Allelic Exclusion of IgH through Inhibition of E2A in a VDJ Recombination Complex Jannek Hauser, Christine Grundström and Thomas Grundström This information is current as of October 2, 2021. J Immunol published online 27 January 2014 http://www.jimmunol.org/content/early/2014/01/25/jimmun ol.1302216 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/01/25/jimmunol.130221 Material 6.DCSupplemental
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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 27, 2014, doi:10.4049/jimmunol.1302216 The Journal of Immunology
Allelic Exclusion of IgH through Inhibition of E2A in a VDJ Recombination Complex
Jannek Hauser, Christine Grundstro¨m, and Thomas Grundstro¨m
A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A. The Journal of Immunology, 2014, 192: 000–000. Downloaded from
he diversity of AgRs is generated by each B and T cell D to J recombination at Igh loci in cells that normally never undergo precursor assembling one variable (V), one joining (J), V(D)J recombination (7). The V segments of the Igh locus have T and, on some receptor genes, a diversity (D) gene segment binding sites for several transcription factors, including E2A and from vast arrays of these segments. This V(D)J recombination of Ig PAX5 (8, 9), and PAX5 is essential in inducing and orchestrating genes starts with IgH. Most IgHs do not become functional because Igh recombination in the V region (10–14). Embryonic kidney cells of the random nature of the fusion of gene segments in V(D)J re- show V to DJ recombination at the Igh loci exclusively when http://www.jimmunol.org/ combination. Therefore, the expression of a pre-BCR with membrane- RAG1/RAG2 and E2A are ectopically coexpressed with PAX5 bound IgH associated with surrogate L chain proteins is a critical (14). Therefore, RAG1, RAG2, PAX5, and E2A are all indis- checkpoint that monitors for functional IgH rearrangement. Later in pensable for VDJ recombination of the Igh locus. the development, the functional rearrangement of Ig L chain gene A key feature of the immune system is the paradigm that one segments is verified by the complete Ab of the BCR. V(D)J re- lymphocyte has only one Ag specificity that can be selected for or combinations are initiated by introduction of dsDNA breaks by the against to enable targeting of the pathogen while avoiding collateral recombination activating gene enzymes RAG1 and RAG2 at re- damage and wasted resources. The monospecificity requires that combination signal sequences (RSSs). The RAG1/RAG2 complex only one allele of genes for AgR chains is made functional. This allelic by guest on October 2, 2021 binds sequence specifically to RSS sequences, and RAG2 specifi- exclusion requires that only one allele at a time undergoes complete cally recognizes histone H3 trimethylated at lysine 4 (H3K4me3). V(D)J recombination. Multiple mechanisms most likely contribute to Recognition of RSSs and H3K4me3 and accessibility of the chro- allelic exclusion, but the relative role of the proposed models remains matin are all necessary for V(D)J recombination (1–4). DNA elements controversial (15–17). The allelic-exclusion system ensures that suc- regulating the accessibility include promoters and enhancers, and cessful V(D)J recombination on one allele shuts off recombination on some transcription factors were shown to play a role (1–4). The the other allele by feedback inhibition to prevent a second successful strongest link with a transcription factor is for E2A, which has the rearrangement (15–17). This feedback inhibition from AgRs has an two splice forms E12 and E47 (3). These bind to “E-box” sequences established major role in allelic exclusion, but little is known about in Ig genes and are required for both RAG expression and Ig re- the molecular mechanism(s) by which it is achieved (15–17). However, combination. E-boxes in the Igk L chain intronic enhancer are expression of RAG1 and RAG2 is lower in pre-B cells expressing the crucial for activation of V-J rearrangement at Igk loci (5), and E12 pre-BCR than in the preceding and succeeding developmental stages, and E47 regulate the sequential rearrangement of the Ig L chain loci suggesting negative feedback of these key recombination proteins (6). Ectopic expression of E2A, together with RAG1/RAG2, induces (18), but the molecular mechanism(s) of the allelic exclusion by feed- back inhibition has remained largely enigmatic. When the main Ca2+-sensor protein calmodulin (CaM) is Ca2+ Department of Molecular Biology, Umea˚ University, SE-901 87 Umea˚, Sweden loaded, it can bind to and inhibit transcriptional activities of E2A Received for publication August 19, 2013. Accepted for publication December 20, (19–21). In this study, we analyzed expression of the two Igh 2013. alleles in B cells expressing CaM-resistant (CaMR) E2A and show This work was supported by grants from the Swedish Research Council and the that they are dramatically defective in allelic exclusion. One Swedish Cancer Society. mechanism of the dependence of allelic exclusion on CaM inhi- Address correspondence and reprint requests to Dr. Thomas Grundstro¨m, Department bition of E2A was shown to be that pre-BCR activation down- of Molecular Biology, Umea˚ University, SE-901 87 Umea˚, Sweden. E-mail address: [email protected] regulates several components of the recombination machinery, The online version of this article contains supplemental material. including RAG1, RAG2, and PAX5, through CaM inhibition of Abbreviations used in this article: B6, C57BL/6; CaM, calmodulin; CaMR, calmod- E2A. Furthermore, data support that E2A, PAX5, and the RAGs ulin resistant; ChIP, chromatin immunoprecipitation; H3K4me3, histone H3 trimethy- are in a complex bound to key sequences on the Igh gene, and they lated at lysine 4; PLA, proximity ligation assay; RSS, recombination signal sequence; instead became bound to CaM after pre-BCR activation. The re- shRNA, short hairpin RNA; TdT, terminal deoxynucleotidyl transferase; WT, wild-type. combination complex is directly released from the key Igh sites Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 through CaM inhibition of E2A.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302216 2 ALLELIC EXCLUSION BY INHIBITION OF E2A
Materials and Methods the human RAG1 promoter, 59-AAGAGCCAGGTGGCAGCTGGAGCTG- 9 9 9 Mice and allelic-exclusion analysis 3 ; the RAG2 promoter, 5 -GCCAAGGCAGGTGGGTCACC-3 ; the RAG enhancer Erag,59-AGAGGGACAGCTGCTTTCTA-39; and the TDT pro- C57BL/6 (B6) male mice heterozygous for E2A knockout (E2A+/2) (22) moter, 59-AGGGCAGCACCTGTGAAGCC-39 (27–30). The control probe were crossed with BALB/c females (Taconic, Ry, Denmark). Total bone from the human GM-CSF promoter was described previously (25). marrow was isolated from resulting E2A+/2 F1 mice at age 6–8 wk and immediately infected with retrovirus expressing wild-type (WT) or CaMR Real-time RT-PCR mutant E12 in culture with complete RPMI 1640 medium, IL-7 (10 ng/ml; RNA was extracted using TRIzol reagent (Invitrogen), and quantitative m PeproTech), and polybrene (10 g/ml). Twenty-four hours postinfection, real-time RT-PCR analysis was performed, as previously described, using bone marrow cells were supplemented with fresh total bone marrow GAPDH as internal control (23). The specificity of the PCR amplifications 3 +/2 (a quarter of the infected amount) from B6 BALB/c F1 E2A mice, and was verified by melt-curve analysis, as described by the manufacturer 3 the mixture was transplanted into lethally irradiated (9.5 Gy) B6 BALB/c (Bio-Rad). Some of the primer pairs used were described previously +/2 3 7 m F1 E2A recipients at age 6–8 wk (up to 2 10 cells in 150–200 l (25, 31). The real-time RT-PCR primer pairs for mouse and human genes HBSS/mouse) by tail-vein injection. Recipient mouse spleen cells were that have not been described previously were as follows: mouse Rag1: collected 2–3 wk posttransplantation. The effect of E12 WT and mutant 59-GACAAAGAAGAAGGTGGAGATG-39 and 59-AGGAAGGTATTGA- expression on allelic exclusion was analyzed by FACS using the IgH 9 9 9 A CACGGATG-3 ; mouse Rag2:5-TTGCCCTACTTGTGATGTTG-3 and allotype-specific Abs IgM -PE (specific for IgH of the BALB/c strain; 9 9 9 B 5 -ACTTGTTGCTTCCTTCTGAC-3 ; mouse TdT:5-GACATCTCCTG- clone DS-1) and IgM -biotin (specific for IgH of the B6 strain; clone AF6- GCTCATCG-39 and 59-TGGTTCTTCGCTGGCAAG-39; mouse Pax5:59- 78), anti–CD19-allophycocyanin (clone 1D3), and streptavidin-PerCP (all AGTCTCCAGTGCCGAATG-39 and 59-TCCGTGGTGGTGAAGATG-39; from BD Biosciences) or streptavidin–PerCP–Cy5.5 (eBioscience). All mouse Ets1:59-CACAACACAAATCCAAGAGTC-39 and 59-GGTGGT- cells were treated with CD16/CD32 Fc Block before staining, and dead AACAGGTCAAGTG-39; human RAG1:59-CCAGCAAAAGAGTGCA- cells were excluded by propidium iodide (both from BD Biosciences). Flow ATGA-39 and 59-TCAGCGACAGAAGATGTTGG-39; human RAG2:59- cytometry was performed with a FACSCalibur, and cell sorting was done 9 9 A+ B+ CAGACAACATTCACAAACAG-3 and 5 -ACATCACAAGTAGGGCA- Downloaded from with a FACSAria III instrument (both from BD Biosciences). IgM IgM G-39;humanTDT:59-GGGAAAAGAAGGGATTACTT-39 and 59-TC- double-positive B cells, as displayed in Fig. 1A, were sorted at 4˚C directly TTTGACGAGGCAATTTGA-39; human FOS:59-GAGCACGGAGGA- m into culture in 500 l complete RPMI 1640 medium supplemented with GGGATGAG-39 and 59-AGTCTCCAGAATGAACTCGCTTT-39; human 20% FCS in a 24-well culture plate containing a layer of S17 feeder cells, OCT2:59-AGTGATCTGGAGGAGCTGGAG-39 and 59-CTCGCGGCA- irradiated at 30 Gy. Approximately 20,000–50,000 double-positive cells TCTATCAACTGG-39; and human IL6: 59-GTCCAGTTGCCTTCTCCCT- were isolated from a total of 10–20 million splenocytes. After sorting, the 39 and 59-GCCTCTTTGCTGCTTTCAC-39. medium was adjusted to 1 ml with 25 mg/ml LPS (Calbiochem) and 10%
FCS, and the cells were cultivated for 3 d before reanalysis by FACS and Proximity ligation assay http://www.jimmunol.org/ immunostaining. All mouse research was carried out under a Swedish project license and was subject to local ethical review. Harvested mouse primary pre-B cells were fixed with 4% paraformalde- hyde, permeabilized with 0.2% Triton X-100, and blocked with 5% FCS in Plasmids, viruses, cell culture, and transfections PBS. Proximity ligation assay (PLA) (32–35) was performed with the Duolink system using in situ PLA kits from Olink Biosciences. In brief, The human pre-B cell line Nalm-6 was maintained in RPMI 1640 medium cells were stained for intracellular proteins using Abs, as listed below. supplemented with 5% FCS and antibiotics. The EBV-based pMEP4 shuttle Secondary Abs conjugated with oligonucleotides (PLA probes) were vector expressing a short hairpin RNA (shRNA) that interferes with E12/E47- subsequently used, according to the manufacturer’s protocol, to generate specific human mRNA and the pMEP4 derivatives encoding WT E12 and the R fluorescence signals only when the two PLA probes were in close prox- CaM mutant m8N47 of E12 were described previously, and the MSCV- imity (#40 nm). The fluorescence signal from each detected pair of PLA
IRES-GFP–based retroviruses were described previously (21, 23, 24). Nalm-6 probes was visualized as a distinct individual red dot in an epifluorescence by guest on October 2, 2021 cells transfected to express pMEP4 derivatives were selected with Hygro- microscope (Nikon 90i). The Abs were used at dilutions that yielded a low mycin B (Roche) for 5 d, as described previously (25). Their pre-BCR was m 9 number of dots/cell to have a low risk for missing dots in the quantifica- stimulated for 5 h, unless otherwise specified, with 5 ggoatF(ab)2 anti- tions. Nuclei were counterstained with DAPI dye. To enhance detection of human IgM (Southern Biotechnologies). For the C-terminal Flag con- infected cells, anti–GFP-FITC Ab (Novus Biologicals) was added during structs, the GFP-encoding DNA of the MSCV-IRES-GFP plasmid was the PLA signal-detection step. replaced by oligonucleotides comprising a tandem repeat of the Flag epitope–coding sequence (21) and restriction enzyme sites for insertion Chromatin immunoprecipitation of full-length cDNAs of RAG2 andPAX5(GenScript).E12WTand CaMR mutant, with or without the C-terminal Flag epitope, were cloned Infected primary mouse pre-B cells from E2A+/2 B6 mice were cross-linked into MSCV-IRES, as described (23). Production of retroviruses was in with 1% formaldehyde for 10 min at room temperature. Cross-linking was 293T cells, as described (23). quenched with a 1/20 volume of 2.5 M glycine, and cells were washed with PBS. Cells were lysed on ice with 250 ml RIPA buffer (50 mM Tris-HCl Activation, infection, and analysis of pre-B lymphocytes from [pH 8], 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxy- mouse bone marrow cholate, and 5 mM EDTA) supplemented with protease inhibitor mixture (Roche Applied Science). The lysate was sonicated at 60% amplitude for Bone marrow cells were collected by flushing femur and tibia of C57BL/6 10 3 10 s on a Vibra-Cell instrument (Sonics) and clarified by centrifugation. mice. The cells were grown on S17 stromal cells with IL-7, and the pre-BCR 9 The average size of sheared chromatin fragments was determined by agarose of pre-B cells, where indicated, was activated by goat F(ab )2 anti-mouse m m gel electrophoresis to be 180 bp. A total of 5 l was saved as input, and the IgM (5 g/ml; 1022-01; Southern Biotechnologies), as previously de- lysate was incubated overnight at 4˚C with mouse anti–Flag-tag Ab (Gen- scribed (25). Pre-B lymphocytes were purified from the bone marrow cells Script). Immunocomplexes were captured for 1 h by 20 mlmagnetic with biotinylated rat Ab to CD25 (7D4; BD Pharmingen) using magnetic Dynabeads Protein A (Invitrogen) at 4˚C. Beads were washed three times anti-biotin MicroBeads, a MidiMACS Separator, and LS columns (all from with RIPA buffer and three times with Tris-EDTA (pH 8) on a magnet Miltenyi Biotec), according to the manufacturer’s recommendations. In- (Invitrogen). Cross-links were reversed with 1% SDS in Tris-EDTA (pH 8) at fections with MSCV-IRES-GFP–based retroviruses were by centrifugation m m 65˚C overnight, and samples were treated with Proteinase K (20 g/ml) for in the presence of polybrene (5 g/ml), as previously described (25), 1 h at 45˚C. DNA was phenol-chloroform extracted, ethanol precipitated, and followed by 18 h of culture in fresh complete medium with elevated IL-7 analyzed by quantitative real-time PCR. Each sample was quantified relative (25 ng/ml). to its input chromatin DNA. The following primers were used for the FACS and DNA-binding analysis chromatin immunoprecipitation (ChIP) analysis of the mouse Igh gene: Em:59-TCAGAACCAGAACACCTGCAGCA-39 and 59-GGTGGGGC- Intracellular immunostaining of harvested cells was performed with 2% TGGACAGAGTGTTTC-39;Hs3b:59-TGGTTTGGGGCACCTGTGC- paraformaldehyde and ethanol, as previously described (26). Flow cytometry TGAG-39 and 59-GGGTAGGGCAGGGATGTTCACAT-39;VHA.1: 59- was performed with a FACSCalibur instrument, and analysis was done with CCTTCGCCCCAATCCACC-39 and 59-CAAGTAACCCTCAAGAGAA- CellQuest software (Becton Dickinson Biosciences). Nuclear extracts were TGGAGACTC-39;VH10: 59-CTCTCTGCCAATGTAGGACCAG-39 and prepared, as previously described (21), from the Nalm-6 human pre-B cell 59-GCCTGAATTTCAGGGTCAGGG-39;VHJ558.47 (2): 59-CTACAAC- line, and EMSAs were performed as described (20). The EMSAs were CAGAAGTTCAAGGGCAA-39 and 59-TCAGGCTGTGATTACAACACTG- performed with probe sequences (E2A binding sites are underlined) from TGT-39; DQ52: 59-AGTCCCTGTGGTCTCTGA-39 and 59-AGCCTCTCTA- The Journal of Immunology 3
