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• W3025964037 abstract "V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger. V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger. Antigen receptor genes are present in the germline as discrete segments that are joined during lymphocyte development by V(D)J recombination (1Gellert M. V(D)J recombination: RAG proteins, repair factors, and regulation.Annu. Rev. Biochem. 2002; 71 (12045092): 101-13210.1146/annurev.biochem.71.090501.150203Crossref PubMed Scopus (609) Google Scholar). V(D)J recombination is initiated by a specialized transposase (2Fugmann S.D. The origins of the Rag genes: from transposition to V(D)J recombination.Semin. Immunol. 2010; 22 (20004590): 10-1610.1016/j.smim.2009.11.004Crossref PubMed Scopus (77) Google Scholar), composed of RAG-1 and RAG-2 subunits, which cleaves DNA at recombination signal sequences (RSSs) that flank the participating gene segments (3Schatz D.G. Swanson P.C. V(D)J recombination: mechanisms of initiation.Annu. Rev. Genet. 2011; 45 (21854230): 167-20210.1146/annurev-genet-110410-132552Crossref PubMed Scopus (356) Google Scholar). There are two classes of RSSs, termed 12-RSS and 23-RSS, in which heptamer and nonamer elements are separated by spacers of 12 bp or 23 bp; DNA cleavage requires the pairing of a 12-RSS with a 23-RSS (3Schatz D.G. Swanson P.C. V(D)J recombination: mechanisms of initiation.Annu. Rev. Genet. 2011; 45 (21854230): 167-20210.1146/annurev-genet-110410-132552Crossref PubMed Scopus (356) Google Scholar). Rearrangement is initiated by nicking of DNA by RAG at the junction of each gene segment with its flanking RSS, followed by transesterification to produce double-strand breaks and assembly of the coding sequence by nonhomologous end joining (1Gellert M. V(D)J recombination: RAG proteins, repair factors, and regulation.Annu. Rev. Biochem. 2002; 71 (12045092): 101-13210.1146/annurev.biochem.71.090501.150203Crossref PubMed Scopus (609) Google Scholar, 4Deriano L. Roth D.B. Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage.Annu. Rev. Genet. 2013; 47 (24050180): 433-45510.1146/annurev-genet-110711-155540Crossref PubMed Scopus (319) Google Scholar). The catalytic and DNA-binding functions reside largely within RAG-1, but RAG-2 is also essential for DNA cleavage, contributing to DNA binding and stabilization of the active site (5Kim M.S. Lapkouski M. Yang W. Gellert M. Crystal structure of the V(D)J recombinase RAG1-RAG2.Nature. 2015; 518 (25707801): 507-51110.1038/nature14174Crossref PubMed Scopus (116) Google Scholar). The carboxyl-terminal quarter of RAG-2, comprising residues 388 through 527, is dispensable for DNA cleavage but carries out several regulatory functions, including the binding of RAG-2 to H3K4me3, an epigenetic mark of active chromatin (6Liu Y. Subrahmanyam R. Chakraborty T. Sen R. Desiderio S. A plant homeodomain in RAG-2 that binds hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement.Immunity. 2007; 27 (17936034): 561-57110.1016/j.immuni.2007.09.005Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 7Matthews A.G. Kuo A.J. Ramon-Maiques S. Han S. Champagne K.S. Ivanov D. Gallardo M. Carney D. Cheung P. Ciccone D.N. Walter K.L. Utz P.J. Shi Y. Kutateladze T.G. Yang W. et al.RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination.Nature. 