Transcriptional control of the recombination activating genes Rag1 and Rag2 in B lymphocytes and non-lymphoid cells
- Author(s): Timblin, Greg Alan
- Advisor(s): Schlissel, Mark S
- Tjian, Robert
- et al.
Expression of the recombination activating genes Rag1 and Rag2 (Rag) is essential for generation of a diverse B cell antigen receptor repertoire necessary for effective adaptive immune responses. The Rag genes are among the first lineage-specific genes expressed in lymphocyte progenitors, and their expression strongly correlates with commitment to the B lineage. Because V(D)J recombination involves generation of DNA breaks by the RAG proteins, Rag expression must be tightly controlled throughout B cell development to maintain genomic integrity. Using Abelson Murine Leukemia Virus-transformed B cells as a model system, we sought to identify factors that repress Rag transcription during pre-B cell differentiation, a period of B cell development marked by rapid proliferation during which the Rag genes are transiently repressed and then reactivated upon differentiation. A screen identified Ebf1 and c-Myb, two well-studied transcription factors in the context of early B cell development, as repressors of Rag transcription in these highly proliferative cells. As we found no evidence for direct Rag repression by Ebf1, we investigated how Ebf1 influences the expression of factors previously implicated in the regulation Rag transcription. We discovered that Ebf1 achieves Rag repression through both negative regulation of the Rag activator Foxo1, and positive regulation of the Rag repressor Gfi1b. In addition to influencing Foxo1 and Gfi1b expression in a similar manner, we found that c-Myb directly binds to the Erag enhancer in the Rag locus and antagonizes Foxo1 binding to this cis regulatory element. Together this work reveals previously unappreciated roles for Ebf1 and c-Myb in the transcriptional repression of the Rag genes during pre-B cell differentiation. In addition to screening for factors that regulate Rag expression during B cell development, we also performed a screen for factors capable of activating the Rag genes in non-lymphoid cells. Given the close connection between Rag expression and B lineage commitment in lymphocyte progenitors, we reasoned that if we were able to induce Rag transcription in non-lymphoid cells such as fibroblast, these cells might be activating a host of B cell-specific genes and undergoing direct reprogramming to the B lineage. While we identified factors that could readily induce Rag expression when overexpressed in primary fibroblasts, we could detect no evidence of a reprogramming event involving activation of additional B lineage genes. Together our work identified novel repressors of Rag transcription in developing B cells, and factors capable of activating Rag transcription in non-lymphoid cells that may inform future fibroblast-to-B cell reprogramming experiments.