CTTCCTCA-39;JH1: 59-GCTCAGGTCACTCAGGTCAGG-39 and 59-GA- Protein expression and binding assay GAATATCTTTTCCCGTTTCAGA-39;JH2: 59-TACTTTGACTACTGGGG- The cDNAs for full-length murine PAX5 and amino acids 431–652 of murine C-39 and 59-CCCTAGTCCTTCATGACC-39;JH3: 59-GCCTGGTTTGCTT- E12 were obtained from Genscript and subcloned into pET21b+(2), a deriv- ACTGG-39 and 59-GACAAAGGGGTTGAATCT-39;JH4: 59-CACCAGGA- ATTGGCATAA-39 and 59-CCTGAGGAGACGGTGACT-39;V1:59-GGTG- ative of pET21b+ (Novagen), in which the N-terminal T7-tag is replaced with AAGCTGGTGGAATCTGGAGGA-39 and 59-CTCCATGTAGAAATCACT- the pelB leader from pET20b+ (Novagen). The His6-tagged proteins were GAAGG-39; V11: 59-GGTGAAGCTGGTGGAATCTGGAGGA-39 and 59- expressed in Escherichia coli BL21(DE3) pLysS. E12-expressing cells were ATAAAACCCAACCACTCAAGTGCCTTTC-39; V13: 59-CAGTGTGAGG- centrifuged and lysed by sonication in 50 mM Tris-HCl (pH 8), 1 M KCl, TGAAGCTGATGGAG-39 and 59-ATCAAAGCCAACCACTCAGGTGACT- 1 mM EDTA, 1 mM DTT, 1 mM PMSF. E12 was purified from the soluble fraction by Ni-NTA agarose chromatography (QIAGEN) under native con- TCC-39;VH7183: 59-GCTGGTGGAGTCTGGGGGA-39 and 59-TGGTATT- ditions, according to the manufacturer’s instructions, followed by purification GTCTCTCGAGATGATGAATCG-39;VHJ558: 59-GTGAAGATGTCCTGC- AAGGCTTCT-39 and 59-GGATTTGTCTGCAGTCAGTGTGGCCTTG-39; on Heparin-Sepharose. E12 was eluted with 20 mM HEPES (pH 8), 0.01% Triton X-100, 0.5 M NaCl, 10% glycerol, and 2 mM DTT. PAX5-expressing VH7183 (2): 59-CTCCAGAGACAATACCAAGAAGACCC-39 and 59-CCC- CCTGCTGGTCCTAGATG-39; VAR34: 59-CCTGGGATGTCACTGATATA- cells were centrifuged, and the pellet was solubilized and lysed by sonication CACTCTG-39 and 59-GTAGTAGCCAGTAAATGAGTAACCAGAAGC-39; in 100 mM NaH2PO4, 10 mM Tris (pH 8), and 8 M urea and centrifuged. The PAX5-containing supernatant was purified by Ni-NTA agarose chroma- VHJ558.47: 59-GCAAGGCTTCTGGATACACATTCAC-39 and 59-CCTTG- tography, according to the manufacturer’s instructions. Purified PAX5 was CCTTTGAACTTCTCATTGTACTTAG-39;VHSM7: 59-CGTTATCCTCAT- TGCTACTACCACC-39 and 59-CCAAGTCCTAACTCTGTCTGAAGAAC-39; renatured by dialysis against 10 mM Tris (pH 8), 1 mM EDTA, and 5 mM DTT, with gradually decreasing concentrations of urea; 0.4 M arginine was VH81X: 59-GAGATGAGATTCTGTCTGTTGTATGCAC-39 and 59-CTGC- AAACAAAGAGTGCTGGTCAG-39;DFL16.1:59-CCCCCAATGCTCTCT- added in the last dialysis without urea. ACA-39 and 59-TATAAACCCCGTGATGCC-39;Cm:59-CACCATTTCCTT- Purified PAX5 dialyzed against 1 M potassium acetate (pH 8.3) was coupled CACCTG-39 and 59-TGTTTTTGCCTCCGTAGT-39;andCd:59-CCATCA- to CNBr-activated Sepharose 4B (GE Healthcare), following the manu- CTTTTTGTCCAT-39 and 59-AGCAAGAGGTGTAAGGTT-39. facturer’s instructions, and using 1 M potassium acetate (pH 8.3) as coupling buffer. Whole-cell extract from 1.5 million mouse B cells or purified WT or Downloaded from The Em and hs3b primers were from (36); the VHA.1 and VH7183 (2) CaMR E12 or ETS1 (0.2 mg) was incubated with 10 ml PAX5-Sepharose or primers were from (11); the VH10, VAR34, VHJ558.47, VHSM7, and VH81X control Sepharose beads in a binding buffer containing 50 mM Tris-HCl primers were from (37); the DQ52, JH1, JH2, JH3, and JH4 primers were from (pH 8), 100 mM NaCl, 1.5% Triton X-100, 1 mM DTT, and protease in- (38); and the V1, V11, V13, VH7183, VHJ558, DFL16.1, Cm,andCd primers were from (14). hibitor mixture tablet without EDTA (Roche) and either 0.1 mM CaCl2 or 0.1mMEGTAwithrotationfor30minatroomtemperature.ThePAX5- Western blot Sepharose and control Sepharose were washed twice with binding buffer, and bound proteins were eluted by boiling in Laemmli loading buffer. Samples Nuclear extracts were prepared as previously described (21), and Western were separated by 12% SDS-PAGE and analyzed by Western blot using Tetra- http://www.jimmunol.org/ blot of purified mouse pre-B cells was performed using SuperSignal His Ab (QIAGEN) for E. coli–produced E12; ETS1/ETS2 Ab (C-275) for Chemiluminescence Substrate (Pierce), according to the manufacturer’s E. coli–produced ETS1; and b-actin Ab (ACTBD11B7), CD19 Ab (E-20), a instructions. mixture of E2A Abs (V-18, H-208, and Yae) (all from Santa Cruz Biotech- nology), and a-tubulin Ab (B-5-1-2; Sigma) for proteins from whole-cell ex- Abs tract. Pixel intensities of E12 bands were quantified by ImageJ software and plotted as percentage of E12 bound to PAX5. Abs and related reagents used included IgMA-PE (DS-1), IgMB-biotin (AF6- 78), CD19-allophycocyanin and CD19-allophycocyanin-H7 (1D3), CD16/CD32 Statistical analysis Fc-block (2.4G2), CD25-biotin (7D4), and SAv-PerCP (all from BD Bio- sciences); SAv–eFluor 660, SAv-allophycocyanin, and SAv-PerCP-Cy5.5 The data are expressed as means 6 SD. The results were analyzed using by guest on October 2, 2021 (eBioscience); anti–PE-TRITC (AP09206TC-N; Acris Abs); terminal deoxy- the Student t test with two-tailed distribution, with the exception that data x2 nucleotidyl transferase (TdT; A1-84083; Affinity BioReagents); anti-goat– in Fig. 1E were analyzed using the Pearson test. allophycocyanin, anti-rabbit–PE, anti-rabbit–allophycocyanin, anti-mouse–PE, and anti-mouse–allophycocyanin (Jackson ImmunoResearch); anti–GFP-FITC Results (NB100-1771; Novus Biologicals); and anti–DYKDDDDK-tag (Flag; A00187; Defective allelic exclusion in B cells expressing CaMR E2A GenScript). The following Abs are labeled with superscript numbers where required to distinguish them when two or more primary Abs against the same To analyze whether inhibition of E2A by CaM was relevant for protein were used in PLA: CaM (N-19), CaM2 (FL-149), E2A (V-18), E2A2 allelic exclusion, we analyzed double expression of the IgH alleles (Yae), E2A3 (H-208), E2A4 (N-649), RAG1 (P-20), RAG12 (H-300), RAG22 3 2 in mice. C57BL/6 (referred to as B6) male mice heterozygous for (D-20), RAG2 (M-300), PAX5 (C-20), PAX5 (E-9), ETS1 (N-276), and SMAD3 E2A knockout (E2A+/2) were crossed with BALB/c females, and (38-Q) (all from Santa Cruz Biotechnology); RAG2 (11825-1-AP; Proteintech +/2 2 3 total bone marrow was isolated from resulting E2A F1 mice and Group); and H3K4me3 (ab8580), H3K4me3 (ab1012), and H3K4me3 R (ab71998) (Abcam). PAX52 Ab was biotinylated when used with PAX5 Ab. infected with retrovirus expressing either WT or CaM mutant of Secondary PLA probes were used as anti-goat/anti-rabbit, anti-goat/anti-biotin, the E12 splice form of E2A together with GFP. Infected bone marrow or anti-rabbit/anti-mouse combinations, depending on each primary Ab pair. cells were transplanted into lethally irradiated B6 x BALB/c F1 E2A+/2 recipients. Recipient mouse spleen B cells were analyzed Single-cell PCR by FACS for BALB/c (IgMA) and B6 (IgMB) IgH allotype expres- + Single GFP+ IgMA+ IgMB+ apparently double-positive B cells of experi- sion. The frequency of live-gated mouse splenic CD19 B cells ment 2 of Fig. 1B (as shown in Fig. 1A) were deposited directly into wells appearing to express both Igh alleles was 1–1.5% for the GFP2 of a PCR plate (Sarstedt) containing 20 ml PCR buffer (Bio-Rad) and 0.5 (i.e., noninfected) control cells from infection with virus expressing mg/ml Proteinase K (PCR grade; Roche). To prepare DNA, plates were incubated at 50˚C for 1 h, followed by heat inactivation at 95˚C for 15 min. WT E2A (Fig. 1A, 1B, Supplemental Fig. 1A), as previously re- Igh rearrangements were detected by two rounds of PCR amplification ported for FACS analysis of double expression of IgH (40). Im- with semi-nested primers (39). In the first round, PCR amplification was portantly, in sharp contrast, both Igh alleles were detected in 7.17 6 performed with 20 ml single-cell lysate in 60 ml total volume containing 0.53% of the cells infected with virus expressing CaMR mutant m 3 m 40 l1 HotStarTaq Master Mix (QIAGEN), 5 g/ml yeast tRNA, 3 mM E2A in the first experiment and in 6.33 6 0.64% of the cells in the final concentration of MgCl2, and 1 mM each first-round primer. In the second round, 2 ml first-round product was added to 10 ml HotStarTaq second experiment (Fig. 1A, 1B, Supplemental Fig. 1A). This dra- Master Mix using the conditions above, but the reactions were in separate matic increase in double-expressing cells also was in sharp con- tubes, each containing a pair of second-round primers (5 mM each). Both trast to the cells infected with virus expressing WT E2A: 1.81 6 rounds of PCR used the following conditions: 95˚C for 15 min; 35 cycles 0.35% and 2.50 6 0.37%, respectively, appeared to double express of 94˚C for 1 min, 60˚C for 1 min, and 72˚C for 2 min; and a final extension m the Igh alleles (Fig. 1A, 1B, Supplemental Fig. 1A). Compared with at 72˚C for 10 min. A total of 5 l of the second-round PCR products were 2 examined on 1.2% agarose gels. Single-cell PCR primers were as described GFP cells from the infection with virus expressing WT E2A, ap- (39). parently double-expressing cells were slightly more frequent both 4 ALLELIC EXCLUSION BY INHIBITION OF E2A
that slightly overexpress WT E2A and due to a slight expression of CaMR E2A in some cells selected as GFP2 after the infections with virus expressing CaMR E2A. Nevertheless, the p values for the difference in the double-expression of the two Igh alleles between the GFP+ cells of the infections with virus expressing WT and CaMR E2A were 0.0003 and 0.00002, respectively, in the two experiments (Fig. 1B), and the p values for the differences in double expression between cells expressing the CaMR E2A and the two GFP2 controls were even lower. The increased frequency of apparent double expression of the Igh alleles in the mutant implies that CaMR E2A either dramati- cally increases allelic inclusion of IgH or, much less likely, through an unknown mechanism radically increases the background of cells that appear double positive in the FACS analysis but are not true double expressors. To discriminate between the alternatives, sorted apparently double-positive cells were cultivated for 3 d and rean- alyzed. Most apparently double-positive cells in FACS analysis are background, because they do not show double expression in the second FACS analysis (40), and this also was the case with the B cells infected with virus expressing WT E2A (Fig. 1C, 1D). Im- portantly, most IgH double-positive cells with CaMR E2A in sharp Downloaded from contrast showed double expression also in the second FACS anal- ysis of IgH expression (Fig. 1C, 1D). Furthermore, we also analyzed the sorted double-positive cells, cultivated for 3 d, that remained apparently double positive in the second FACS analysis, by intra- cellular immunostaining after the second sorting. Most apparently double-positive cells after this second sorting of WT E2A–infected http://www.jimmunol.org/ cells were background because they were not double stained, whereas most resorted double-positive cells with CaMR E2A instead showed both IgMA and IgMB immunostaining (Supplemental Fig. 1B). Thus, the mutation of E2A increased true double expression and not the background. To further discriminate between the two alternatives, VDJ recombination of the Igh locus of sorted GFP+ (i.e., infected) apparently double-positive cells, selected as shown in Fig. 1A and Supplemental Fig. 1D, was analyzed by single-cell by guest on October 2, 2021 FIGURE 1. CaMR E2A leads to allelic inclusion of IgH. B6 male mice PCR. Approximately two thirds of cells with both alleles detected heterozygous for E2A knockout (E2A+/2) were crossed with BALB/c that were infected with virus expressing WT E2A, and the control females, and total bone marrow was isolated from resulting E2A+/2 F1 mice cells, had only one VDJ recombined allele, whereas the other allele and infected with retrovirus coding for either WT or CaMR mutant E12 was DJ recombined (Fig. 1E, Supplemental Fig. 1C). In contrast, splice form of E2A, together with GFP, which resulted in 5–10% of infected 75% of the cells expressing CaMR E2A had both alleles VDJ re- GFP-expressing cells (data not shown). Twenty-four hours postinfection, the combined (Fig. 1E, Supplemental Fig. 1C). The p values for this bone marrow cells were supplemented with fresh total bone marrow from 2 difference between the mutant and the WTs were #0.00016. Thus, B6 3 BALB/c F1 E2A+/ mice, and each cell mixture was transplanted into 3 +/2 A CaM resistance of E2A dramatically increases allelic inclusion of IgH. a lethally irradiated B6 BALB/c F1 E2A mouse. ( ) Recipient spleen R B cells were analyzed 2 wk posttransplantation for BALB/c and B6 allotypes of Next, we analyzed whether CaM E2A increases allelic inclu- IgH to study the effect of expression of WT and CaMR mutant E12 on sion only when overexpressed or whether it also inhibits the allelic +/2 allelic inclusion of IgH. Typical FACS plots of the GFP2 controls (upper exclusion at normal expression levels. E2A mice have 50% panels) and the GFP-expressing retrovirus-infected cells (GFP+, lower panels) reduced expression of E2A (22). FACS analysis showed increased of live-gated recipient mouse splenic CD19+ B cells. (B) Quantification of E2A expression level with increased GFP fluorescence level (Sup- experiments as in (A) from five mice (Experiment 2) and as in Supple- plemental Fig. 1F). Interestingly, significantly increased allelic inclu- mental Fig. 1A from three mice (Experiment 1). Results are mean 6 SD. sion was seen in the CaMR mutant over a broad range of expression C A+ B+ A ( ) Apparently IgM IgM double-positive cells shown in ( )weresorted levels, and the peak level of allelic inclusion in the mutant was m + to culture and activated with LPS (25 g/ml) for 3 d. Surviving GFP cells around the normal WT level of E2A (Supplemental Fig. 1E, 1F). Thus, were reanalyzed for IgMA and IgMB expression, revealing a much higher CaMR E2A strongly increases allelic inclusion of IgH at normal percentage (indicated in the upper right quadrant) of double-positive cells in the CaMR E2A–infected samples. (D) Quantification of experiments, as E2A expression levels. We also noted that strong overexpression R in (C). Results are mean 6 SD (n = 3). (E) Single-cell PCR analysis of Igh of either WT or CaM E2A distorted the control of VDJ recom- A rearrangements in apparent IgMA+ IgMB+ double-positive cells in (A) and bination, leading to a high level of cells not expressing IgM or Experiment 2 of (B) summarized from Supplemental Fig. 1C. GFP+ IgMA+ IgMB (Supplemental Fig. 1F), which further supports that E2A has IgMB+ single B cells were sorted directly into PCR plates and analyzed for a critical role in the control of VDJ recombination. These strongly VHDJH rearrangements by two-round semi-nested PCR. WT splenic B GFP-fluorescent and heavily E2A-overexpressing cells were ex- cells of B6 3 BALB/c F1 mice also were analyzed as controls. cluded from the analyses in Fig. 1 and Supplemental Fig. 1B, 1C (as shown in Supplemental Fig. 1D). These cells were present among cells infected with virus expressing WT E2A and among 2 wk posttransplantation (Supplemental Fig. 1D–F) but were al- GFP2 cells from infections with virus expressing CaMR E2A most completely absent 1 wk later (Fig. 1B, left panel, Supplemen- (Fig. 1A, 1B, Supplemental Fig. 1A). These slight increases might tal Fig. 1A), indicating that cells heavily overexpressing E2A are be due to a slightly less efficient inhibition of E2A in some cells rapidly selected away. The Journal of Immunology 5
