Temporal Regulation of Ig Gene Diversification Revealed by Single-Cell Imaging1

Rearranged Ig V regions undergo AID-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions; but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G1 phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-YFP transfectants, we now demonstrate that AIDYFP can stably localize to the nucleus in G1 phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 PolyLacO-λR cells, in which polymerized lactose operator tags the rearranged λR gene, we show that both the repair polymerase Polη and the multifunctional factor MRE11/RAD50/NBS1 localize to λR, and that λR/Polη colocalizations occur predominately in G1 phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of γ-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G1 phase. AID deaminates single-stranded DNA, and restriction of mutagenesis to G1 phase would contribute to protecting the genome from offtarget attack by AID when DNA replication occurs in S phase.