Dual reporter system to dissect cis- and trans-effects influencing the mutation rate in a hypermutating cell line

Activation induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching in the immunoglobulin genes of B-lymphocytes. AID expression by itself is sufficient to induce a GC-basepair biased mutator phenotype in lymphoid and non-lymphoid cell lines. Nevertheless a network of cis-regulatory elements and additional trans-factor proteins seems to govern the molecular mechanism of somatic hypermutation. To address the nature of mutation rate changes observed in the hypermutating pre-B cell line 18-81, we extended our previously described green fluorescent protein (GFP) reversion-system. Introducing an additional mutation reporter transgene enables us to discriminate between cis- and trans-factor caused alterations in the mutator phenotype. We show here that in cell line 18-81 the mutation rate declines upon prolonged periods of cell culture. The gradual loss of the mutator phenotype in cell line 18-81 is due to the downregulation of endogenous AID expression and can be reconstituted by overexpression of human AID protein. A correlation between AID mRNA levels and mutation rates is evident and even small changes in AID expression levels cause a significant effect on the mutability of the reporter transgenes.