Beyond endonuclease - understanding the effect of RAG1 on chromatin and it`s implication on V(D)J recombination control

The RAG endonuclease initiates V(D)J recombination at the antigen receptor (AgR) loci by introducing DNA breaks adjacent to V,D and J segments of the variable loci. RAG ability to induce DNA breakage necessitates a tight regulation to ensure its activity to the AgR. Our studies suggested that RAG is doubly anchored to chromatin. The first anchor is mediated by interaction with promoter’s histone mark - H3K4me3. The second is yet unknown, but our data points out that this interaction is mediated by the RAG-RING domain - that confers E3 Ubiquitin ligase functionality - and marked H3K27Ac as a candidate for this interaction. Other studies showed that the RING domain of RAG– can interact and ubiquitylate Histone 3. This RAG-mediated H3 ubiquitylation on one hand, and the previously demonstrated ability of H3-Ub to facilitate H3 acetylation and promoter activation on the other hand, provides a possible link between RAG localization and activation. In this project we sought to explore this link, and to identify the effect of RAG-mediated ubiquitylation on chromatin. Our preliminary results – obtained by mapping of chromatin accessibility in WT pre-B cells and cells with RAG mutant that lacks the E3-ligase functionality, demonstrate that RAG can shape the chromatin structure in its binding regions, suggesting a novel role of RAG as a chromatin modulator, that is dependent on its E3-ligase functionality, but independent of its endonuclease activity. Such function may have a role both in its targeting to the AgR and its off- target activity in lymphoid cancers.