Transcriptional Regulation of the V(D)J Recombinase (Rag)

Tight regulation of RAG activity is required for proper development of the adaptive immune system as well as prevention of genomic instability. Foxo1 and GFI family members are crucial transcriptional regulators of Rag expression. To identify the regulatory pathways activating Foxo1 for Rag expression in B cells, we generated a panel of Foxo1 mutants and assayed their ability to upregulate Rag expression. We discovered a novel residue, serine at amino acid 215 (S215), on Foxo1 that is required for transactivating Rag expression, but not most other Foxo1-regulated genes. S215 modulates Foxo1 activity, at least in part, by regulating Foxo1 binding to the Rag locus. We also identified MK5 as an activator of Rag transcription, likely by phosphorylating S215 and thereby activating Foxo1. Further, we sought to study the negative regulators of Rag transcription. Previously in our lab, GFI1B was identified as a repressor of Rag transcription. However, Gfi1b-deficient mice have no defect in Rag expression. We thus hypothesized that GFI1, a member of the same transcription factor family, may compensate for loss of GFI1B. To test this hypothesis, we generated conditional knockout mice for both Gfi1 and Gfi1b. Deleting both Gfi1 and Gfi1b in primary B cell cultures resulted in an upregulation of Rag expression. Moreover, both GFI1 and GFI1B bind directly to the Rag locus. Together, these data indicate that both family members serve redundant functions in Rag repression in developing B cells. Lastly, to study whether GFI family proteins play a role in Rag repression outside the lymphoid lineage, we utilized a V(D)J recombination reporter mouse to study the effect of Gfi1 and Gfi1b deletion in other hematopoietic lineages. We observed aberrant Rag expression in plasmacytoid dendritic cells (pDCs) when GFI family proteins were deleted in ex vivo cultures. Microarray analysis revealed that GFI family proteins regulate a diverse set of genes in pDCs, but not a lymphoid-specific transcriptional program. Together, this study identified a novel pathway and elucidated the functions of a positive and a negative regulator of Rag expression