Title In Vivo Analysis of Aicda Gene Regulation: A Critical Balance between Upstream Enhancers and Intronic Silencers Governs

The Aicda gene, encoding activation-induced cytidine deaminase (AID) is strongly transcribed in activated B cells to diversify immunoglobulin genes, yet low-level expressions upon physiological/pathological stimuli in various cells have been reported. Mutagenic nature of AID has shown to be involved in tumor development. Here, by using a transgenic strategy with bacterial artificial chromosomes (BAC), we examined the in vivo function of Aicda regulatory elements which cluster in two regions, namely approximately 8-kb upstream of the transcription start site (region 4) and the first intron (region 2). Deletion of either of regions completely abolished the expression of Aicda-BAC-reporters, indicating critical roles of these elements. Furthermore, we found that the selective deletion of two C/EBP binding sites in region 4 inactivates its enhancer activity in spite of the presence of the intact NF-κB-, STAT6and Smad-binding-sites. On the other hand, the selective deletion of E2Fand c-Myb-binding-sites in region 2 increased the frequency of the cells with active Aicda promoter in germinal center B cells, indicating that E2F and c-Myb function as silencer in vivo. Interestingly, the silencer deletion did not cause the ectopic activation of Aicda promoter, indicating that specific stimulation of the enhancer is required for Aicda activation. In summary, the precise regulation of the Aicda promoter appears to dependent on a coordinated balance of activities between the enhancer and silencer elements.