A RAG1 Mutation Found in Omenn Syndrome Causes Coding Flank Hypersensitivity: A Novel Mechanism for Antigen Receptor Repertoire Restriction This information is current as of September 25, 2021. Serre-Yu Wong, Catherine P. Lu and David B. Roth J Immunol 2008; 181:4124-4130; ; doi: 10.4049/jimmunol.181.6.4124 http://www.jimmunol.org/content/181/6/4124 Downloaded from References This article cites 35 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/181/6/4124.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A RAG1 Mutation Found in Omenn Syndrome Causes Coding Flank Hypersensitivity: A Novel Mechanism for Antigen Receptor Repertoire Restriction1 Serre-Yu Wong,2 Catherine P. Lu,2 and David B. Roth3 Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunode- ficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoim- mune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively Downloaded from impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity. The Journal of Immunology, 2008, 181: 4124–4130. menn syndrome (OS)4 is a severe immune deficiency T cells, leading to impaired self tolerance (12, 13). Second, the that is, peculiarly, accompanied by both autoimmune diminished production of T cells likely fosters homeostatic prolif- http://www.jimmunol.org/ and allergic symptoms (1, 2). Patients usually present by eration in the periphery, which could lead to T 2 skewing O H the age of 6 mo, suffering recurrent and opportunistic infections, as (14–16). well as hepatosplenomegaly, lymphadenopathy, erythrodermia, The reduced V(D)J recombinase activity underlying most cases eosinophilia, and elevated serum IgE. T cells from OS patients of OS may stem from defects affecting any of several distinct steps have a highly restricted, oligoclonal repertoire, and B cells are in the recombination reaction. The RAG complex first recognizes generally absent from peripheral blood (3–7). The disease is fatal Ag receptor V, D, and J coding segments by binding to recombi- without bone marrow transplantation. nation signal sequences (RSSs) that border each segment and syn- OS is typically caused by mutations in the RAG1 and RAG2 apsing a pair of RSSs. The recombinase then generates single- genes, which encode the lymphoid-specific components of the strand nicks precisely between each RSS and the adjacent coding by guest on September 25, 2021 V(D)J recombinase responsible for generating TCR and Ig diver- flank (the sequence of the coding segment directly 5Ј of the RSS). sity in developing T and B lymphocytes (8–11). Most OS-causing These nicks liberate 3Ј-hydroxyl groups that are used by the RAG mutant RAG proteins display severely decreased recombination proteins to attack the other strand, forming a covalently sealed activity in vitro, and it is thought that this creates a “bottleneck” in DNA hairpin structure at each coding flank and a blunt double- lymphocyte differentiation, resulting in production of the restricted strand break at each RSS (17). The hairpins are then opened and TCR repertoire characteristic of OS (8). joined together to form a coding joint, while the signal ends form It is easy to see how defective RAG mutants could cause im- a signal joint (18). OS-causing RAG mutants can be defective in munodeficiency, but it has been more difficult to elucidate the con- the binding, nicking, hairpin formation, or joining steps of V(D)J nection between reduced V(D)J recombination and the immuno- recombination (8, 9, 19, 20). regulatory defects characteristic of OS. Recent work in mouse The prevailing pathogenetic model for OS holds that hypomor- models bearing RAG mutations offers some suggestions. First, the phic RAG mutations severely reduce recombinase activity uni- constrained repertoire may cause a relative deficiency in regulatory formly, across all Ag receptor gene segments. According to this model, the observed repertoire restriction is secondary to the ex- Program in Molecular Pathogenesis, Helen L. and Martin S. Kimmel Center for