Although hundreds of miRNAs are present in the mammalian genome, genetic studies addressing their physiological roles are at an early stage. In the present work, we address the function of miR-155 in the context of antibody diversification by 1) identifying this particular miRNA as upregulated in B cells stimulated to undergo CSR, 2) by bioinformatically identifying AID as a putative target, and 3) by genetically mutating the target sequence in the 3′UTR of AID to allow the gene to escape miR-155 control. These experiments are the first to identify a target of a miRNA in vivo by manipulating not the expression of the miRNA itself but rather, by genetically disrupting the association between a miRNA and its putative target gene.
Most genes contain multiple predicted target sites for several different miRNAs. Deletion of just one of these targeting miRNAs can result in protein upregulation in a dose-dependent manner, leading to the hypothesis that multiple microRNAs synergistically fine-tune the expression of a single target gene (Xiao et al., 2007
). Surprisingly, the AID 3′UTR is predicted to contain only one miRNA target site, binding miR-155. We have shown that mutation of the seed region of this site leads to dramatic protein overabundance and disruption of proper temporal protein downregulation. We cannot exclude the possibility that the specific point mutations introduced at the miR-155 target site could result in additional miR-155 independent effects (for example, alteration of RNA secondary structure, mRNA stability, or disruption of a binding site for some unknown regulatory factor). However, the previously-reported miR-155 knockout mouse also showed upregulated AID expression (though to a lesser degree than observed in our AID-GFP-Mut mouse, to be addressed below)(Vigorito et al., 2007
), and we believe that the phenotypes observed in the AID-GFP-Mut mouse are explained by a model in which miR-155 directly regulates AID expression. In addition, levels of AID expression are stringently regulated in B cells such that mice heterozygous for AID show clear signs of haploinsufficiency (R. Casellas, manuscript submitted). These observations together with our results suggest that, especially when protein levels are already limiting, a single miRNA can contribute far more significantly to control of protein expression than previously appreciated.
Ablating miR-155 control of AID expression leads to increased protein expression in switching cells, which undergo significantly higher levels of CSR (). It also leads to increased protein expression in germinal center cells (). However, increased AID expression does not lead to more SHM in the Ig locus, in accordance with previous work by the Honjo group (Muto et al., 2006
), who have shown that transgenic AID overexpression does not lead to an increase in SHM. Our data presented here, and the data published by Muto et al., support the notion of the existence of a limiting factor that targets AID to the Ig locus so that in its presence, excess AID cannot access the locus (though in its absence it can, possibly in a stochastic fashion).
Excess AID in germinal center cells, however, appears to increase hypermutation in other loci such as bcl-6
, which are known secondary targets of the SHM apparatus. Excess AID is also likely to increase the rates of chromosomal translocations associated with errant hypermutation (Ramiro et al., 2006
; Ramiro et al., 2004
). Therefore, lack of miR-155 control of AID expression may be causal for B cell lymphomagenesis. Indeed, Kluiver et al. have recently documented a lack of miR-155 expression in primary cases of B cell Burkitt lymphomas, which constitutively express AID (Kluiver et al., 2006
In addition to overabundance of AID, we find that lack of miR-155 control leads to persistence of AID expression in post-germinal center B cells, thus effectively marking a novel subset of circulating B cells as recent emigrants from the germinal center. We also find that persistent AID expression is associated with specific defects in affinity maturation. It is possible that persistent AID expression supports ongoing mutation in the Ig locus well after cells exit the germinal center with an affinity-matured antigen receptor, effectively destroying the properly-selected antigen-specific Ig repertoire. However, this scenario is not strongly supported by our data, emphasizing again that specific co-factors may be required to target AID to the Ig locus. Instead, we observe multiple low-affinity B cell clones in the blood of animals where the AID gene is not subject to miR-155 control, suggesting a defect in positive selection of properly matured B cells. Though the mechanism of positive selection and affinity maturation are not well understood, it is thought that B cells cycle between the dark zone of the germinal center, where mutation occurs, to the light zone, where their newly minted receptors are substrates for positive selection. Eventually, high-affinity B cells are thought to emerge after multiple rounds of recycling through the germinal center. Our data would support a scenario where B cells overexpressing AID may not be allowed to “recycle” into a germinal center for proper affinity maturation. We could speculate on the existence of a mechanism for cellular sensing of AID levels that would form a feedback loop between proper AID extinction and GC cycling. However, this would be difficult to experimentally demonstrate as little is presently known about the cellular and molecular parameters governing the mechanism of proper selection and affinity maturation.
The previously described miR-155 knockout mouse models (Rodriguez et al., 2007
; Thai et al., 2007
) highlighted the immense contribution of miR-155 –mediated regulation to various aspects of vertebrate immunity. The phenotypes therein, specifically – modest increase in AID expression, decreased in vivo
CSR to IgG1, and impaired affinity maturation – likely reflect the composite deregulation of at least 60 genes (Vigorito et al., 2007
). In contrast to global ablation of miR-155 regulation, we describe here specific disruption of a single miR-155 : target interaction. In our mouse model, we observe significant AID-GFP deregulation, increased in vitro
CSR, and impaired affinity maturation – effects which may not be apparent in the more complex context of total miR-155 deficiency.
As the central catalyst for antibody diversification processes, AID has been shown to be regulated at the level of transcription (Dedeoglu et al., 2004
; Gonda et al., 2003
; Sayegh et al., 2003
) (R. Casellas, manuscript submitted), by nucleo-cytoplasmic trafficking (Ito et al., 2004
; McBride et al., 2004
), and by phosphorylation (Basu et al., 2005
; Basu et al., 2007
; McBride et al., 2006
; Pasqualucci et al., 2006
). Here we reveal an additional miRNA-mediated pathway of AID regulation, which controls AID expression levels in germinal center B cells and ensures proper extinction of AID expression as cells affinity mature and exit the secondary lymphoid organs. Thus, miR-155 plays an important role in the molecular restraint of AID, an enzyme that confers great immunological benefit, but must be tightly regulated to limit its mutagenic potential.