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A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data

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A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data This information is current as Ang Cui, Roberto Di Niro, Jason A. Vander Heiden, Adrian of October 1, 2021. W. Briggs, Kris Adams, Tamara Gilbert, Kevin C. O'Connor, Francois Vigneault, Mark J. Shlomchik and Steven H. Kleinstein J Immunol published online 5 October 2016 http://www.jimmunol.org/content/early/2016/10/05/jimmun ol.1502263 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/10/05/jimmunol.150226 Material 3.DCSupplemental 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 October 1, 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 5, 2016, doi:10.4049/jimmunol.1502263 The Journal of Immunology A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Immunoglobulin Sequencing Data Ang Cui,* Roberto Di Niro,† Jason A. Vander Heiden,* Adrian W. Briggs,‡ Kris Adams,‡ Tamara Gilbert,‡ Kevin C. O’Connor,x,{ Francois Vigneault,‡ Mark J. Shlomchik,† and Steven H. Kleinstein*,{,‖ Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific l+ germinal center B cells and sequenced the unexpressed k L chains using k next-generation methods. Most of these sequences had out-of-frame junctions and were presumably uninfluenced by selection. Downloaded from Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, http://www.jimmunol.org/ suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns. The Journal of Immunology, 2016, 197: 000–000. omatic hypermutation (SHM) is a process that diversifies C/G base pair (phase I) or at neighboring base pairs (phase II) (2). BCRs by introducing point mutations into Ig genes at a high Although stochastic, SHM is biased by the local DNA sequence S rate (1). SHM is initiated when activation-induced cytidine context and preferentially introduces mutations at specific DNA deaminase (AID) is recruited to the Ig locus and converts cytosine motifs (hot spots) while avoiding others (cold spots) (3–5). SHM (C) to uracil (U). Error-prone DNA repair pathways are then ac- plays a crucial role in the B cell immune response and immune- by guest on October 1, 2021 tivated, resulting in somatic mutations either at the AID-targeted mediated disorders. The analysis of mutation patterns and distri- butions has been widely used to infer selective processes involved in such responses (6). However, the analysis of SHM patterns can *Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511; †Department of Immunology, University of be confounded by the intrinsic biases of SHM targeting, driving Pittsburgh, Pittsburgh, PA 15213; ‡AbVitro Inc., Boston, MA 02210; xDepartment of the need for accurate characterization of targeting patterns that { Neurology, Yale School of Medicine, New Haven, CT 06511; Human and Transla- reflect inherent SHM properties in the absence of Ag-driven tional Immunology Program, Yale School of Medicine, New Haven, CT 06511; and ‖Departments of Pathology and Immunobiology, Yale School of Medicine, New selection (7, 8). Haven, CT 06511 The SHM process can be quantitatively characterized by a ORCIDs: 0000-0002-1087-8568 (A.C.); 0000-0002-1474-310X (J.A.V.H.); 0000- targeting model, consisting of a mutability model, which specifies 0002-7056-419X (K.C.O.); 0000-0003-4957-1544 (S.H.K.). the relative mutation frequency of DNA microsequence motifs, and Received for publication October 21, 2015. Accepted for publication August 22, a substitution model, which describes the specific nucleotide 2016. substitution frequencies at the mutated sites (9–13). These models This work was supported by the National Institutes of Health (Grants R03AI092379 can serve as a background distribution for statistical analysis of and R01AI104739 to S.H.K., Grant R01AI43603 to M.J.S.), the Myasthenia Gravis Foundation of America, the National Institute of Allergy and Infectious Diseases mutation patterns in Ig sequences, improving the ability to detect (Awards R01AI114780 and U19 AI056363 to K.C.O.), the Natural Sciences and deviations in SHM pathways related to diseases or identify se- Engineering Research Council of Canada (NSERC PGS-M to A.C.), and the National Library of Medicine (Grant T15LM07056 to J.A.V.H.). The Yale University Biomed- lected mutations that drive Ag specificity and affinity maturation ical High Performance Computing Center is supported by National Institutes of (7, 8). However, modeling these intrinsic biases has been limited Health Grants RR19895 and RR029676-01. by the lack of large sets of Ig sequences that have undergone SHM The content is solely the responsibility of the authors and does not necessarily in vivo in the absence of selection pressures. Previous work has represent the official views of the National Institutes of Health. focused on studying mutations in intronic regions or in Ig se- Address correspondence and reprint requests to Dr. Steven H. Kleinstein, Yale School quences that were determined to be nonfunctional (e.g., because of of Medicine, 300 George Street, Suite 505, New Haven, CT 06511. E-mail address: [email protected] an out-of-frame junction) (9–11, 13). However, intronic regions The online version of this article contains supplemental material. have limited diversity and a different base composition from ex- Abbreviations used in this article: GC, germinal center; NP, (4-hydroxy-3-nitro- onic V(D)J regions, and some mutations in nonfunctional se- phenyl)acetyl; RS, replacement and silent; RS5NF, RS, 5-mer, nonfunctional; S5F, quences may be subject to selection pressures if the sequences silent, 5-mer, functional; S5NF, silent, 5-mer, nonfunctional; SHM, somatic hyper- were rendered nonfunctional during the affinity maturation mutation; UID, unique identifier; UNG, uracil-DNA glycosylase. process. Another strategy to determine targeting models in- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 volves using mutations that do not alter the amino acid sequence www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502263 2 MOUSE SOMATIC HYPERMUTATION TARGETING MODEL (i.e., silent or synonymous mutations), which presumably are not barcoded on the 59 end in a template switchlike reaction using an oligo- subject to selection pressures. We previously used this strategy to nucleotide harboring a semidegenerate 17-nt-long unique identifier (UID) construct the human silent, 5-mer, functional (S5F) SHM targeting barcode and a universal flanking sequence. The cDNA was purified using . streptavidin bead pulldown and then subjected to a first round of 12 cycles model from 800,000 mutations in functional Ig sequences (12). of PCR. The human samples used a human C region primer mix composed Despite the high resolution of this S5F model, the mutability of of Ig H chain C region, Ig k L chain C region, and Ig l L chain C region some DNA motifs could not be estimated directly because they do flanked by another universal sequence (19–21), and the mouse samples k l not yield silent mutations. used a mouse primer mix composed of Ig L chain C region and Ig L chain C region (21). A second round of 12 cycles of PCR was then Modeling and analysis of SHM would also benefit from a clear performed using universal overhang from both ends to add the required understanding of whether equivalent models can be used across Illumina sequencing and clustering adapters. The samples were then L and H
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