Identification of a PU.1-regulated repressive region in Aicda gene locus
Abstract
Activation-induced cytidine deaminase (AID, encoded by Aicda) plays a primary role in producing mutations during somatic hypermutation and DNA breaks during class switch recombination, producing antibody diversity. Our laboratory generated a murine B cell-specific PU.1/Spi-B knockout, and whole-exome sequencing of their leukemias revealed C>T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 negatively regulates Aicda during B cell development. Using ChIP-seq, two regulatory regions (R1 & R2) within the first intron of Aicda were identified to bind PU.1, and R1 was targeted with CRISPR-Cas9 mutagenesis. ChIP-qPCR and sequencing experiments revealed that the mutations created were heterozygous and dampened PU.1’s ability to bind. In addition, larger deletions in the R1 region resulted in an up-regulation of Aicda expression in response to lipopolysaccharide. However, alteration of the PU.1 binding site resulted in no change in expression, suggesting that PU.1 does not act in a silencer fashion.