A RAG1 Mutation Found in Omenn Syndrome Causes Coding Flank : 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

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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 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, , erythrodermia, The reduced V(D)J recombinase activity underlying most cases , 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

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23D 279 AC AC TW 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 CA GT CA GT

D E

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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 CA GT 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). No known OS mutations, however, map to this region, nor tag) (33). Reporter substrates were transfected into RMP41 CHO cells, as have coding flank-sensitive mutants been described elsewhere in described (33). The GFP V(D)J recombination reporter substrates have the RAG coding sequences. been described (33). Briefly, a poly(A) sequence between 12-RSS and 23-RSS elements were cloned into pEGFP-N1 (Clontech) between the Given the close proximity of all known coding flank-sensitive CMV promoter and GFP gene. RAG-mediated recombination results in mutants to a catalytic amino acid (D600), we reasoned that coding deletion of the poly(A) sequence and expression of GFP. In the coding joint flank hypersensitivity might also result from mutations in other substrate, RSSs are oriented such that GFP is expressed after coding joint regions of RAG1 that are directly involved in catalysis. We now formation, while in the signal joint substrate, inversion of both RSSs allows expression after signal joint formation. Cells were harvested 48 h after report that the RAG1 R972Q mutation, located in the primary se- transfection, trypsinized, spun at 1200 rpm for 5 min, and resuspended in quence near the catalytic amino acid E962 is hypersensitive to 50 ␮l PBS with 0.5% BSA and 5 mM EDTA. Cells were analyzed by flow certain coding flank sequences. This mutation is found at the or- cytometry using an LSR II flow cytometer (BD Biosciences) and FlowJo thologous human RAG1 residue, R975Q, in a patient with OS (9) software. and was serendipitously discovered in mice that display an OS-like Altered coding flank substrates phenotype (14). Our results provide the first evidence of an OS mutation that has a primary (rather than secondary) effect on the Coding flank sequences at both 12-RSS and 23-RSS of fluorescent reporter repertoire and support an alternative model for the molecular coding joint and signal joint substrates, as described above, were changed to all 16 permutations by a site-directed mutagenesis kit (Stratagene). pathogenesis of OS and perhaps other disease states involving lymphopenia-associated autoimmunity (29, 30). Substrate-specific integrated cell lines RMP41 CHO cell lines bearing integrated recombination substrates were Materials and Methods developed as previously described (33). Stable substrate-integrated cells ϫ 5 RAG protein purification (1 10 ) were plated per well in 24-well plates and transfected 20 h later with 100 ng each of RAG1 and RAG2 expression plasmids using Fugene6 Experiments were performed with recombinant GST-tagged mouse core reagent (3:2 ratio of FuGENE 6/DNA). Cells were harvested and analyzed RAG1 and core RAG2. RAG1 and RAG2 proteins are copurified from as described above. Chinese hamster ovary (CHO) cells (as described in Ref. 27) using GST affinity resin beads (GE Healthcare). Three different protein purifications Results for each mutant were made, and mutant phenotypes were consistent be- R972Q displays coding flank hypersensitivity in vitro tween different preparations. Human RAG1 R975, orthologous to murine RAG1 R972, is mu- In vitro cleavage assay tated to glutamine in a patient with OS (9). This mutation shares Oligonucleotides and cleavage reactions were described previously (31, two features with mutations in the 606–611 region of RAG1: it 32). Reaction products were detected and quantified using a Phosphor- lies near a catalytic amino acid (E962) (34), and the R972A/ Imager and ImageQuant software. K973A mutant exhibits a specific defect in hairpin formation (19). 4126 OS RAG MUTANT IS HYPERSENSITIVE TO CODING FLANKS