Bi- tremely low throughput of productive Ag receptor rearrangements ology and Medicine at the Skirball Institute for Biomolecular Medicine, and Depart- and selective expansion of those few mature cells that encounter ment of Pathology, New York University School of Medicine, New York, NY 10016 their cognate Ags. We considered an alternative hypothesis: cer- Received for publication June 2, 2008. Accepted for publication July 3, 2008. tain RAG mutations might directly affect the ability of particular The costs of publication of this article were defrayed in part by the payment of page coding segments to undergo recombination, resulting in a primary charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. defect in repertoire generation. Two lines of evidence make such a hypothesis worth considering. First, the coding flank sequence is 1 This study was supported by National Institutes of Health Grant AI36420 (to D.B.R.) and by the Irene Diamond Foundation (to D.B.R.). well known to affect the efficiency of recombination by the wild- 2 S.-Y.W. and C.P.L. contributed equally to this work. type RAG proteins (21–25). Second, a certain group of RAG1 3 Address correspondence and reprint requests to Dr. David B. Roth, Department of mutants shows dramatic hypersensitivity to certain coding flank Pathology, New York University School of Medicine, 540 First Avenue, New York, sequences: these mutations cluster near a known catalytic amino NY 10016. E-mail address: [email protected] acid (D600), mapping to the region between amino acids 606– 4 Abbreviations used in this paper: OS, Omenn syndrome; CHO, Chinese hamster 611, and include a deletion-insertion mutation (known as “D32”) ovary; RSS, recombination signal sequence. and two point mutations, H609L and K608A (19, 26–28). All of Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 these mutant proteins are severely defective for hairpin formation www.jimmunol.org The Journal of Immunology 4125 A B C 12RSS AC TG HP/(HP+nicks) % AC 1 9 7 2 / 9 7 3 * 3 100 1 / 9 7 100 R 9 7 2 Q TG R 9 7 2 Q D 3 2 D 3 2 9 7 2 AC AC WT 0.8 80 800.8 TG WT 0.6 23RSS 60 600.6 * HP% 400.4 Nick% 12RSS 400.4 TG * 0.2 * 20 200.2 AC TG TG 0 0 00 AC WT D32 972/3 972Q WT D32 972/3 972Q * WT D32 972/3 R972Q WT D32 972/3 R972Q WTWT D32 D32 972/3 972/3R972Q 972QWT WT D32 D32 972/3 972/3R972Q 972Q 23RSS 15Lane AC TG AC TG D E 12RSS AC TG HP/(HP+pre-nicks) % * AC 9 7 2 / 9 7 3 / 9 7 3 1001 R 9 7 2 Q TG Q AC AC R 9 7 2 2 D 3 2 9 7 2 0.8 WT 80 TG D 3 WT 23RSS 600.6 12RSS 400.4 * TG * AC 200.2 Downloaded from TG TG AC * 00 23RSS WTWT D32 D32 972/3 972/3 R972Q 972Q WT D32 D32 972/3 972/3 R972Q 972Q 1 5 Lane AC TG FIGURE 1. R972Q RAG1 mutant shows preferences for coding flank sequence in vitro. A, Cleavage assay using oligonucleotide substrates as depicted on the left, with either AC (nonpreferred) or TG (preferred) flank. Only one strand of each oligo pair was 5Ј radiolabeled (*, strand in black) and was thus detectable after electrophoresis. B, Quantitative analysis of gel in A, showing the percentage of hairpin formation. Error bars represent SEM of three experiments. C, Quantitative analysis of gel in A. The percentage of hairpins and nicks are shown in dark gray and light gray, respectively, and the length http://www.jimmunol.org/ of each bar represents total nicking activity, as nicks were converted into hairpins. D, Cleavage assay using oligonucleotide substrates with the same sequence as in A, except these contain a preformed nick. E, Quantitative analysis of gel in D, showing the percentage of pre-nicks converted to hairpins. at certain coding flank sequences, leading to the deduction that the Cellular V(D)J recombination assays region of RAG1 between amino acids 606 and 611 contacts the Wild-type and mutant murine full-length RAG1 and wild-type full-length coding flank and is directly involved in hairpin formation (19, murine RAG2 were expressed from a modified pEBG vector (lacking GST by guest on September 25, 2021 26–28).