H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase John Bettridgea,b, Chan Hyun Nac,d, Akhilesh Pandeyc,d, and Stephen Desiderioa,b,1 aDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; bInstitute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cMcKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and dDepartment of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited by Frederick W. Alt, Boston Children’s Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, and approved December 27, 2016 (received for review September 26, 2016) V(D)J recombination is initiated by the recombination-activating cleft and participates in formation of the active site (11). Although gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. residues 387 through 527 of RAG-2 are dispensable for DNA The susceptibility of gene segments to cleavage by RAG is associated cleavage in vitro, this region supports several regulatory functions with histone modifications characteristic of active chromatin, in- in vivo, including binding of RAG-2 to H3K4me3 (7, 8, 12). This cluding trimethylation of histone H3 at lysine 4 (H3K4me3). Binding function is mediated by a noncanonical plant homeodomain (PHD) of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates finger that spans residues 415 through 487 (12, 13). Engagement of substrate binding and catalysis, which are functions of RAG-1. This H3K4me3 by the PHD finger promotes recombination in vivo (7, 8) has suggested an allosteric mechanism in which information re- and synthetic peptides bearing the H3K4me3 modification stimu- late cleavage of RSS substrates by RAG in vitro (14–16), consistent garding occupancy of the RAG-2 PHD is transmitted to RAG-1. To with the interpretation that H3K4me3 is an allosteric activator of determine whether the conformational distribution of RAG is altered the V(D)J recombinase. by H3K4me3, we mapped changes in solvent accessibility of cysteine This interpretation was reinforced by the identification within thiols by differential isotopic chemical footprinting. Binding of RAG-2 of an autoregulatory region, the presence of which was H3K4me3 to the RAG-2 PHD induces conformational changes in revealed by second site mutations that rescue the activity of RAG-1 within a DNA-binding domain and in the ZnH2 domain, which RAG-2 lacking a functional PHD finger (15). Disruption of this acts as a scaffold for the catalytic center. Thus, engagement of autoregulatory region is associated with constitutive increases in RSS H3K4me3 by the RAG-2 PHD is associated with dynamic conforma- binding affinity, catalytic rate, and recombination frequency, thus tional changes in RAG-1, consistent with allosteric control by mimicking the stimulatory effects of H3K4me3 (15). These obser- active chromatin. vations support a model in which RAG activity is suppressed by an autoinhibitory domain whose action is relieved by active chromatin. DNA recombination | genomic plasticity | allosteric control | epigenetic Allosteric activation is usually accompanied by a change in the modification | immune development distribution of accessible protein conformations in the ligand- bound state (17). We asked whether the conformational distribu- tion of RAG is altered upon binding of H3K4me3 to the RAG-2 ll forms of DNA processing—replication, transcription, re- — PHD finger. To quantify effects of H3K4me3 on the conformations Acombination, and repair use allosteric regulation, often as of RAG-1 and RAG-2 we carried out differential isotopic chemical a basis for molecular discrimination but also to establish a se- footprinting of solvent-exposed cysteine thiols, in combination with quence of interactions or to bias the outcome of a reaction. In many instances the allosteric ligand is a specific DNA structure, as Significance in Cre-mediated recombination, in which the Holliday junction intermediate effects allosteric conformational changes that switch active and inactive Cre monomers (1). In other instances the al- Accessibility of antigen receptor gene segments to recombination- losteric ligand is a DNA-bound protein array, as in λ integration, activating gene (RAG), the V(D)J recombinase, is correlated with which is driven to completion by a flanking DNA-protein array that marks of active chromatin. One mark, histone H3 at lysine 4 biases the conformation of λ-integrase (2). (H3K4me3), binds to a plant homeodomain (PHD) in the RAG-2 V(D)J recombination, the process by which antigen receptor subunit; mutations that abolish binding of the PHD to H3K4me3 genes are assembled, is also subject to allosteric control, but in this also impair V(D)J recombination. Engagement of H3K4me3 by case the allosteric ligand is a specific chromatin mark rather than a RAG-2 enhances substrate binding and catalysis, which are func- DNA structure. V(D)J recombination is initiated by recombination- tions