30 | Chapter 2 RAGulation: the intricate regulation of RAG expression and activity RAG1 and RAG2 proteins are regulated on multiple levels. In the next paragraphs, various levels of RAG regulation are outlined. Cell cycle and post-translational regulation The cell cycle is an important regulator of RAG2 expression, ensuring that RAG2 is only expressed in the G0/G1 phase, whereas it is almost undetectable in the S and the G2/M phases of the cell cycle. The RAG2 availability is modified by cyclin-dependent kinases (CDK), which initiate the rapid proteasomal degradation when entering the S phase. RAG2 is phosphorylated at Thr-490, which is a consensus substrate sequence for CDK2, CDK2, and CDK4, providing the conserved degradation signal116. Consequently, V(D)J recombination occurs exclusively in the G1 phase. In contrast to the cell cycle-dependent oscillation of RAG2, the levels of RAG1 remain rather consistent throughout the cell cycle117. Though not cell-cycle-dependent, post-translational modifications of RAG1 have also been described. The N-terminus of RAG1 contains a functional E3 ubiquitin ligase RING domain118. This domain has been shown to be involved in the ubiquitination of histone H3 as well as RAG1 itself. Recently, it was found that mouse mutants carrying the P326G mutation in the RAG1 RING domain accumulate RAG1 protein, likely due to the impaired ability to degrade RAG1 through autoubiquitylation119,120. Interestingly, we observed that DNA damage negatively impacts the protein stability of RAG1. Specifically, in human pre-B cell line harboring BCR-ABL1 translocation, co-treatment with the DNA damaging agent neocarzinostatin (NCS) and the protein synthesis inhibitor cycloheximide (CHX) resulted in a shorter RAG1 half-life compared to treatment with CHX alone, possibly due altered post-translational modification of RAG1121. Spatial and temporal regulation The subcellular spatial regulation of RAG proteins has been shown to be an important regulator of V(D)J recombination. In T cells, the different regions of T-cell receptor b (Tcrb) were shown to interact with the nuclear lamina and at the same time, the RAG2 abundance in the nuclear periphery was found to be decreased. The spatial distributions of RAG2 and the Tcrb locus may thus be involved in the regulation of V(D)J recombination by limiting accessibility, and may even be one of the mechanisms enforcing allelic exclusion122. Not only RAG2 but also RAG1 has been shown to be spatially regulated; in mouse pre-B cells RAG1 localized with nucleolar markers and disruption of nucleoli lead to increased V(D)J recombination activity. The nucleolar sequestration of RAG1, controlled by N-terminal regions of RAG1, contributes to the regulation of the V(D)J recombination activity123. In addition, PAX5, known mostly for being an important B-cell commitment factor controlling transcription, also regulates the spatial positioning of chromatin towards the central nucleus regions
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