Transcription‐dependent AID targeting to immunoglobulin variable regions

Two mechanisms that increase antibody diversity, somatic hypermutation (SHM) and class switch recombination (CSR), depend upon the protein activation‐induced deaminase (AID), which can convert cytosine to uracil in single‐strand DNA (ssDNA) regions in vitro . Recently, our lab and others have shown that RNA polymerase II (RNAPII) accumulated in mouse switch regions due to the specialized DNA‐RNA structures termed R‐loops which form using G‐rich repetitive sequences in switch regions. R‐loops inhibit RNAPII processivity and may allow for increased access of AID to ssDNA. However, the variable region does not form R‐loops, so it is unclear how AID is targeted there. We have used chromatin immunoprecipitation (ChIP) and nuclear run‐on assays to determine RNAPII distribution in the VB1‐8 knock‐in mouse variable region. ssDNA patches were detected by treatment with sodium bisulfite, and mutations were mapped across the entire variable region. The hypothesis is that there may be secondary structure in the variable region which will pause the transcriptional machinery. Furthermore, the ssDNA regions could provide substrates for AID's deamination activity.