Inhibition of expression of components of the recombination Furthermore, none of the Id mRNA or protein levels shows an machinery upon pre-BCR activation dependent on CaM increase after pre-BCR stimulation either in primary pre-B cells or sensitivity of E2A Nalm-6 cells (25, 41) that can account for the relatively fast reduc- tions in the mRNAs and proteins in this study. A possible reason for the defect in allelic exclusion when E2A is 2+ CaMR is that sensitivity of E2A to CaM is needed for the signal To examine whether Ca signaling from the pre-BCR inhibited from the pre-BCR that arrests further recombination. To test this the expression of recombination components through CaM in- hypothesis, we characterized the effects of pre-BCR signaling on hibition of E2A, we first used an EBV-based shuttle vector that the expression of proteins relevant for the recombination. We used stably directs synthesis of an shRNA interfering with human E2A mouse bone marrow–derived pre-B cells grown on limiting numbers mRNA. This shRNA plasmid reduced the E2A mRNA and pro- ∼ of stromal cells. We showed previously that the pre-BCR is not tein levels in Nalm-6 cells by 85%, as previously reported (25). signaling constitutively at these conditions and is inducible upon The shRNA expression plasmid reduced the expression of RAG1, RAG2, and TdT mRNAs by 70–80% (Fig. 2D), which confirmed activation by cross-linking with Ab against the m H chain (25, 41). that the expression of these genes is E2A dependent. These re- The pre-BCR stimulation efficiently induced increased intracel- ductions were specific, because the shRNA expression plasmid did lular Ca2+ concentration within ,1 min (Supplemental Fig. 2), and not affect the expression of the pre-BCR–regulated genes FOS, several of the effects of pre-BCR stimulation on gene expression OCT2, and IL6 (Fig. 2D). Importantly, stimulation of the pre-BCR are through Ca2+ signaling (25, 41). Quantitative RT-PCR showed did not inhibit RAG1, RAG2, or TdT expression further in the that the pre-BCR stimulation reduced the mRNA levels of RAG1, presence of the shRNA against E2A (Fig. 2D), indicating that the RAG2, and TdT, which adds nucleotides at the RAG-generated inhibitions by pre-BCR engagement were entirely dependent on DNA ends, as well as those of the PAX5 and ETS1 transcription Downloaded from inhibition of E2A. The reduction in human E2A mRNA could be factors, between 2- and 4-fold within 2 h (Fig. 2A). The levels of complemented by cotransfection with the expression plasmid for the proteins also were reduced in most cases, but with a delay. the E12 isoform of mouse E2A with which the shRNA does not These reductions were $2-fold within 5–8 h (Fig. 2A, Supplemental interfere. This complementation fully restored the expression levels Fig. 3A). However, the level of E2A mRNA was reduced to a of RAG1, RAG2, and TdT mRNA and their sensitivity to pre-BCR much lesser extent, and the E2A protein level was not reduced by stimulation (Fig. 2D). Importantly, anti-m had no effect on any of the pre-BCR stimulation (Fig. 2A, Supplemental Fig. 3A). To study http://www.jimmunol.org/ these genes when complementation was performed instead with whether Ca2+ signaling is important for the identified downregu- the CaMR mutant of E12 (Fig. 2D). This loss of effect of pre-BCR lation, we inhibited all Ca2+ signaling with the Ca2+ chelator BAPTA- engagement was specific, because the mutation had no effect on AM and blocked IP receptor Ca2+ channels with TMB-8 and/or 3 the inhibition of the control genes (Fig. 2D). Thus, CaM resistance blocked L-type Ca2+ channels with nifedipine. For RAG1, RAG2, of E2A specifically causes the expression of the genes for the TdT, PAX5, and ETS1, the reduction in mRNA level after pre- components of the recombination process to be unaffected by pre- BCR stimulation was partially or completely blocked by TMB-8 BCR stimulation. To further examine the importance of CaM in- or nifedipine, and it was completely blocked by the Ca2+ chelator hibition of E2A, we infected primary pre-B cells with retroviruses or by combining the channel blockers (Fig. 2B). Thus, the regu- R
encoding WT or CaM E12, together with GFP. It was not pos- by guest on October 2, 2021 lation of these proteins by the pre-BCR is dependent on Ca2+ 2+ sible to study the effects of overexpression of CaM, because no signaling. The Ca -dependent downregulation of RAG1, RAG2, infected mouse pre-B cells with substantial overexpression of this and TdT is not species specific, because we obtained similar results protein were obtained (data not shown), presumably because CaM using the human pre-B cell line Nalm-6 (Supplemental Fig. 3B, 3C). overexpression affects viability and/or differentiation of pre-B cells. Inhibitors of other signaling pathways from AgRs (PD98059, Infection with both WT and mutant E12 resulted in, on average, MEK1/2 inhibitor; bisindolylmaleimide, PKC inhibitor) showed that 2+ only a slight increase in total E2A protein compared with vector- Ca plays a dominant, but not exclusive, role in pre-BCR regulation infected cells (detected by FACS with E2A Ab) in this system, as of these genes. PAX5 and ETS1 mRNAs could not be analyzed in previously reported (25). Stimulation of the pre-BCR of the pri- this way, because their levels remained unchanged upon pre-BCR mary pre-B cells infected with retrovirus expressing WT E12 stimulation as a result of unknown causes, perhaps coupled to the or with vector control for 7 h resulted in significant (p # 0.01) fact that Nalm-6 is a tumor cell line. ∼2-fold inhibition of RAG1, RAG2, TdT, and PAX5 protein expres- We determined whether activation of the pre-BCR affected the sion (Fig. 2E). Importantly, in cells infected with virus expressing amount of protein that could bind to E2A binding sites in the RAG1, CaMR E12, the protein levels became completely resistant to pre- RAG2,andTDT promoters, as well as the RAG enhancer Erag,by 2+ BCR stimulation for the RAGs and partially resistant for TdT and EMSA of nuclear extracts in the absence of Ca . No reduction in PAX5 (Fig. 2E). Thus, CaMR E12 renders the expression of the the amount of any of the E2A–DNA complexes was seen during 5 h genes for components of the recombination machinery resistant to m of anti- treatment (Fig. 2C). This was expected, because pre-BCR pre-BCR stimulation, implying that the inhibition of their expres- stimulation did not have much effect on the expression of E2A sion is through CaM inhibition of E2A. mRNA or protein in primary pre-B cells (Fig. 2A) or in Nalm-6 cells (25) (data not shown). The shifted bands were identified as Inducible in vivo proximities of the recombination-coupled E2A–DNA complexes using neutralizing Abs (Supplemental Fig. components E2A, RAG1, RAG2, and PAX5 with CaM and loss 3D). The binding of E2A to the RAG1, RAG2, TDT,andErag of proximities with H3K4me3 upon pre-BCR stimulation probes, but not binding of other transcription factors to a control The downregulation of recombination-coupled components was rel- probe, was much reduced under conditions that enable Ca2+ loading atively modest and took many hours, which suggested the presence of of CaM (Fig. 2C). Id proteins are inhibitors of DNA binding of E2A another effect of pre-BCR stimulation to achieve efficient arrest of and other E-proteins. However, induction of Id protein did not Igh recombination. To further analyze the regulation of the recom- contribute to the rapid inhibition of recombination-coupled genes bination, we performed PLAs, which enable visualization and quan- and proteins upon pre-BCR stimulation, because the number of tification of protein interactions. Primary mouse pre-B cells were stained E2A–DNA complex with either of the RAG/TdT sites was not re- with Abs against different combinations of two target proteins and duced upon pre-BCR stimulation in the absence of Ca2+ (Fig. 2C). subsequently with secondary Abs conjugated with oligonucleotides 6 ALLELIC EXCLUSION BY INHIBITION OF E2A
