RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination

Abstract

Nuclear processes such as transcription, DNA replication, and recombination are dynamically regulated by chromatin structure. Transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent posttranslational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 -an essential component of the RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly 1 -contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease. +To whom correspondence should be addressed: oettinger@frodo.mgh.harvard.edu; ogozani@stanford.edu. * These authors contributed equally to the work Note added in proof: While this work was under review, another study also reported that the RAG2 PHD finger binds to methylated H3K4 30 .Atomic coordinates and structure factors of the RAG2 PHD -H3K4me3 peptide complex have been deposited in the Protein Data Bank with the accession code of 2v89. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Since RAG2 contains a noncanonical plant homeodomain (PHD) finger 6,7 -a module that can mediate interactions with chromatin 8-10 -we asked whether a polypeptide encompassing the RAG2 PHD finger (RAG2 PHD : aa 414-527) can recognize modified histone proteins. In an in vitro screen of peptide microarrays containing ~70 distinct modified histone peptides, we found that RAG2 PHD specifically binds to histone H3 trimethylated at lysine 4 (H3K4me3) ( Fig. 1a ; Fig. S1; data not shown). The specificity of this interaction was confirmed by peptide pulldown assays ( Fig. 1b ; Fig. S2; Fig. S3). RAG2 has a C-terminal extension of 40 aa that is essential for phosphoinositide (PtdInsP)-binding 7 (aa 488-527), but this region is dispensable for H3K4me3-binding as the minimal PHD finger alone (aa 414-487) is sufficient for H3K4me3-recognition (Fig. 1c). In addition, the acidic hinge region of RAG2 (aa 388-412), previously implicated in...

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

“…4A, panel d). Thus, both mouse and human PHD domains displayed a preference for H3K4me3, confirming published results and thereby validating our experimental procedure [ 23,24,27 ]. This strongly suggests that the sea urchin SpRAG2L PHD domain indeed differs from its vertebrate counterpart in its selectivity for binding to H3K4me2.…”

“…Recent reports showed that the murine RAG2 PHD domain binds specifically to H3K4me3, and suggested that this interaction is required for high levels of V(D)J recombination [ 24,25,27 ]. The same binding specificity has been reported for other PHD domains, including that found in of human ING2 [ 23 ].…”

“…These can be categorized as "writers" (adding modifications), "erasers" (removing such modification), or "readers" (recognizing such modifications) [ 21 ]. One chromatin mark that correlates well with accessible areas in antigen receptor gene loci is hypermethylation of H3K4 [ 24,27,[35][36][37]. Recently, strong experimental evidence was provided that the RAG2 PHD domain is a histone code reader, rendering the recombinase highly active when in contact with nucleosomes displaying H3K4me3 [ 24,27 ].…”

“…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].…”

Structural insights into human KAP1 PHD finger–bromodomain and its role in gene silencing

“…Recent reports on the recognition of unmodified histone H3 by the BHC80 PHD finger in gene repression 17 and the simultaneous methyllysine and methylarginine recognition of histone H3 by the RAG2 PHD finger in V(D)J recombination 18,19 exemplify the functional versatility of a conserved structural fold. Integrated functions in histone interactions have also been shown for tandem modules of the same fold, such as the double bromodomain in human transcriptional proteins BRD2 and BRD4 (ref.…”