Dual Mechanism of Rag Gene Repression by c-Myb during Pre-B Cell Proliferation

Developing B lymphocytes undergo clonal expansion following successful immunoglobulin heavy chain gene rearrangement. During this proliferative burst, expression of the Rag genes is transiently repressed to prevent the generation of double-stranded DNA (dsDNA) breaks in cycling large pre-B cells. The Rag genes are then reexpressed in small, resting pre-B cells for immunoglobulin light chain gene rearrangement. We previously identified c-Myb as a repressor of Rag transcription during clonal expansion using Abelson murine leukemia virus-transformed B cells. Nevertheless, the molecular mechanisms by which c-Myb achieved precise spatiotemporal repression of Rag expression remained obscure. Here, we identify two mechanisms by which c-Myb represses Rag transcription. First, c-Myb negatively regulates the expression of the Rag activator Foxo1, an activity dependent on M303 in c-Myb's transactivation domain, and likely the recruitment of corepressors to the Foxo1 locus by c-Myb. Second, c-Myb represses Rag transcription directly by occupying the Erag enhancer and antagonizing Foxo1 binding to a consensus forkhead site in this cis-regulatory element that we show is crucial for Rag expression in Abelson pre-B cell lines. This work provides important mechanistic insight into how spatiotemporal expression of the Rag genes is tightly controlled during B lymphocyte development to prevent mistimed dsDNA breaks and their deleterious consequences.