(PLA probes) to generate a strong red fluorescence dot when the two PLA probe–marked proteins are in close proximity (#40 nm) (Fig. 3A). PLA is a binary assay in which the number of dots is pro- portional to the number of proximities in the cell (32–35). We obtained proximity for all pairs of CaM and one of the recombination-coupled proteins analyzed: E2A, RAG1, RAG2, and PAX5. Anti-m treat- ment for 30 min increased the number of proximities detected by $2-fold, and the increases remained for 5 h (Fig. 3, Supplemental Fig. 4A, 4B). Instead, the pre-BCR stimulation reduced proximities that were found between H3K4me3, which is a binding site for RAG2, and E2A, RAG1, RAG2, and PAX5 $3-fold (Fig. 3, Supplemental Fig. 4A, 4B). The background was minimal in control cells lacking the probe-marked protein, as well as when one or both of the pri- mary Abs were omitted (Supplemental Fig. 4). The pre-BCR effect on each proximity was analyzed with similar results using two or more different Ab pairs (distinguished by superscript numbers on the Abs in Figs. 3, 4, Supplemental Fig. 4). The results show that, upon pre-BCR stimulation, all of the analyzed recombination-coupled pro- teins quickly leave the active chromatin that is targeted by RAG2. The findings indicate that the recombination-coupled proteins are in Downloaded from a complex, so we also studied the proximities between these proteins. We found proximities between each pair of PAX5, RAG1, RAG2, and E2A. Importantly, these proximities remained after stimulation of the pre-BCR receptor (Fig. 3B, Supplemental Fig. 4A–C). The small reductions in some of the proximities after 5 h are not more than expected, considering the reductions in the levels of most of the http://www.jimmunol.org/ proteins (Fig. 2A). Taken together, the data suggest the existence of a recombination complex consisting of at least E2A, RAG1, RAG2, and PAX5 that remains intact when CaM binds to the complex after pre-BCR stimulation. This suggested complex is on H3K4me3- modified chromatin before pre-BCR stimulation, but it leaves this modified chromatin upon the stimulation that activates CaM to join the complex. Some of the pairs of H3K4me3 and/or a RAG protein yielded more than two specific PLA dots in some of the cells (Fig. by guest on October 2, 2021 3A, Supplemental Fig. 4A–C). This finding that the number of specific PLA dots can exceed the number of Igh alleles implies that some of the detected proximities occur at other RAG-binding H3K4me3 mod- ification sites than at the Igh locus. Therefore, we analyzed inter- FIGURE 2. Downregulation of key components of the recombination actions of E2A, PAX5, and RAG with the Igh locus (see below). process after pre-BCR stimulation dependent on CaM sensitivity of E2A. (A) Levels of RAG1, RAG2, TdT, PAX5, E2A, and ETS1 mRNA and protein In vivo regulation of the proximities of E2A, PAX5, and the after stimulation of the pre-BCR of primary bone marrow–derived mouse RAG proteins through CaM inhibition of E2A m pre-B cells by anti- were determined by quantitative real-time RT-PCR and To further study the regulation of the suggested recombination Western blotting, respectively. Levels before addition of anti-m were set as complex, we performed PLA of primary pre-B cells infected with 100%. Results are mean 6 SD (n $ 3). With the exception of E2A, the reductions were significant for all mRNA after 1 h of anti-m (p , 0.012) and for all proteins after 5 h of anti-m (p , 0.047). (B) Calcium signaling–de- pendent downregulation of the indicated genes after pre-BCR activation. Anti-m was added to one half of BAPTA-AM–treated (15 mM), TMB-8– mRNA. Nalm-6 cells were transfected with pMEP4 vector expressing treated (50 mM), nifedipine-treated (50 mM), or TMB-8 plus nifedipine– shRNA against human E2A mRNA or the empty shuttle vector or with the treated or untreated primary pre-B cells for 3 h, followed by quantitative shRNA expression vector plus the corresponding pMEP4 vector express- real-time RT-PCR. Values represent normalized mRNA levels with anti-m as ing WT or CaMR mutant of mouse E12, all together with CMV-EBNA the percentage of the levels without anti-m. Results are mean 6 SD (n =3). expression plasmid. Where indicated (shaded bars), cells were treated with (C) No change in DNA binding activity of E2A in the absence of Ca2+ after anti-m for 5 h before the harvest. Levels of mRNA were determined by pre-BCR activation with anti-m (left panel) and inhibition of formation of quantitative real-time RT-PCR. Results are mean 6 SD (n = 3). (E) Loss of the E2A–DNA complex in the presence of Ca2+ (right panel). Amounts of anti-m sensitivity of RAG1, RAG2, TdT, and PAX5 expression by ex- E2A–DNA complexes (indicated by arrow) using nuclear extracts (NE) from pression of CaMR E12. Changes are shown in the indicated protein levels the Nalm-6 human pre-B cell line and probes containing E2A-binding sequences upon pre-BCR stimulation with anti-m for 7 h in mouse primary pre-B from the human RAG1 promoter, RAG2 promoter, the RAG enhancer Erag, cells grown with IL-7 on S17 stromal cells and infected with retrovirus the TdT promoter, and a control probe from the human GM-CSF promoter encoding WT E12 or CaMR mutant of E12, together with GFP. Proteins (25) (nucleotides 262 to 225), which binds many transcription factors were quantified by intracellular immunostaining and flow cytometry. Cells (including AML1/RUNX, ETS, NFAT, and JUN/FOS proteins), were ana- with a large increase in GFP fluorescence were considered retrovirus lyzed by EMSA in the presence of the chelator EDTA (0.5 mM) or Ca2+ (0.5 infected and were used to calculate average protein levels. The expression mM CaCl2). (D) Effects of a shuttle vector that expresses shRNA targeting levels are calculated as the percentage of the level of the corresponding human E2A mRNA and of complementation by expression of WT or mutant cells not treated with anti-m. Data are mean 6 SD (n =4).*p , 0.05, mouse E12 on the expression of RAG1, RAG2, TdT, Fos, Oct-2, and IL-6 **p , 0.01. The Journal of Immunology 7 Downloaded from
