Epigenetic Regulation of Aicda Transcription in B Cells

Abstract

<p>Activation-induced cytidine deaminase (AID), encoded by the Aicda gene, is indispensable for secondary antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR). It is expressed predominantly in germinal center (GC) B cells, where it deaminates cytosine to uracil in the DNA of immunoglobulin (Ig) genes, triggering mutagenesis (SHM) or deletional recombination events (CSR). However, when misregulated, AID can also mutate non-Ig genes, including proto-oncogenes, thereby contributing to genomic instability and cancer. Due to its potentially deleterious effects, AID expression must be tightly controlled. At the transcriptional level, Aicda is regulated by four highly conserved cis-regulatory regions (Regions 1-4). Region 1 contains the promoter, and is responsible for basal transcription of Aicda. Region 2 contains both enhancer and silencer elements, but functions as a negative regulatory region, restricting AID expression to antigen-activated B cells. Region 3 is reported to be essential for normal AID expression, but its role is otherwise unclear. Region 4, which contains two STAT6 sites essential for its function, provides enhancer activity in response to cytokine stimulation and is responsible for the high levels of AID expression found in GC B cells. Epigenetic mechanisms could add another layer of control to the regulation of Aicda; however, little research has been done in this regard. In this study, I investigated the role of DNA methylation in the cis-regulation of Aicda transcription. Through bisulfite sequencing of splenic mouse B cell DNA, I demonstrate that Region 4 is highly methylated in naïve B cells, but becomes demethylated in activated B cells. This is in contrast to Region 2, which I found to be constitutively unmethylated, and Region 1, which only becomes partially demethylated. Using quantitative methylation-specific PCR (qMSP), I show that loss of methylation in Region 4 correlates positively with cell division number, consistent with a passive mechanism of demethylation. I also show that demethylation in Region 4 cannot be achieved by cell proliferation alone, and that it depends upon induction of AID expression (via IL-4 stimulation). However, bisulfite sequencing of CH12F3-2 cell DNA shows that demethylation by itself is insufficient to activate AID expression. Taken together, the data suggests that IL-4 induces STAT6 to bind to its cognate sites in Region 4 and inhibit DNMT1, likely through chromatin reorganization, triggering passive demethylation during the induction of Aicda transcription.</p>