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Deletion of the RAG2 C Terminus Leads to Impaired Lymphoid Development in Mice

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Deletion of the RAG2 C Terminus Leads to Impaired Lymphoid Development in Mice Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice Yoshiko Akamatsu*†‡, Robert Monroe‡§¶, Darryll D. Dudley§, Sheryl K. Elkin*, Frank Ga¨ rtner§ʈ, Sadiqur R. Talukder†**, Yousuke Takahama†**, Frederick W. Alt§, Craig H. Bassing§††‡‡, and Marjorie A. Oettinger*††‡‡ *Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; §The Howard Hughes Medical Institute, Children’s Hospital, Harvard Medical School, Center for Blood Research, Boston, MA 02115; †Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan; and **RIKEN Research Center for Allergy and Immunology, Tokushima 770-8503, Japan Contributed by Frederick W. Alt, November 19, 2002 The recombination-activating gene (RAG)1 and RAG2 proteins required for activity, because full-length RAG1 and RAG2 are comprise the lymphocyte-specific components of the V(D)J recom- largely insoluble. However, the activities of these mutant ‘‘core’’ binase and are required for the assembly of antigen-receptor proteins differ from those of the full-length RAGs when assayed variable-region genes. A mutant truncated RAG2 protein (‘‘core’’ with extrachromosomal substrates in transfected cells. In this RAG2) lacking the C-terminal 144 amino acids, together with core context, the mutant core RAG proteins support V(D)J recom- RAG1, is able to mediate the basic biochemical steps required for bination with reduced efficiency and with different levels and V(D)J recombination in vitro and in transfected cell lines. Here we types of recombination products (9–15). examine the effect of replacing the endogenous RAG2 locus in mice Although not required for the biochemistry of V(D)J recom- with core RAG2. These mice generate substantial numbers of B and bination, the noncore regions of RAG1 and RAG2 are con- T cells, demonstrating that the core RAG2 protein retains signifi- served throughout evolution. For example, sequence conserva- cant in vivo function. However, core RAG2 mice display a reduction tion across the entire RAG2 protein from pufferfish to humans in the total number of B and T cells, reflecting impaired lymphocyte is Ϸ60%, with conservation of the noncore C-terminal region development at the progenitor stage associated with reduced slightly higher than the core domain (16). Therefore, the non- chromosomal V(D)J recombination. We discuss potential roles of core regions of RAG1 and RAG2 may serve important acces- the RAG2 C terminus in mediating rearrangement of endogenous sory and͞or regulatory functions in chromosomal V(D)J recom- antigen-receptor loci. bination. Consistent with this notion, studies of Abelson murine leukemia virus-transformed pre-B cells have suggested that the uring B and T cell development, the Ig and T cell receptor noncore regions of RAG1 are important for IgH locus DH-to-JH D(TCR) variable-region genes are assembled from compo- rearrangement (17), whereas the C terminus of RAG2 is more nent germ line variable (V), diversity (D), and joining (J) gene important for VH-to-DJH rearrangement than for DH-to-JH segments through a process known as V(D)J recombination rearrangement (18). The C terminus of RAG2 is predicted to (1–3). Each V, D, and J gene segment is flanked by a recom- fold into a plant homeodomain (PHD) (19, 20), a motif found in bination signal sequence (RSS) composed of conserved hep- a number of chromatin-associated proteins including some that tamer and nonamer sequences separated by either a 12- or 23-bp have chromatin-modifying activities (21–24). spacer. V(D)J recombination is initiated by the lymphoid- To investigate potential in vivo function of the RAG2 noncore specific recombination-activating gene (RAG) 1 and 2 proteins, region in chromosomal V(D)J recombination and normal lym- which recognize a pair of 12 and 23 RSSs and introduce a DNA phocyte development, we have generated and characterized double-strand break between each RSS and flanking coding mice containing specific replacement of the full-length endog- sequence. The RAG-liberated ends are held in a complex with RAG1 and RAG2 until resolved into precise recombination enous RAG2 gene with a gene encoding the mouse core RAG2 signal and imprecise coding joins via the generally expressed protein. nonhomologous end-joining DNA-repair factors (1). Materials and Methods During lymphocyte development, V(D)J recombination is highly regulated in the contexts of lineage specificity, develop- Generation of Core RAG2 Mice. For information about the gener- mental-stage specificity, and allelic exclusion (4, 5). In addition, ation of core RAG2 mice, see Supporting Materials and Methods, developing lymphocytes use strategies that require productive which is published as supporting information on the PNAS web V(D)J rearrangements for continued developmental progres- site, www.pnas.org. sion (4, 5). For example, Ig heavy chain (IgH) variable-region genes are assembled in progenitor (pro) B cells via an ordered Southern Blotting and PCR. Standard methods were used for the process in which DH-to-JH rearrangement occurs on both alleles isolation of genomic DNA and for Southern hybridization (25). before VH-to-DJH rearrangement. Productive VH-to-DJH re- ␮ arrangements produce cell-surface expression of IgH chains Abbreviations: TCR, T cell receptor; RSS, recombination signal sequence; RAG, recombina- that signal expansion and differentiation of pro-B cells to the tion-activating gene; IgH, Ig heavy chain; PHD, plant homeodomain; PE, phycoerythrin; pre-B cell stage. This expression of IgH ␮ chains also signals the CyC, CyChrome; FACS, fluorescence-activated cell sorter; dpc, days postconception; DN, double-negative. cessation of further VH-to-DJH rearrangement via feedback ‡Y.A. and R.M. contributed equally to this work. regulation. Pro-B cells that first assemble nonproductive VH-to- ††C.H.B. and M.A.O. are co-senior authors of this work. DJH rearrangements proceed to rearrange their second allele. Both RAG1 and RAG2 are required for V(D)J recombina- ¶Present address: Department of Pathology, Stanford University, Stanford, CA 94305. tion, because RAG1-orRAG2-deficient mice exhibit a complete ʈPresent address: Albert Ludwigs University Medical Center, Department of Medicine- block in lymphocyte development at the progenitor stage (6, 7). Hematology͞Oncology, Freiburg, Germany. In addition, RAG1 and RAG2 together are sufficient to initiate ‡‡To whom correspondence may be addressed at: (C.H.B.) Children’s Hospital, Enders Building, Room 870, 300 Longwood Avenue, Boston, MA 02115. E-mail: cbassing@ V(D)J recombination in vitro (8). Nearly all in vitro studies of enders.tch.harvard.edu; or (M.A.O.) Department of Molecular Biology, Massachusetts RAG function have used the minimal regions of RAG1 (amino General Hospital, 50 Blossom Street, Boston, MA 02114. E-mail: [email protected]. IMMUNOLOGY acids 384–1,008 of 1,040) and RAG2 (amino acids 1–383 of 527) harvard.edu. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237043100 PNAS ͉ February 4, 2003 ͉ vol. 100 ͉ no. 3 ͉ 1209–1214 Downloaded by guest on September 23, 2021 Fig. 2. VH-to-DJH rearrangement is reduced in developing B cells from core RAG2 mice. (A) PCR analysis of DH-to-JH rearrangement in fetal liver genomic DNA from RAG WT (ϩ͞c) and core (c͞c) littermates is shown with serial dilutions from 14.5-dpc embryos (Right). (Left) PCR amplification of C␮ is included as a loading control and shown at the bottom. (B) PCR analysis of VH-to-DJH rearrangement of VH7183 and VHQ52 segments with serial dilutions for VH7183 rearrangements from 15.5-dpc embryos (Right). PCR products were probed with the JH4 probe. B cells is reduced substantially in the core RAG2 mouse Fig. 1. B cell development of core RAG2 mice is impaired. (A) Representative (approximately one-third of the number of WT cells) (Fig. 1A). FACS analysis of bone marrow cells from RAG2 WT (ϩ͞Ϫ), core (c͞Ϫ), and The CD43͞B220 stain shows an increased percentage of knockout (KO, Ϫ͞Ϫ) mice stained with CyC-anti-B220 and either PE-anti-IgM CD43hi͞B220lo (pro-B) cells in the core RAG2 mouse and a or FITC-anti-CD43. (B) FACS analysis of peripheral splenic B cells from RAG2 WT reduction in the percentage of CD43lo͞B220int (pre-B and ϩ͞Ϫ ͞Ϫ Ϫ͞Ϫ ( ), core (c ), and knockout ( ) mice stained with CyC-anti-B220 and immature B cells) and CD43Ϫ͞B220hi (mature B cells) compared PE-anti-IgM. The percentage of B220ϩIgMϩ cells of total lymphocytes is shown. The percentages of B220 and CD43 staining are calculated on the basis with WT (Fig. 1A). The absolute numbers of pro-B cells are of the total number of B220ϩ cells in each sample. equivalent in core RAG2 and WT mice (data not shown). This FACS profile indicates that core RAG2 mice exhibit impaired development at the pro-B cell stage. Peripheral lymphoid com- IgH and TCR␤ rearrangements were amplified by PCR with partments such as spleen and lymph nodes were also examined, ϩ primers as described (18, 26). and they too contain decreased numbers of IgM B cells in the core RAG2 mice (Ϸ50% of WT controls) (Fig. 1B), which is Flow Cytometry. Single-cell suspensions from each tissue were consistent with the decreased rate of B cell production in bone stained with FITC-, phycoerythrin (PE)-, and CyChrome (CyC)- marrow. conjugated antibodies (PharMingen) and analyzed by a FACScali- bur or FACSVantage SE (Becton Dickinson). Data were analyzed Reduced V(D)J Recombination of the IgH Locus. B cell development with CELLQUEST (Becton Dickinson) software. Cell sorting was in RAG-deficient mice is arrested completely at the pro-B cell Ϫ performed on a MoFlo machine (Cytomation, Fort Collins, CO). stage with virtually no CD43lo͞B220int or CD43 ͞B220hi cells (Fig. 1A) (6, 7). Thus, we investigated whether the impairment Results in B cell development in core RAG2 mice is a reflection of Core RAG2 Mice Exhibit a Partial Arrest in B Cell Development.
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