2007; 450 (18033247): 1106-111010.1038/nature06431Crossref PubMed Scopus (375) Google Scholar, 8Ramon-Maiques S. Kuo A.J. Carney D. Matthews A.G. Oettinger M.A. Gozani O. Yang W. The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18993-1899810.1073/pnas.0709170104Crossref PubMed Scopus (164) Google Scholar). V(D)J recombination is preceded by the appearance of noncoding transcripts at the unrearranged locus (9Van Ness B.G. Weigert M. Coleclough C. Mather E.L. Kelley D.E. Perry R.P. Transcription of the unrearranged mouse Cκ locus: sequence of the initiation region and comparison of activity with a rearranged Vκ-Cκ gene.Cell. 1981; 27 (6101210): 593-60210.1016/0092-8674(81)90401-3Abstract Full Text PDF PubMed Scopus (133) Google Scholar, 10Yancopoulos G.D. Alt F.W. Developmentally controlled and tissue-specific expression of unrearranged VH gene segments.Cell. 1985; 40 (2578321): 271-28110.1016/0092-8674(85)90141-2Abstract Full Text PDF PubMed Scopus (526) Google Scholar) and the deposition of H3K4me3 (6Liu Y. Subrahmanyam R. Chakraborty T. Sen R. Desiderio S. A plant homeodomain in RAG-2 that binds hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement.Immunity. 2007; 27 (17936034): 561-57110.1016/j.immuni.2007.09.005Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 7Matthews A.G. Kuo A.J. Ramon-Maiques S. Han S. Champagne K.S. Ivanov D. Gallardo M. Carney D. Cheung P. Ciccone D.N. Walter K.L. Utz P.J. Shi Y. Kutateladze T.G. Yang W. et al.RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination.Nature. 2007; 450 (18033247): 1106-111010.1038/nature06431Crossref PubMed Scopus (375) Google Scholar, 11Chakraborty T. Chowdhury D. Keyes A. Jani A. Subrahmanyam R. Ivanova I. Sen R. Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus.Mol. Cell. 2007; 27 (17803947): 842-85010.1016/j.molcel.2007.07.010Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12Goldmit M. Ji Y. Skok J. Roldan E. Jung S. Cedar H. Bergman Y. Epigenetic ontogeny of the Igk locus during B cell development.Nat. Immunol. 2005; 6 (15619624): 198-20310.1038/ni1154Crossref PubMed Scopus (137) Google Scholar, 13Jung D. Giallourakis C. Mostoslavsky R. Alt F.W. Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus.Annu. Rev. Immunol. 2006; 24 (16551259): 541-57010.1146/annurev.immunol.23.021704.115830Crossref PubMed Scopus (433) Google Scholar, 14Morshead K.B. Ciccone D.N. Taverna S.D. Allis C.D. Oettinger M.A. Antigen receptor loci poised for V(D)J rearrangement are broadly associated with BRG1 and flanked by peaks of histone H3 dimethylated at lysine 4.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 11577-1158210.1073/pnas.1932643100Crossref PubMed Scopus (164) Google Scholar, 15Subrahmanyam R. Du H. Ivanova I. Chakraborty T. Ji Y. Zhang Y. Alt F.W. Schatz D.G. Sen R. Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions.Nat. Immunol. 2012; 13 (23104096): 1205-121210.1038/ni.2447Crossref PubMed Scopus (30) Google Scholar), which binds to a plant homeodomain (PHD) finger spanning residues 415 through 487 of mouse RAG-2 (8Ramon-Maiques S. Kuo A.J. Carney D. Matthews A.G. Oettinger M.A. Gozani O. Yang W. The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18993-1899810.1073/pnas.0709170104Crossref PubMed Scopus (164) Google Scholar, 16Callebaut I. Mornon J.P. The V(D)J recombination activating protein RAG2 consists of a six-bladed propeller and a PHD fingerlike domain, as revealed by sequence analysis.Cell. Mol. Life Sci. 1998; 54: 880-89110.1007/s000180050216Crossref PubMed Scopus (98) Google Scholar). Engagement of H3K4me3 promotes recombination in vivo (6Liu Y. Subrahmanyam R. Chakraborty T. Sen R. Desiderio S. A plant homeodomain in RAG-2 that binds hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement.Immunity. 2007; 27 (17936034): 561-57110.1016/j.immuni.2007.09.005Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 7Matthews A.G. Kuo A.J. Ramon-Maiques S. Han S. Champagne K.S. Ivanov D. Gallardo M. Carney D. Cheung P. Ciccone D.N. Walter K.L. Utz P.J. Shi Y. Kutateladze T.G. Yang W. et al.RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination.Nature. 