B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of the antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins introduce DNA double-strand breaks (DSBs) at antigen receptor gene segments, and is completed when the DSBs are sensed and repaired by non-homologous end-joining. This process is subjected to many layers of regulation, but an elementary means to constrain V(D)J recombination is to control the level of the RAG proteins themselves. Work in this laboratory suggests that RAG1 levels are controlled by a RAG1 interacting protein we identified called Vpr binding protein (VprBP; also called DCAF1), which exerts its control through two different mechanisms: first, by promoting timely proteasome-dependent degradation of RAG1 through VprBP?s association with a Cul4-DDB1 E3 ubiquitin ligase complex; and second, by regulating Rag transcriptional induction under conditions that stimulate V(D)J recombination. The details underlying the second mechanism remain unclear. However, recent studies have identified VprBP as potentially mediating direct interactions with a key transcription factor required for Rag expression, called FoxO1, in a manner that is sensitive to FoxO1 acetylation status. These findings lead us to hypothesize that VprBP(DCAF1) mediates acetylation-dependent association with FoxO1 to regulate Rag transcriptional induction. To test this hypothesis, we will (i) establish whether VprBP mediates acetylation-dependent association with FoxO1 and influences its localization; and (ii) test whether FoxO1 acetylation regulates FoxO1 deposition to the Rag locus, Rag transcriptional activation, and V(D)J recombination, in a VprBP-dependent manner. Establishing the molecular basis for VprBP-FoxO1 interactions, their reliance on acetylation, and the influence of acetylation on Rag expression would define a new physiological role for VprBP in V(D)J recombination, and exposes potential sources for altered immune repertoire and genomic instability caused by dysregulated Rag expression. This work would also provide a paradigm for understanding how VprBP regulates FoxO1-dependent transcriptional activation of its target genes, which would further broaden the scientific interest of the project and potentially reveal new avenues to therapeutically regulate FoxO1-dependent gene expression.
V(D)J recombination is a DNA rearrangement process that is responsible for generating a diverse population of B and T cells that together provide highly specific immunity to pathogenic microorganisms. The proposed research will investigate whether a protein called DCAF1(VprBP) binds and regulates the activity of a key transcription factor, called FoxO1, that is required for the expression of the RAG proteins that initiate V(D)J recombination. This work will not only illuminate a novel role for DCAF1(VprBP) in regulating V(D)J recombination, but because VprBP and FoxO1 are broadly expressed, this work will also shed important insight into how VprBP generally regulates transcriptional activation of FoxO1-dependent genes, which may be therapeutically exploited to modulate the expression of FoxO1-regulated genes in disease states.