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FIGURE 4. CaM E12 blocks pre-BCR stimulation–induced changes of http://www.jimmunol.org/ in vivo proximity of E2A, PAX5, and RAG proteins with CaM and H3K4me3, and E12 interacts directly with PAX5. (A) Mouse pre-B cells were infected with retrovirus expressing WT or CaMR mutant of E12, as in Fig. 2E. Infected cells were identified by their GFP fluorescence in microscopy and used to quantify the numbers of proximities (dots)/cell. The Abs were used at dilutions that yielded a low number of dots/cell to have a low risk for missing dots in the quantifications. The number of dots/cell in pre-B cell samples before stimulation of the pre-BCR were for all pairs—E2A-CaM (4.4 dots/cell for WT and 9.5 dots/cell for CaMR), RAG1-CaM (4.0 dots/cell for WT E2A and 11.0 dots/cell for CaMR E2A), RAG2-CaM (8.7 dots/cell for WT E2A and by guest on October 2, 2021 20.2 dots/cell for CaMR E2A), PAX5-CaM (11.1 dots/cell for WT E2A and 24.9 dots/cell for CaMR E2A)—set as 100%. The results are mean 6 SD FIGURE 3. Inducible in vivo proximities of the recombination-cou- (n =3).(B) E12 binds directly to PAX5. His6-tagged full-length PAX5 pled components E2A, RAG1, RAG2, and PAX5 with CaM and rapid was produced in E. coli, and the purified PAX5 was immobilized on reduction of the proximities with H3K4me3 upon pre-BCR stimulation. cyanogen bromide–activated Sepharose 4B. Binding to the PAX5- A ( ) The pre-BCR of mouse primary pre-B cells grown with IL-7 on S17 Sepharose is shown for proteins from whole-cell extracts of mouse B cells m stromal cells was stimulated with anti- for the indicated times. The (upper panels) and for purified proteins (middle and lower panels). The detection of the proximal location of each protein pair analyzed (visu- whole-cell extract from 1.5 million cells or 0.2 mg of purified His6-tagged 3 R alized as red dots) was done by in situ PLA (original magnification 60). WT or CaM E12 (aa 431–652) or ETS1 from expression in E. coli was B The controls are shown in Supplemental Fig. 4. ( ) Summary of PLA allowed to interact with 10 ml of PAX5-Sepharose for 30 min. The results on regulation of proximities of the recombination-coupled com- incubations of whole-cell extract were in the presence of 0.1 mM EGTA, ponents E2A, RAG1, RAG2, and PAX5 with CaM, with H3K4me3, and and those of purified proteins were in the presence of either 0.1 mM with each other from (A) and Supplemental Fig. 4A–C. Different Abs EGTA or 0.1 mM CaCl2. After washing, the bound proteins were detected against the same protein are distinguished by superscript numbers. by Western blot against the protein or, in the case of E. coli–produced E12 proteins, against the His -tags. Preloads were 10% of the amount of protein R 6 retrovirus expressing either WT or CaM E12, together with GFP. extract, E12, or ETS1 added to the binding reactions. Negative controls As for noninfected cells, the proximity between CaM and each were with empty Sepharose (Seph.) and Protein-A Sepharose (Prot.A-Seph.) of E2A, RAG1, RAG2, and PAX5 was induced upon pre-BCR and without addition of protein or with addition of ETS1 instead of E12 to stimulation for 30 min in the cells infected with virus express- PAX5-Sepharose (PAX5-Seph.). Quantification of the E12 bands from three ing WT E12 (Fig. 4A). Importantly, all of these inductions were to nine experiments were plotted as the percentage of E12 bound to PAX5 completely lost in cells infected with virus expressing the CaMR (bottom panel). Results are mean 6 SD. The multiple bands in the panel E12 mutant (Fig. 4A). Proximity between H3K4me3 and each of with E2A from cell extract are due to alternative splicing and multiple E. coli E2A, RAG1, RAG2, and PAX5 was inhibited upon pre-BCR phosphorylation sites in E2A, and the lower band in the panels with – produced E12 is due to partial protease cleavage N-terminal to the bHLH stimulation for 30 min in the cells infected with virus express- domain during E. coli production. *p , 0.05, **p , 0.01, ***p , 0.001. ing WT E12 (Fig. 4A), as shown for noninfected cells. The smaller reductions in the infected cells compared with noninfected ones might reflect that the higher level of E2A, and, thereby, less with virus expressing CaMR E12 (Fig. 4A). These data support CaM relative to E2A, could reduce the efficiency of the inhibi- that CaM binds to E2A in a complex of E2A, RAG1, RAG2, and tion. In contrast to cells infected to express WT E2A, no reduction PAX5 after pre-BCR stimulation, thus inhibiting the DNA binding in the proximity between H3K4me3 and E2A, RAG1, RAG2, or of E2A and, thereby, making the recombination complex leave the PAX5 was seen after pre-BCR stimulation in the cells infected H3K4me3-modified chromatin. 8 ALLELIC EXCLUSION BY INHIBITION OF E2A
The interactions in vivo between the RAGs, PAX5, E2A, and RAG1 and RAG2 binding sites (Fig. 5C). Taken together, these data CaM could all be direct; alternatively, one or more interactions strongly support the notion that E12, RAG2, and PAX5 interact could be indirect through another protein in the suggested complex. in vivo in a complex on the Igh locus through the binding sites of Zhang et al. (14) reported a direct interaction between the RAG1/ these proteins, and we infer that RAG1 is also part of this protein RAG2 complex and PAX5, and we (19–21) reported and charac- complex. The fraction of anti-Flag immunoprecipitated DNA varied terized the direct interaction between E2A and CaM. However, no for the analyzed probe sites for the different Flag-labeled proteins interaction between PAX5 and E2A has been reported. To analyze (Fig. 5). This probably occurs, at least in part, because the detection whether PAX5 and E2A can interact directly with each other, we of the interaction of the suggested complex with the chromatinized recombinantly expressed full-length PAX5, the E12 splice form of Igh DNA varies because it can bind with any of its DNA-binding E2A (C-terminal 222 aa), and ETS1 in E. coli, purified the pro- domains and the H3K4me3-binding domain of RAG2. The levels of teins, and determined the ability of E12 and the control protein to cross-linking in ChIP depend on the specific amino acid side chain bind to PAX5. We also analyzed the binding of E2A and other that is located at different points of close contact of the protein with proteins from a mouse B cell extract to the PAX5 Sepharose. E2A a reactive group at the base at the DNA. Thus, the efficiency of ChIP from the whole-cell extract showed very efficient binding to the can change when either the nucleotide sequence at the site and/or the PAX5 Sepharose, which was in sharp contrast to the poor binding protein(s) binding there is changed or has a changed conformation. of b-actin, a-tubulin, and CD19 control proteins (Fig. 4B). The Furthermore, in this case, the accessibility for the Ab, and, thereby, E. coli–produced E12 bound to PAX5 Sepharose beads but not to the efficiency of the ChIP, might be different when the Tag is on control Sepharose beads with Protein A or without coupled protein, different proteins in the suggested complex. In addition, all protein and E12 bound PAX5 Sepharose much more than did ETS1 control protein (Fig. 4B). The binding of E12 to PAX5 was equally effi- Downloaded from cient in the presence of Ca2+ and when Ca2+ was absent, because of the presence of the Ca2+ chelator EGTA, and the CaMR mutation of E12 did not affect the efficiency of the binding to PAX5 (Fig. 4B). In summary, the RAG1/RAG2 complex can interact directly with PAX5, PAX5 can interact directly with E2A, and E2A can interact directly with CaM. http://www.jimmunol.org/
