Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination

Abstract

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS. Introduction V(D)J recombination is the site-specific DNA rearrangement process that assembles the B cell receptor and TCR genes during lymphoid development. Recombination is initiated by the lymphoid-specific Rag1 and Rag2 recombinase (1, 2), which introduces double-strand DNA breaks at recombination signal sequences (RSSs) flanking variable (V), diversity (D), and junction (J) gene segments spread along the Ig and TCR loci. Subsequently, the ubiquitous nonhomologous end-joining (NHEJ) machinery, which includes Ku70/Ku80, DNA-PKcs, Artemis, XRCC4, LigaseIV, and Cernunnos-XLF, repairs those lesions to form V(D)J coding joints and signal joints (3, 4). The recombination process is tightly regulated, occurring at specific stages of development and in specific cell types (e.g., Ig and TCR genes are rearranged in B and T cells, respectively). This process takes place in a temporal manner, with Ig heavy chain rearrangements preceding Ig light chain rearrangements and D-to-J rearrangements preceding V-to-DJ rearrangements (5).V(D)J recombination is critical for proper immune function and, when impaired, generally results in the arrest of both B and T cell development, leading to severe combined immunodeficiency (SCID). Accordingly, mutations in Rag1, Rag2, or NHEJ factors have been described in patients characterized by a complete absence

Human RAG mutations: biochemistry and clinical implications

“…Moreover, the W416L, C446W, W453R and C478Y mutant proteins are associated with a marked decrease in D H –J H and Vκ–Jκ rearrangements when introduced into Abelson virus-transformed Rag2 −/− pro-B cells 63 . Interestingly, the W416L, C446W and W453R RAG2 mutants showed abnormal subcellular localization, being partially retained in the cytoplasm 63 . Overall, these data conclusively demonstrate the crucial role of the RAG2 PHD in V(D)J recombination.…”

“…Among the 13 missense mutations in the non-core region of RAG2 that have been reported in patients with SCID and Omenn syndrome, 12 affect the RAG2 PHD, and for many of these mutations the structural and functional implications have been studied in vitro 63 . In particular, C446W and C478Y destabilize the RAG2 protein.…”

Reciprocal regulation of Rag expression in thymocytes by the zinc-finger proteins, Zfp608 and Zfp609

“…This ZORI defect is similar to that observed in Omenn Syndrome. 16 ZORI mice and Omenn syndrome cases both have limited levels of V(D)J recombination due to either reduced Rag expression or the presence of hypomorphic mutations in Rag resulting in reduced Rag activity, respectively. This points to the fine balance of Rag expression and timing required for normal development and function of the immune system.…”

Omenn syndrome does not live by V(D)J recombination alone

“…Mutations in the RAG2 gene, although less frequent, have been reported in parallel [2, 22,25]. Biochemical analysis of these mutations supports the structural model of a six-bladed b propeller with a C terminus containing a noncanonical plant homeodomain (PHD) finger.…”

“…This region directly interacts with histones [26] and in particular with hypermethylated histone H3 at lysine 4, which is crucial for efficient V(D)J recombination [27 ]. Point mutations in the PHD affect the subcellular localization and stability of Rag2, rendering them unable to reach their DNA target [25]. Furthermore, it is important to note that the mutation of a single critical residue within the RAG2-PHD finger, tryptophan 453, is associated with Omenn syndrome.…”