I am interested in understanding how immunoglobulin (Ig) genes are diversified at the molecular and cellular levels. Three processes of Ig gene diversification (somatic hypermutation, gene conversion, and class switch recombination) are triggered by activation-induced deaminase (AID). One important question is how the Ig genes are specifically targeted and regulated for diversification within the nucleus. To investigate this, we are using a derivative of the chicken DT40 B cell line, in which a polymerized lactose operator (PolyLacO) is inserted upstream of the diversifying Igλ gene. We have imaged the tagged Igλ genes by tethering lactose repressor (LacI) fused to GFP or RFP, and analyzed colocalizations of the Igλ genes with factors key to the process: for example, active RNA polymerase II, E2A regulatory factor and the MRE11/RAD50/NBS1 nuclease complex. With our research, we hope to reveal important details regarding the spatiotemporal aspects of the Ig gene diversification.
