E2A Acts in cis in G1 Phase of Cell Cycle to Promote Ig Gene Diversification1

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, AID. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis-activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged IgλR gene in the chicken DT40 B cell line, which carries out constitutive Ig gene diversification. By analysis of a DT40 derivative in which polymerized lactose operator tags the rearranged λR gene, we show that E2A must function in cis to promote diversification, and that stimulation of diversification in cis depends on the E2A activation domains. By direct imaging, we show that λR/E2A colocalizations are most prominent in G1 phase of cell cycle. We further show that expression of the E2A antagonist Id1 prevents λR/E2A colocalizations in G1 phase, and impairs diversification but not transcription of λR. Thus, E2A acts in cis to promote Ig gene diversification, and G1 phase is the critical window for E2A action.