In vivo analysis of Aicda gene regulation: a critical balance between upstream enhancers and intronic silencers governs appropriate expression

The Aicda gene encodes activation-induced cytidine deaminase (AID). Aicda is strongly transcribed in activated B cells to diversify immunoglobulin genes, but expressed at low levels in various other cells in response to physiological or pathological stimuli. AID's mutagenic nature has been shown to be involved in tumor development. Here, we used a transgenic strategy with bacterial artificial chromosomes (BACs) to examine the in vivo functions of Aicda regulatory elements, which cluster in two regions: in the first intron (region 2), and approximately 8-kb upstream of the transcription start site (region 4). Deleting either of these regions completely abolished the expression of Aicda-BAC reporters, demonstrating these elements' critical roles. Furthermore, we found that selectively deleting two C/EBP-binding sites in region 4 inactivated the enhancer activity of the region despite the presence of intact NF-κB-, STAT6- and Smad-binding sites. On the other hand, selectively deleting E2F- and c-Myb-binding sites in region 2 increased the frequency of germinal-center B cells in which the Aicda promoter was active, indicating that E2F and c-Myb act as silencers in vivo. Interestingly, the silencer deletion did not cause ectopic activation of the Aicda promoter, indicating that Aicda activation requires enhancer-specific stimulation. In summary, precise regulation of the Aicda promoter appears to depend on a coordinated balance of activities between enhancer and silencer elements.