of RAG-1. We show that H3K4me3 acts through the PHD to activating gene (RAG)-1 and RAG-2, which together cleave DNA induce conformational changes in an autoinhibitory domain re- at recombination signal sequences (RSSs) flanking the participating siding within RAG-2 as well as in substrate-binding and catalytic gene segments (3). There are two classes of RSS, termed 12-RSS regions of RAG-1. Our data suggest that H3K4me3 promotes and 23-RSS, in which heptamer and nonamer elements are sepa- displacement of the autoinhibitory domain as well as concomitant rated by spacers of 12 bp or 23 bp; physiological DNA cleavage conformational alterations in RAG-1 that favor increased substrate requires the pairing of a 12-RSS with a 23-RSS (3). V(D)J re- affinity and catalytic rate. combination acts in an ordered, locus-specific fashion during lym- phoid development. The accessibility of gene segments to V(D)J Author contributions: J.B., A.P., and S.D. designed research; J.B., C.H.N., and S.D. per- recombination is positively correlated with transcription at the formed research; J.B. and A.P. contributed new reagents/analytic tools; J.B., C.H.N., and unrearranged locus and with histone modifications characteristic of S.D. analyzed data; and J.B., C.H.N., and S.D. wrote the paper. active chromatin, including hypermethylation of histone H3 at ly- The authors declare no conflict of interest. sine 4 (H3K4me3) (4–10). This article is a PNAS Direct Submission. RAG-1 and RAG-2 are 1,040 and 527 aa residues long, re- Freely available online through the PNAS open access option. spectively. The catalytic core and DNA-binding functions are largely 1To whom correspondence should be addressed. Email: [email protected]. contained within RAG-1 (11). RAG-2, which is also essential for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DNA cleavage activity, comprises part of a putative DNA-binding 1073/pnas.1615727114/-/DCSupplemental. 1904–1909 | PNAS | February 21, 2017 | vol. 114 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1615727114 Downloaded by guest on October 1, 2021 mass spectrometry (18). Strikingly, binding of H3K4me3 to RAG-2 RAG-2 (14, 15). In addition to full-length, wild-type RAG-2, we is accompanied by robust increases in the solvent accessibility of used RAG-2(W453A), in which the PHD finger cannot bind RAG-1 within the dimerization and DNA-binding domain (DDBD) H3K4me3 (7). These RAG-2 variants, tagged at the amino termi- and in the ZnH2 domain, which acts as a scaffold for the catalytic nus with MBP, were coexpressed individually with maltose binding center (11). These H3K4me3-induced changes in solvent accessi- protein (MBP)-tagged cR1ct and RAG tetramers were purified by bility are abolished by mutation of the RAG-2 PHD finger. Our a protocol that removes endogenous H3K4me3 (15) (Fig. S1A). observations provide direct evidence that engagement of H3K4me3 TheactivefractionsoftheseRAGpreparations, as determined by by the RAG-2 PHD finger is associated with discrete changes in the burst kinetics (Fig. S1 B and C)rangedfrom5.3to5.4%(Fig. S1D), conformational distribution of RAG-1, consistent with the stimula- consistent with earlier observations (15). tory effects of H3K4me3 on RSS binding and cleavage by RAG. Equivalent amounts of each active RAG tetramer were assayed in vitro for coupled cleavage of a radiolabeled 12-RSS in the Results and Discussion presence of unlabeled 23-RSS substrate and increasing amounts of Information indicating engagement of H3K4me3 by the RAG-2 a histone H3-derived peptide containing trimethylated lysine 4 PHD finger must be communicated within the RAG tetramer in (H3K4me3) or unmethylated lysine 4 (H3K4me0). As previously such a way that substrate affinity and catalytic rate are increased. reported (15), the activity of wild-type RAG was stimulated in a An allosteric ligand can be thought to act on an ensemble of protein dose-dependent fashion by H3K4me3, but not by H3K4me0, native states by inducing a shift in the energetic distribution of those whereas the RAG-2(W453A) mutation abolished responsiveness states and their associated protein conformations (17). In accor- to H3K4me3 (Fig. S2 A and B). We proceeded to use these dance with this view, binding of H3K4me3 to the RAG-2 PHD preparations in chemical footprinting and partial proteolysis assays. finger may be accompanied by a shift in the distribution of RAG native states, so as to favor conformations associated with increased Mapping Solvent Accessibility of RAG Cysteine Thiols by Differential DNA binding and catalysis. We sought to obtain physical evidence Isotopic Chemical Footprinting. The chemical footprinting procedure for the induction of such conformational changes by H3K4me3. is outlined in Fig. 1A. Two protein conformations are depicted, one Conformational states are expected to differ with respect to the in the absence (Fig.