2007; 450 (18033247): 1106-111010.1038/nature06431Crossref PubMed Scopus (375) Google Scholar) and stimulates coupled cleavage of RSS substrates in vitro (17Lu C. Ward A. Bettridge J. Liu Y. Desiderio S. An autoregulatory mechanism imposes allosteric control on the V(D)J recombinase by histone H3 methylation.Cell Rep. 2015; 10 (25543141): 29-3810.1016/j.celrep.2014.12.001Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 18Grundy G.J. Ramon-Maiques S. Dimitriadis E.K. Kotova S. Biertumpfel C. Heymann J.B. Steven A.C. Gellert M. Yang W. Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.Mol. Cell. 2009; 35: 217-22710.1016/j.molcel.2009.06.022Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19Shimazaki N. Tsai A.G. Lieber M.R. H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations.Mol. Cell. 2009; 34 (19524534): 535-54410.1016/j.molcel.2009.05.011Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), consistent with allosteric activation. In agreement with this interpretation, binding of H3K4me3 by the RAG-2 PHD finger is associated with changes in the conformational distribution of RAG-1 (20Bettridge J. Na C.H. Pandey A. Desiderio S. H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase.Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28174273): 1904-190910.1073/pnas.1615727114Crossref PubMed Scopus (15) Google Scholar). The requirement for H3K4me3 recognition can be bypassed by mutations within an inhibitory domain in RAG-2, whose disruption is associated with constitutive increases in RSS binding affinity, catalytic rate, and recombination frequency, mimicking the stimulatory effects of H3K4me3 (17Lu C. Ward A. Bettridge J. Liu Y. Desiderio S. An autoregulatory mechanism imposes allosteric control on the V(D)J recombinase by histone H3 methylation.Cell Rep. 2015; 10 (25543141): 29-3810.1016/j.celrep.2014.12.001Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Association of RAG-1 and RAG-2 with the IgH locus in B cell progenitors depends on recognition of H3K4me3 by the PHD (21Teng G. Maman Y. Resch W. Kim M. Yamane A. Qian J. Kieffer-Kwon K.R. Mandal M. Ji Y. Meffre E. Clark M.R. Cowell L.G. Casellas R. Schatz D.G. RAG represents a widespread threat to the lymphocyte genome.Cell. 2015; 162 (26234156): 751-76510.1016/j.cell.2015.07.009Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Ji Y. Resch W. Corbett E. Yamane A. Casellas R. Schatz D.G. The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci.Cell. 2010; 141 (20398922): 419-43110.1016/j.cell.2010.03.010Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); disruption of the inhibitory domain permits association of RAG with the IgH locus in the absence of H3K4me3 binding (23Ward A. Kumari G. Sen R. Desiderio S. The RAG-2 inhibitory domain gates accessibility of the V(D)J recombinase to chromatin.Mol. Cell. Biol. 2018; 38: e00159-1810.1128/MCB.00159-18Crossref PubMed Scopus (8) Google Scholar). Thus, the inhibitory domain blocks access of RAG to the IgH locus except when H3K4me3 is engaged by RAG-2. Taken together these observations suggest the following model for the association of RAG with antigen receptor loci. 1) In the absence of H3K4me3, RAG has low affinity for the RSS. 2) When the RAG-2 PHD finger is engaged by H3K4me3, an allosteric signal is propagated to RAG-1, which acquires high affinity for the RSS. 3) The binding of RAG to a nearby RSS stabilizes its association with the locus. As a test of this model, we sought to identify mutations in the RAG-2 PHD finger that separate binding of H3K4me3 from allosteric activation of RAG-1. Here, we employ a strategy of phylogenetic substitution to identify amino acid residues within the PHD finger, but distinct from the H3K4me3 binding site, that function in transmission of an allosteric