The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2

Abstract

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatindependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.J recombination is the site-specific DNA rearrangement that assembles antigen receptor genes from dispersed arrays of V, D, and J gene segments. Recombination is initiated by the lymphoid-specific recombination activating gene (RAG) 1 and RAG2 recombinase, which recognizes and cleaves the recombination signal sequences (1). V(D)J recombination is tightly regulated, occurring in a preferred temporal order and only in specific cell types and developmental stages. Ig heavy chain rearrangement precedes light chain rearrangement and Ig heavy-chain D to J joining precedes V to DJ recombination. In addition, Ig genes are fully rearranged only in B cells (not T cells), and T cell receptor genes are assembled in T but not B cells (1). Overexpression of RAG1 and RAG2 in nonlymphoid cells is sufficient to induce recombination of an artificial extrachromosomal substrate but does not support V(D)J recombination of endogenous loci (2). Therefore, the accessibility of these loci to the recombinase must be regulated (3). A large body of evidence suggests that the regulation of chromatin structure is involved in the regulation of V(D)J recombination (4-6).

Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation

“…Since PHD domains have been shown to be capable of interacting with trimethylated H3K4, the association of the RAG2 PHD domain with H3K4 was recently tested. The crystal structures of RAG2-PHD alone and complexed with methylated H3K4 have been determined [60]. These studies have demonstrated that in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site.…”

The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

“…This raised the possibility that the RAG2 PHD domain enables the binding of the RAG complex to nucleosomes with H3K4me3 modifications whose chromosomal locations correlate with active gene expression and V(D)J recombinase accessibility. Recent reports showed that murine RAG2 is indeed able to interact with trimethylated histone tails, and this ability is important for efficient V(D)J recombination [24][25][26][27].…”

Exploring the binding diversity of intrinsically disordered proteins involved in one‐to‐many binding

“…6(D)]. 69 Histone demethylase LSD1 regulates transcription by demethylating Lys5 of histone H3 [ Fig. 6(E)].…”