A Wild-type 23D Q279R 12RSS MMM MMMMMM * AC Time Matched TG (M) AC * TG 23RSS

12RSS * * AC Mismatched GA (MM) AC GA * 23RSS 1 6 11 16 21 26 Lane

B 2.52.5 Downloaded from

2.02 WT-MMWT-MM WT-M

oitar skcin / PH / skcin oitar WT-M 1.51.5 D32-MMD32-MM D32-MD32-M R972Q-MMR972Q-MM 1 http://www.jimmunol.org/ 1.0 R972Q-MR972Q-M

0.50.5

00 5 10204080 5 10 20 40 80 Time (min) by guest on September 25, 2021 FIGURE 2. Hairpin formation defect of coding flank-sensitive RAG1 mutant can be rescued by oligonucleotide substrate that contains mismatched base pairs. A, Oligonucleotide substrates containing matched (M) or mismatched (MM) base pairs as depicted on the left. Triangles above gel indicate increasing incubation times: 5, 10, 20, 40, and 80 min. Reaction products are depicted on the right (from top to bottom): uncleaved substrates, hairpins, and nicks. B, Quantitative analysis of gel shown in A. Reactions performed with matched substrates are shown in solid lines and mismatched substrates in dashed lines.

We asked whether R972Q might also show hypersensitivity to mutants formed little or no hairpins at a pre-nicked AC flank (lanes certain coding flank sequences. We purified the R972Q mutant as 2–4), although they efficiently convert a pre-nicked TG flank to well as wild-type RAG1, D32, and R972A/K973A proteins as con- hairpin form, as expected (lanes 6–8). Data from three experi- trols, and examined the ability of these proteins, in conjunction ments are quantified in Fig. 1E, confirming that hairpin formation with wild-type RAG2, to cleave oligonucleotide substrates con- by the mutants is not enhanced by providing a pre-nicked substrate taining either AC or TG coding flanks (Fig. 1A). As expected, and is essentially the same as with uncleaved substrates (Fig. 1B). wild-type RAG proteins catalyzed robust nicking and hairpin for- These results localize the coding flank sensitivity of these RAG1 mation with both substrates (lanes 1 and 5). D32 was specifically mutants to the hairpin formation step. defective for hairpin formation with AC, but not TG, coding flanks Furthermore, as previously found for the D32 mutant (27), hair- (lanes 2 and 6) (26), and R972A/K973A was severely defective for pin formation by R972Q was rescued by oligonucleotide substrates hairpin formation on both substrates, with a slight increase in containing two mismatched bases at both coding flanks (Fig. 2 and activity at a TG flank (lanes 3 and 7). Interestingly, R972Q was data not shown), indicating that, at certain coding flanks, R972Q severely impaired at the AC flank but formed hairpins at wild- renders the RAG recombinase unable to generate the DNA distor- type levels on the TG flank (lanes 4 and 8). We define this tion required for hairpin formation, as suggested previously for sequence-dependent effect on hairpin formation as coding flank mutants in the 606–611 region (26, 27). hypersensitivity. Quantitative analyses of the efficiency of hairpin formation from R972Q is hypersensitive to coding flank sequence in cells multiple experiments confirmed these observations (Fig. 1B). Regard- We next examined the behavior of the wild-type and mutant RAG1 less of the coding flank sequence, total nicking activity (the sum of proteins in cells. Previous work has shown that the 2 bp proximal nicking and hairpin formation) was not affected by the mutations (Fig. to the cleavage site are the major determinant of sequence hyper- 1C). To confirm that the coding flank sequence specifically affects sensitivity of D32 (26). We therefore constructed a set of extra- hairpinning, we turned to pre-nicked substrates (Fig. 1D). Wild- chromosomal fluorescent reporter substrates (33) bearing each of type RAG proteins performed similar levels of hairpin formation at the 16 possible permutations of the two coding flank base pairs the pre-nicked AC and TG substrates (lanes 1 and 5), whereas the (Fig. 3A) and used them to examine coding flank sensitivity of the The Journal of Immunology 4127 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Wild-type and R972Q RAG1 display distinct coding flank preferences during V(D)J recombination in a cell culture assay. A, Sche- matic of V(D)J recombination assay for coding joint formation. Rearrangement of plasmid results in GFP fluorescence. Coding flank sequences are marked N. A total of 16 coding flank sequence permutations were generated and tested. Substrates contained the same coding flank adjacent to both 12-RSS (‚) and 23-RSS (Œ). B–D, Plasmids containing RAG1 (wild-type, D32, or R972Q, repectively), wild-type RAG2, and each of 16 coding joint (filled bars) substrates or 16 signal joint (open bars) substrates were transfected into CHO cells and analyzed by FACS for GFP expression. Percentage of GFP-positive cells is graphed in the left panel. The graph on the right depicts the same data normalized over the mean. Error bars represent SEM of three or more experiments. Horizontal bars indicate mean coding joint formation across all coding flanks. 4128 OS RAG MUTANT IS HYPERSENSITIVE TO CODING FLANKS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 4. Omenn syndrome mutants display weak, hypomorphic activity with all coding flank sequences. Plasmids containing RAG1 R621C, R621H, E719K, C897X, or Y909C and wild-type RAG2 plus each of 16 coding joint substrates were transfected into CHO cells and analyzed by FACS. Percentage of GFP-positive cells is shown. Error bars represent SEM of three or more experiments.