signal to RAG-1. It remains unclear how information regarding the binding of H3K4me3 by RAG-2 is transmitted to RAG-1. We hypothesized that such information is conveyed through a path involving amino acid residues within the PHD finger other than those that engage H3K4me3. To test this hypothesis, we sought to identify separation-of-function mutations in the RAG-2 PHD finger that impair V(D)J recombination but spare binding to H3K4me3. To decrease the complexity of the search, we employed a phylogenetic approach, reasoning that RAG-2 PHD fingers from species distantly related to the mouse might retain the ability to bind H3K4me3 but be sufficiently different in sequence so that transmission of the allosteric signal to mouse RAG-1 might be impaired. RAG-2 of the brownbanded bamboo shark, Chiloscyllium punctatum, is phylogenetically distant from that of the mouse (24Brinkmann H. Venkatesh B. Brenner S. Meyer A. Nuclear protein-coding genes support lungfish and not the coelacanth as the closest living relatives of land vertebrates.Proc. Natl. Acad. Sci. U.S.A. 2004; 101 (15037746): 4900-490510.1073/pnas.0400609101Crossref PubMed Scopus (156) Google Scholar). In the interval spanning residues 414 through 485, the RAG-2 PHD finger of M. musculus differs from that of C. punctatum at 25 positions (Fig. 1A). When mapped onto a crystal structure of the mouse PHD finger (7Matthews A.G. Kuo A.J. Ramon-Maiques S. Han S. Champagne K.S. Ivanov D. Gallardo M. Carney D. Cheung P. Ciccone D.N. Walter K.L. Utz P.J. Shi Y. Kutateladze T.G. Yang W. et al.RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination.Nature. 2007; 450 (18033247): 1106-111010.1038/nature06431Crossref PubMed Scopus (375) Google Scholar), these differences largely reside opposite the H3K4me3 binding pocket (Fig. 1B) and are potentially free to participate in an allosteric interface. The similarity of the M. musculus and C. punctatum PHD fingers in the region of the H3K4me3 binding site suggested that the shark PHD finger would also be capable of binding H3K4me3. To test this, we constructed a RAG-2 chimera in which the mouse PHD finger was replaced by the PHD finger from C. punctatum. Lysates of HEK293T cells expressing the WT murine RAG-2 or the murine RAG-2 bearing the C. punctatum PHD finger were incubated with a bead-bound peptide corresponding to residues 1–21 of histone H3, trimethylated at lysine 4 (H3K4me3). Both WT murine RAG-2 and the mouse-shark RAG-2 chimera bound to the H3K4me3 peptide (Fig. 1C, right, lanes 1 and 3). As expected, a W453A mutation in the H3K4me3 binding pocket of mouse RAG-2 abolished binding to H3K4me3 (Fig. 1C, right, lane 2). In addition, the KMT2D lysine methyltransferase, which carries multiple PHD fingers with specificity distinct from that of RAG-2, failed to bind H3K4me3 (Fig. 1C, right, lane 4). We next asked whether the C. punctatum PHD finger could replace its murine counterpart in an assay for allosteric activation of coupled DNA cleavage by RAG. The version of RAG-1 used in these assays, cR1ct, lacks the amino-terminal noncore region and is more soluble than the WT protein but remains responsive to H3K4me3 (17Lu C. Ward A. Bettridge J. Liu Y. Desiderio S. An autoregulatory mechanism imposes allosteric control on the V(D)J recombinase by histone H3 methylation.Cell Rep. 2015; 10 (25543141): 29-3810.1016/j.celrep.2014.12.001Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 25Grundy G.J. Yang W. Gellert M. Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 22487-2249210.1073/pnas.1014958107Crossref PubMed Scopus (46) Google Scholar). Full-length WT mouse RAG-2 and chimeric RAG-2 bearing the C. punctatum PHD finger, both tagged at the amino terminus with MBP, were coexpressed individually with MBP-tagged cR1ct, and RAG tetramers were purified by a protocol that removes endogenous H3K4me3 (20Bettridge J. Na C.H. Pandey A. Desiderio S. H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase.Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28174273): 1904-190910.1073/pnas.1615727114Crossref PubMed Scopus (15) Google Scholar). Equivalent amounts of each RAG tetrameric complex were assayed in vitro for coupled cleavage of a radiolabeled 12-RSS substrate in the presence of an unlabeled 23-RSS substrate and increasing amounts of a histone H3-derived peptide containing trimethylated lysine 4 (H3K4me3) or unmethylated lysine 4 (H3K4me0). As reported previously (17Lu C. Ward A. Bettridge J. Liu Y. Desiderio S. An autoregulatory mechanism imposes allosteric control on the V(D)J recombinase by histone H3 methylation.Cell Rep. 2015; 10 (25543141): 29-3810.1016/j.celrep.2014.12.001Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 19Shimazaki N. Tsai A.G. Lieber M.R. H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for translocations.Mol. Cell. 2009; 34 (19524534): 535-54410.1016/j.molcel.2009.05.011Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 25Grundy G.J. Yang W. Gellert M. Autoinhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 22487-2249210.1073/pnas.1014958107Crossref PubMed Scopus (46) Google Scholar), formation of nicked product was stimulated in a dose-dependent manner by H3K4me3 but not by H3K4me0 (Fig. 1D). In contrast, the chimeric version of RAG-2 failed to be stimulated by H3K4me3 (Fig. 1D). Similar results were obtained with independent protein preparations. Taken together, these observations indicated that replacement of the mouse RAG-2 PHD finger with the PHD finger from C. punctatum produces a protein in which the H3K4me3 binding is retained while the ability to support allosteric activation of RAG-1 is lost. We went on to test whether the binding of H3K4me3 could be separated from the ability of RAG-2 to support V(D)J recombination in vivo. An initial assay for recombination of an extrachromosomal substrate (17Lu C. Ward A. Bettridge J. Liu Y. Desiderio S. An autoregulatory mechanism imposes allosteric control on the V(D)J recombinase by histone H3 methylation.Cell Rep. 2015; 10 (25543141): 29-3810.1016/j.celrep.2014.12.001Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 23Ward A. Kumari G. Sen R. Desiderio S. The RAG-2 inhibitory domain gates accessibility of the V(D)J recombinase to chromatin.Mol. Cell. Biol. 2018; 38: e00159-1810.1128/MCB.00159-18Crossref PubMed Scopus (8) Google Scholar, 26Hesse J.E. Lieber M.R. Gellert M. Mizuuchi K. Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals.Cell. 1987; 49 (3495343): 775-78310.1016/0092-8674(87)90615-5Abstract Full Text PDF PubMed Scopus (271) Google Scholar) suggested that the mouse-shark chimeric RAG-2 (PHDPUNC) was impaired in its ability to support V(D)J recombination, as its activity was similar to that of RAG-2(W453A), which cannot bind H3K4me3, and of a chimeric RAG-2 mutant bearing the corresponding mutation, W455A in the C. punctatum PHD finger (Fig. S1). We proceeded to a more extensive analysis, with the goal of identifying mutations that impair V(D)J recombination while disrupting neither protein expression nor the ability to bind H3K4me3. Cassettes encoding WT RAG-2 and a series of variants, fused at the amino terminus to the FLAG epitope and at the carboxyl terminus to poly-histidine and Myc tags (Fig. 2A), were introduced into the retroviral vector pCLIP2a (27Pomerantz J.L. Denny E.M. Baltimore D. CARD11 mediates factor-specific activation of NF-κB by the T cell receptor complex.EMBO J. 2002; 21 (12356734): 5184-519410.1093/emboj/cdf505Crossref PubMed Scopus (173) Google Scholar); these