AID is critical for immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM). Here we showed that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda promoter. This site functioned in synergy with a conserved Sp-NF-κB-binding site. HoxC4 was preferentially expressed in germinal center B cells and was upregulated by CD154:CD40 engagement, lipopolysaccharide and interleukin-4. HoxC4 deficiency resulted in impaired CSR and SHM, due to decreased AID expression and not other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4 −/− B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM.CSR and SHM are critical for the maturation of antibody responses to foreign and selfantigens. CSR recombines switch (S) region DNA located upstream of constant heavy chain (C H ) region exons, thereby changing immunoglobulin (Ig) C H regions and endowing antibodies with new biological effector functions. SHM introduces mainly point mutations in Ig variable regions, thereby providing the structural substrate for selection of higher affinity antibody mutants by antigen. In spite of the recent advances made in the identification of some factors involved in CSR and SHM, the intimate mechanisms of these processes remain elusive. Both CSR and SHM require activation-induced cytidine deaminase (AID), which is expressed by activated B cells, mainly in germinal centers (GCs) of peripheral lymphoid organs1,2. AID initiates CSR and SHM by deaminating dC residues to yield dU:dG mispairs in DNA3-8. These dU:dG mispairs trigger DNA repair processes entailing introduction of mutations in V(D)J regions or DNA breaks, including doublestranded DNA breaks, which lead to non-classic non-homologous end-joining and CSR3,5,9-14.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence should be addressed to P.C. (pcasali@uci.edu). 1 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS H.Z. and S.-R.P. contributed equally to this work; S.-R.P., H.Z., Z.P., J.Z., T.M., E.J.P. and A.A.-Q. performed the experiments; Z.X. helped designing experiments, discussed the results and read and provided comments on the manuscript; H.Z. designed the experiments, analyzed the data and prepared the manuscript; P.C. designed the experiments, analyzed the data, supervised the work and prepared the manuscript. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe mechanisms governing the transcriptional regulation of the gene encoding AID (AICDA in the human and Aicda in the mouse) remain to be elucidated. A conserved region in the first intron of Aicda containing two E-boxes, the consensus sequence for E2A (http:// www.signa...

Class switch DNA recombination (CSR) is the mechanism that diversifies the biological effector functions of antibodies. Activation-induced cytidine deaminase (AID), a key CSR player, targets IgH switch (S) regions, which contain 5′-AGCT-3′ repeats in their core. How AID is recruited to S regions remains unclear. Here we show that 14-3-3 adaptor proteins play an important role in CSR. 14-3-3 proteins specifically bind 5′-AGCT-3′ repeats, are upregulated in B cells undergoing CSR and are recruited together with AID to the S regions involved in CSR events (Sμ→Sγ1, Sμ→Sγ3 or Sμ→Sα). Moreover, blocking 14-3-3 by difopein, deficiency in 14-3-3γ or expression of a dominant negative 14-3-3σ mutant impaired recruitment of AID to S regions and decreased CSR. Finally, 14-3-3 proteins interact directly with AID and enhance AID-mediated in vitro DNA deamination, further emphasizing the important role of these adaptors in CSR.

Transforming growth factor (TGF)‐β1 is well established as a critical IgA isotype switching factor and Smad molecules have been reported to act as transducers and transcriptional factors in the expression of TGF‐β1‐targeted genes. We examined the involvement of Smad proteins in TGF‐β1‐induced IgA expression. First, we found that TGF‐β1 significantly increases endogenous germ‐line (GL) α transcripts by LPS‐stimulated CH12.LX.4933 (μ+) B lymphoma cells. To investigate its signaling mechanisms, the lymphoma cell line was transfected with pFL3 that contains the TGF‐β‐responsive element of the GLα promoter, and stimulated with TGF‐β1. Similar to endogenous GLα transcripts, TGF‐β1 induces GLα promoter activity and overexpression of Smad3 markedly enhances the promoter activity. This activity is further augmented by cotransfected Smad4. On the other hand, Smad7 substantially abrogates the synergistic effect of Smad3/4 onGLα promoter activity. In addition, overexpression of Smad3/4 enhances TGF‐β1‐induced endogenous GLα transcripts in normal spleen B cells. Finally, in the presence of TGF‐β1, overexpression of Smad3/4 selectively increases both surface IgA expression and IgA production. The results from the present study indicate that Smad3, Smad4, and Smad7, at least in part, serve as mediators linking TGF‐β1 to transcriptional regulation of IgA switching related gene and regulation of IgA class switching.