Epigenetic Modulation of Class-Switch DNA Recombination to IgA by miR-146a Through Downregulation of Smad2, Smad3 and Smad4

IgA is the predominant antibody isotype at intestinal mucosae, where it plays a critical role in homeostasis and provides a first line of immune protection. Dysregulation of IgA production, however, can contribute to immunopathology, particularly in kidneys in which IgA deposition can cause nephropathy. Class-switch DNA recombination (CSR) to IgA is directed by TGF-β signaling, which activates Smad2 and Smad3. Activated Smad2/Smad3 dimers are recruited together with Smad4 to the IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in the unfolding of CSR to IgA. Epigenetic factors, such as non-coding RNAs, particularly microRNAs, have been shown to regulate T cells, dendritic cells and other immune elements, as well as modulate the antibody response, including CSR, in a B cell-intrinsic fashion. Here we showed that the most abundant miRNA in resting B cells, miR-146a targets Smad2, Smad3 and Smad4 mRNA 3’UTRs and keeps CSR to IgA in check in resting B cells. Indeed, enforced miR-146a expression in B cells aborted induction of IgA CSR by decreasing Smad levels. By contrast, upon induction of CSR to IgA, as directed by TGF-β, B cells downregulated miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to the Iα promoter for activation of germline Iα-Cα transcription. Deletion of miR-146a in miR-146a–/– mice significantly increased circulating levels of steady state total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a–/– mice, the elevated systemic IgA levels were associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA as well as kidney IgA deposition, a hallmark of IgA nephropathy. Increased germline Iα-Cα transcription and CSR to IgA in miR-146a–/– B cells in vitro proved that miR-146a-induced Smad2, Smad3 and Smad4 repression is B cell intrinsic. The B cell-intrinsic role of miR-146a in the modulation of CSR to IgA was formally confirmed in vivo by construction and OVA immunization of mixed bone marrow μMT/miR-146a–/– chimeric mice. Thus, by inhibiting Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.

Rad52 mediates class-switch DNA recombination to IgD

While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig class remains unknown. In B cells, IgD is expressed together with IgM as transmembrane receptor for antigen through alternative splicing of long primary VHDJH-Cμ-s-m-Cδ-s-m RNA, which also underpins the secreted form of IgD. IgD is also expressed through class switch DNA recombination (CSR), as initiated by AID-mediated double-strand DNA breaks (DSBs) in Sμ and σδ and resolution of such DSBs by a yet unknown alternative endjoining (A-EJ) mechanism. This synapses Sμ with σδ region DSB resected ends leading to insertion of extensive S-S junction microhomologies, unlike the Ku70/Ku86-dependent NHEJ which resolves DSB blunt ends in CSR to IgG, IgA and IgE with little or no microhomologies. We previously demonstrated a novel role of DNA annealing homologous recombination Rad52 protein in 'short-range' microhomology-mediated synapsis of intra-Sδ region DSBs. This led us to hypothesize that Rad52 is also involved in the short-range microhomology-mediated A-EJ recombination of Sμ with σδ. We found that induction of IgD CSR by T-dependent or T-independent stimuli downregulated Zfp318 (the suppressor of Cδ-s-m transcription termination), promoted Rad52 phosphorylation, recruitment of Rad52 to Sμ and σδ leading to Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52-/- B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Further, Rad52 knockdown in human B cells virtually abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and lupus patients. Thus, Rad52 mediates CSR to IgD by synapsing Sμ-σδ resected DSB ends through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in mammalian DNA repair.