included RAG-2(PHDPUNC), the chimera bearing the C. punctatum PHD finger in the context of mouse RAG-2. As negative controls, we tested RAG-2(W453A) and RAG-2(PHDPUNC,W455A) (Fig. 2A), in which an invariant tryptophan residue involved in H3K4me3 binding was mutated to alanine. We previously showed that disruption of the RAG-2 inhibitory domain permits V(D)J recombination to override the requirement for recognition of H3K4me3 (17Lu C. Ward A. Bettridge J. Liu Y. Desiderio S. An autoregulatory mechanism imposes allosteric control on the V(D)J recombinase by histone H3 methylation.Cell Rep. 2015; 10 (25543141): 29-3810.1016/j.celrep.2014.12.001Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 23Ward A. Kumari G. Sen R. Desiderio S. The RAG-2 inhibitory domain gates accessibility of the V(D)J recombinase to chromatin.Mol. Cell. Biol. 2018; 38: e00159-1810.1128/MCB.00159-18Crossref PubMed Scopus (8) Google Scholar). Therefore, it was also of interest to assay RAG-2(D/E352-405A) and RAG-2(D/E352-405A,PHDPUNC) (Fig. 2A), in which the acidic residues within the inhibitory domain were mutated to alanine. We constructed an additional set of mutants in which groups of amino acid residues in the C. punctatum PHD finger were mutated to the corresponding residues in the mouse. Using the three-dimensional structure of the mouse PHD finger as a guide, we assigned these sequence differences to five groups, each of whose residues are closely clustered on the surface opposite the H3K4me3 binding site (Fig. 2, B and C), reasoning that one or more alterations on this surface might disrupt recombination but spare binding to H3K4me3. These spatially clustered mutations, as depicted in Fig. 2B, were termed Group 1 (K418T, S420C, S421P, A422T, and N424D), Group 2 (M425V, N426D, V427I, I429T, E431V, and Y433F), Group3 (N460D, A462E, S464R, Q465T, Q468H, and F469L), Group 4 (Q471E, E472G, N473S, and T474N), and Group 5 (F445Y, S448H, F477Y, and Y482V), with the numbering following that of the mouse sequence. To assay recombination quantitatively, we employed a fluorescent assay for recombination of an integrated substrate (28Gapud E.J. Lee B.S. Mahowald G.K. Bassing C.H. Sleckman B.P. Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites.J. Immunol. 2011; 187 (21742970): 1826-183410.4049/jimmunol.1101388Crossref PubMed Scopus (16) Google Scholar). Retroviral constructs encoding the RAG-2 variants described in Fig. 2A, as well as constructs encoding the clustered shark-to-mouse mutants, were used to infect the cell line R2K3, a RAG-2-deficient, Abelson murine leukemia virus-transformed B progenitor (28Gapud E.J. Lee B.S. Mahowald G.K. Bassing C.H. Sleckman B.P. Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites.J. Immunol. 2011; 187 (21742970): 1826-183410.4049/jimmunol.1101388Crossref PubMed Scopus (16) Google Scholar). R2K3 contains an integrated substrate for V(D)J recombination whose inversional rearrangement results in the expression of GFP. Following transduction of RAG-2, recombination is induced by STI-571; rearrangement of the integrated substrate is detected by flow cytometry. Cells transduced with WT RAG-2 exhibited increasing levels of GFP fluorescence, approaching a recombination frequency of 80%, over a period of at least 96 h following arrest with STI-571 (a representative analysis at 96 h is shown in Fig. 3A; the results of three technical replicate assays at 48 h and 96 h are shown in Fig. 3B). As expected, recombination was impaired by the W453A mutation, which disrupts binding of H3K4me3 (Fig. 3, A and B). We also observed little recombination in cells transduced with the RAG-2(PHDPUNC) chimera or with RAG-2(PHDPUNC,W455A) (Fig. 3, A and B). Notably, the RAG-2(D/E352-405A,PHDPUNC) chimera, in