mutants in transiently transfected CHO cells. As measured by the sequence preferences shown by R972Q over all 16 coding proportion of GFP-positive cells, wild-type RAG proteins cat- flanks differs significantly from wild-type RAG1 ( p Ͻ 0.00001, alyzed recombination over a 30-fold range that varied according paired t test). Based on these data, we conclude that R972Q is to the sequence of the last two bases of the coding flank (Fig. hypersensitive to certain coding flank sequences. 3B, left panel). D32 shows a distinct profile of coding flank We asked whether other OS RAG1 mutations near known active preferences, in agreement with previous reports (26). In com- site residues and/or the coding flank binding region (including parison with wild-type RAG1, R972Q displays strikingly dif- R621C, R621H, E719K, C897stop, and Y909C) (8–10) might ferent coding flank preferences, with similar effects on both confer coding flank hypersensitivity. All of these mutants dis- signal and coding joints. These data are in agreement with our played extremely low activity across all 16 coding flank sequences, in vitro results, which demonstrate that coding sequence affects with no coding flank hypersensitivity (Fig. 4). Note that the scales the efficiency of cleavage. of the graphs in Fig. 4 are the same as those in the left panels of To facilitate comparisons between wild-type and mutant Figs. 3, B–D. This behavior is in sharp contrast to R972Q, which RAG proteins, we normalized the data obtained from the re- displays much more robust recombination (average of 6%), but porter substrates to the mean levels of coding and signal joints with hypersensitivity to certain flank sequences (Fig. 3D, left observed over all coding flanks (ϳ20% GFP-positive cells for both with wild-type RAG proteins). This analysis revealed that panel). the efficiency of recombination by wild-type RAG1 over most The coding flank hypersensitivity of R972Q was recapitu- coding flanks is close to the mean. In contrast, both D32 and lated using chromosomally integrated substrates, as shown in R972Q display reduced levels of V(D)J recombination overall Fig. 5. These data illustrate that the sequence sensitivity of (ϳ5% for coding joints and ϳ6% for signal joints), and the R972Q is not limited to plasmid substrates but also holds in the individual substrates show a much wider variance from the context of chromatin, suggesting that R972Q is hypersensitive mean (Fig. 3, B–D, right panels). In sharp contrast with wild- to coding flanks in vivo. Taken together, these data support two type RAG1, R972Q strongly prefers a few coding flanks, espe- distinct mechanisms for the pathogenesis of OS derived from cially CA, CG, and TA, but shows very poor recombination at RAG mutants: uniformly hypomorphic activity and selective GG and GC flanks (Fig. 3D, right panel). The distribution of coding flank hypersensitivity. The Journal of Immunology 4129 Downloaded from http://www.jimmunol.org/

