis coupled with selection can in principle be used to optimize affinity or specificity of a variety of target proteins, including B and T cell receptors. However the complex regulation of AID has previously thwarted attempts to do so effectively. AIDF193A-mCherry-CDT1 and analogous AID derivatives should be useful in strategies that seek to evolve antibodies and other proteins ex vivo by iterative cycles of hypermutation and selection. Cell cycle tags may also be useful for optimizing activities of enzymes that act on DNA or RNA in the nuclease, including CRISPR/Cas9 and other nucleases used for genome engineering and gene correction.
AID ( Activation Induced Deaminase ) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram the genome in early development . AID is potentially highly mutagenic , as it deaminates C to U within single-stranded regions . Here we show that AID abundance is regulated by cell cycle , and that high levels of nuclear AID are tolerated only in G1 phase . These results identify an unanticipated role for spatiotemporal regulation in balancing demands of AID-initiated mutagenesis and its potentially pathological outcomes .