which the inhibitory domain is neutralized, supported recombination to a level near to that of WT RAG-2 or RAG-2(D/E352-405A) (Fig. 3, A and B, IDNEUT,PHDPUNC), indicating that the chimeric protein can interact functionally with RAG-1 to support recombination when the requirement for H3K4me3 binding is bypassed. Among the back-mutated mutants, RAG-2(PHDPUNC,Group1) and RAG-2(PHDPUNC,Group4) showed sharply diminished recombination activity with respect to WT RAG-2, while RAG-2(PHDPUNC,Group2), RAG-2(PHDPUNC,Group3), and RAG-2(PHDPUNC,Group5) supported recombination at frequencies about 38–75% that of WT (Fig. 3, A and B). Because any impairment of recombination could result from decreased protein accumulation, we examined expression of each of the RAG-2 variants at 96 h after arrest by STI-571, with the goal of identifying mutant proteins that are robustly expressed but fail to support efficient V(D)J recombination. Accumulation of RAG-2(PHDPUNC) was reduced to less than one tenth that of WT RAG-2 (Fig. 3C; Fig. S2) and could not be rescued by overexpression of RAG-1 in a cotransfection assay (Fig. S3). Of the additional mutant proteins that exhibited impaired recombination in the R2K3 assay, only RAG-2(PHDPUNC,Group1) was expressed at a level similar to those of RAG-2 variants that retained recombination activity (Fig. 3C; Fig. S2). Thus, the Group 1 mutation permitted near-normal accumulation of the RAG-2(PHDPUNC) chimeric protein but was associated with a large reduction in recombination activity. To confirm the effects of the RAG-2 mutations described above on V(D)J recombination, the R2K3 rearrangement assay was performed in biological triplicate, with each biological assay consisting of three technical replicates (Fig. 3D). Of note, cells transduced with RAG-2(PHDPUNC,Group1) exhibited a large and statistically significant decrease in recombination frequency, compared with cells transduced with WT RAG-2 or RAG-2(PHDPUNC,Group2) (Fig. 3D, Group 1), while neutralization of the inhibitory domain robustly conferred recombination activity on the RAG-2(PHDPUNC) chimera (Fig. 3D, IDNEUT,PHDPUNC). We considered that the impairment of recombination activity observed for the RAG-2(PHDPUNC,Group1) mutant might result from disruption of intramolecular interactions that propagate the allosteric signal initiated by the binding of RAG-2 to H3K4me3. To test this interpretation, we asked 1) whether the defect in recombination observed for RAG-2(PHDPUNC,Group1) could be reversed by an additional back-mutation within the C. punctatum PHD finger; and 2) whether RAG-2(PHDPUNC,Group1) retains the ability to bind H3K4me3. Because RAG-2(PHDPUNC,Group2) robustly supported V(D)J recombination, the Group 2 back-mutation was an attractive candidate for rescue of RAG-2(PHDPUNC,Group1). We observed that a mouse-shark RAG-2 chimera bearing combined Group 1 and Group 2 mutations exhibited a robust increase in recombination frequency, relative to the Group 1 mutant (Fig. 4, A and B, compare Group 1 to Groups 1+2). In this assay, the expression levels of RAG-2(PHDPUNC,Groups1+2), RAG-2(PHDPUNC,Group1), and WT RAG-2 differed by about 2-fold or less (Fig. 4C). Taken together, the results described above indicate that the Group 1 mutation enhances accumulation of the RAG-2(PHDPUNC) chimera without permitting recombination and that additional back-mutation of the Group 2 cluster confers recombination activity on the Group 1 mutant. Consistent results were obtained with a similar recombination assay in which RAG-2 variants were transduced using the pCST virus (Fig. S4). To establish that RAG-2(PHDPUNC,Group1) is indeed a separation-of-function mutant, we" @default.
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