FIGURE 5. Coding flank preferences of wild-type and R972Q RAG1 are recapitulated on integrated substrates. The percentage of GFP-positive cells is shown for cells containing integrated coding flank substrates transfected with plasmids containing RAG1 (wild-type or R972Q, respectively) and wild-type RAG2. The graph on the right depicts the extrachromosomal substrate data from Fig. 3 for the same coding flank sequences. Error bars represent SEM of three or more experiments. by guest on September 25, 2021 Discussion those few coding segments that have preferred coding flank Our data provide two new insights into the functions of the re- sequences. combinase and the pathogenesis of OS. First, in addition to the Our analysis of murine TCR␤ coding flank sequences reveals a canonical coding flank-sensitive region (606–611), we have found number of coding flanks predicted to be poorly utilized by R972Q, that a mutation in the C-terminal coding flank binding region of including GG and GC at both D␤ 23-RSSs. This should result in RAG1 (amino acids 889–974) (35), R972 (near the catalytic a severe restriction of the TCR␤ repertoire as well as a substantial E962), also affects coding flank preferences at the hairpin forma- block to development because of inefficient D-J joining. We tion step. Of note, the R972 residue is part of a previously sug- also identified many IgH coding flanks that should be poor sub- gested hairpin-forming motif in RAG1, YKE962FR972K (31). In- strates for R972Q (both in mice and in humans), but given the terestingly, a RAG2 mutant, K38A/R39A, appears to be sensitive presence of at least some preferred coding flanks at V, D, and J to coding flank sequences at the nicking step in vitro, although, segments, we speculate that R972Q might skew the repertoire unlike D32 and R972Q, it is severely defective for coding and without causing such a severe block in differentiation. This signal joint formation (20, 32) (S.-Y.W., unpublished observa- is in agreement with the clinical data from the OS patient bearing tions). Taken together, these findings suggest that multiple regions the corresponding R975Q mutation, which shows an almost nor- of the RAG recombinase contact and selectively cleave coding mal peripheral B cell count, which is much higher than that usually flank DNA during V(D)J recombination. observed in OS (9). This clinical observation is also consistent Second, our results separate RAG mutants found in OS patients with our finding that R972Q is substantially more active (on most into two distinct classes: hypomorphic mutants that show severely coding flanks) than other OS RAG mutants. ϩ diminished recombination across all 16 dinucleotide coding flanks, Toshio Hirano’s group showed that V␤ segment usage in CD4 ϩ and at least one coding flank-sensitive mutant that is defective only and CD8 cells is skewed in the R972Q mouse compared with at some coding flank sequences. These findings lead us to propose wild-type mouse (14). This analysis was performed on T cells that two pathogenetic mechanisms for the TCR repertoire restriction have already passed through TCR selection and therefore not rep- characteristic of OS. In one scenario, hypomorphic RAG mutants resentative of all V(D)J recombination events. However, it is in- restrict the repertoire secondary to their crippled activity at all teresting that the “best” and “worst” V␤ coding flanks for R972Q, coding segments. The remaining recombination activity still al- TG at V␤8.1/2 and GC at V␤12, were among the more highly and lows a few, random V(D)J recombination events to occur; certain less represented segments, respectively, compared with wild-type of these are amplified by exposure to Ags. In an alternative sce- mice, providing some confirmation that the activity we describe for nario, supported by the data reported herein, repertoire restriction R972Q recapitulates its activity in vivo. Additionally, the variabil- by R972Q (and possibly other, as yet unknown coding flank-sen- ity of V␤ segment usage among mice bearing a RAG2 mutation, sitive mutants) is primary, a direct result of recombination only at R229Q, was much higher than that observed in the R972Q mouse 4130 OS RAG MUTANT IS HYPERSENSITIVE TO CODING FLANKS

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