Binding of E12, RAG, and PAX5 at the Igh locus is sensitive to CaM inhibition of E2A We used retrovirus-infected primary pre-B cells and performed ChIP to analyze the effects of CaM resistance of E12 on the lo- calization of E12, RAG, and PAX5 in the Igh locus before and after pre-BCR stimulation. We added a C-terminal sequence encoding a tandem Flag-epitope to the full-length E12 (E12-Flag) in the by guest on October 2, 2021 retrovirus. Lysates of infected pre-B cells were subjected to ChIP against the Flag-epitope. The recovered DNA was quantified by PCR using primer pairs amplifying regions of the Igh locus con- taining binding sites for E2A (E-box sequences), RAG1/RAG2 (RSS), RAG2 (H3K4me3-rich sequences), and PAX5. E2A has a strong preference for E-boxes with the sequence CAGCTG, but there are also preferences for the surrounding nucleotides, and there are only a few E2A sites/million bp in the genome (42). As expected, we observed ChIP signals for the E-box–containing Igh enhancers Em and hs3b (36) in cells expressing WT E12-Flag over the background of cells infected with empty vector (Fig. 5A). Importantly, anti-Flag signals for E12-Flag also were above the vector background for most amplicons containing RAG1, RAG2, FIGURE 5. ChIP of E2A, PAX5, and RAG proteins at the Igh locus. or PAX5 binding sites but not for control sequences having Mouse pre-B cells were infected with empty retrovirus (vector) or viruses expressing E12-Flag (A), E12 plus RAG2-Flag (B), or E12 plus PAX5-Flag acetylated (i.e., active) chromatin without a binding site for E2A, (C) on the same MSCV-IRES virus and subjected to ChIP using the Flag- RAG, or PAX5 (Fig. 5A). Thus, E2A binds not only to E2A sites but epitope of the tagged protein. The recovered DNA was analyzed by quan- also to many RAG and PAX5 sites. Next, we added a C-terminal titative real-time PCR using primer pairs amplifying regions of the Igh locus tandem Flag-epitope to RAG2 andPAX5inretroviruses.E12 containing binding sites for E2A (E-box sequences), RAG1/RAG2 (RSS), remained without the Flag-tag, thereby allowing expression of both RAG2 (H3K4me3-rich sequences), and PAX5 (PAX5-consensus sequences) E12 and either flagged RAG2 or flagged PAX5 from the same virus. or acetylated active chromatin. The E2A and PAX5 sites were confirmed by We did not construct the corresponding retrovirus for RAG1 be- ChIP (8, 14, 36). The amplified VHA.1, VH10, and VhJ558.47(2) segments cause of the large size of its cDNA. As expected, cells infected with contain the RSS where V and DJ segments are known to join after cleavage virus expressing RAG2-Flag plus E12 gave anti-Flag signals over by bound RAG1/RAG2 and, thus, are known RAG binding sites, and RAG the background of empty virus in the ChIP for most of the RAG1 binding to the DQ52 and JH1–JH4 sites was verified by ChIP (38). The identical amount of DNA, in mass, was used in the analysis of all samples and RAG2 binding sites (Fig. 5B). Importantly, signals also were from the same mouse, all samples were treated identically, and all quantifi- strong for the E-box–containing hs3b, as well as three of five PAX5 cations were from the early log phase of the PCR. Three mice were analyzed, binding sites (Fig. 5B). Finally, cells infected with PAX5-Flag plus and all PCR samples from these three mice were measured in triplicate. The E12 gave anti-Flag signals much over the vector control for all five figure shows the amount of PCR product obtained for each site after immu- PAX5 binding sites (Fig. 5C). Importantly, specific signals also were noprecipitation with anti-Flag (expressed as & of the amount of PCR product obtained for the E-box–containing Em and hs3b, as well as all of the before immunoprecipitation). All data are mean 6 SD. The Journal of Immunology 9 contacts in a complex will not be cross-linked when precipitating sites), and PAX5 binding sites (Fig. 6). These reductions were a large complex of proteins on DNA; therefore, differential stability statistically significant (p , 0.035 in all cases) and remained of noncross-linked protein interactions in the complex, in vivo or approximately equally large after 2 h. Thus, pre-BCR stimulation during the ChIP procedure, also could affect the strength of the ChIP rapidly reduces the binding of E2A to the Igh locus both on E2A signals. Nevertheless, it is notable that Em did not precipitate binding sites and on RAG and PAX5 sites where the localization significantly more than the vector control in the RAG-Flag ChIP of E2A must be indirect. Importantly, the reductions in anti-Flag (Fig. 5B). This could be a result of the technical reasons described ChIP signals were abolished both on the E2A binding sites and on above that the combination of Flag on RAG and the E-box site of sites for RAG1, RAG2, and PAX5 by the expression of CaMR Em gives weak signals for the complex. However, it is also possible E12-Flag. All of the differences between WT and mutant E12 that there is less RAG protein at this site compared with sites where were statistically significant after 30 min of pre-BCR stimulation RAG-Flag gives significant ChIP signals. Our data suggesting that (Fig. 6). These results suggest that the recombination complex E2A, PAX5, and RAG are together in a complex at many sites on the leaves the Igh locus upon pre-BCR stimulation through CaM inhi- Igh locus does not necessarily imply that all of these proteins are bition of E2A. All of the reductions in ChIP signal after pre-BCR present in stoichiometric amounts at all of the sites. In that case, it stimulation when expressing E12 plus RAG2-Flag were 2–10-fold would make sense if less RAG is located at Em, or if the binding of and were highly statistically significant (after 30 min all p , 0.025; RAG is less stable during the ChIP procedure at Em,comparedwith after2hallp # 0.014), with the exception of the hs3b site where DNA segments that have RAG binding sites (RSS and/or H3K4me3) no significant reduction was obtained (Fig. 6). A possible reason for and are RAG cleavage sites. this exception is that other proteins binding together with E2A at To analyze the effects of pre-BCR stimulation and the role of this DNase-hypersensitive site might retain RAG2 there also when CaM inhibition of E2A, we infected the pre-B cells with retrovirus inhibition by CaM reduces the DNA binding of E2A. Importantly, Downloaded from expressing either WT or CaMR E12 and performed anti-Flag ChIP all reductions in ChIP signals were completely or almost completely before and after pre-BCR stimulation. We analyzed those Igh abolished by expression of CaMR E2A (Fig. 6). The difference DNA sequences that resulted in signals clearly over background in between expression of WT and mutant E12 was significant (p # Fig. 5. In cells infected with the retrovirus expressing WT E12- 0.046) on all sites, with the exception of hs3b, after 30 min of pre- Flag, 30 min of pre-BCR stimulation reduced the ChIP signal with BCR stimulation (Fig. 6). ChIP analyses postinfection with virus anti-Flag between 2- and 4-fold for all E2A binding sites, RSS expressing E12 plus PAX5-Flag resulted, similarly to E2A-Flag http://www.jimmunol.org/ sites (RAG1/RAG2 binding sites), H3K4me3 sites (RAG2 binding and RAG1-Flag, in $2-fold reductions for the WT E12 that were by guest on October 2, 2021
FIGURE 6. The binding of E2A, PAX5, and RAG proteins to the Igh locus is sensitive to CaM inhibition of E2A. Mouse pre-B cells were infected with retrovirus constructs, as in Fig. 5, using either WT or CaMR E12. The pre-BCR of infected cells was stimulated with anti-m for the indicated times and then subjected to ChIP using the Flag-epitope of the tagged protein and analyzed by quantitative real-time PCR, as in Fig. 5. The figure shows the amount of PCR product obtained for each site after immunoprecipitation with anti-Flag compared with the amount of PCR product before immunoprecipitation. These values for anti-Flag–immunoprecipitated DNA are expressed relative to the immunoprecipitated DNA from the corresponding unstimulated cells expressing either WT or CaMR E12 that were set as 100% (bars not shown). All data are mean 6 SD (n = 3). *p , 0.05, **p , 0.01, ***p , 0.001. 10 ALLELIC EXCLUSION BY INHIBITION OF E2A statistically significant (all p # 0.024 after 30 min; all p # 0.038 ductions in PAX5, RAG1, and RAG2 were ∼2-fold, and the re- after 2 h). Importantly, the reductions in ChIP signals were com- ductions in their pair-wise proximities with each other and with pletely abolished by coexpression of the CaMR E2A with PAX5- E2A also were relatively modest. Nevertheless, the identified Flag (Fig. 6). The differences between WT and mutant E12 were downregulation of recombination-coupled components are likely statistically significant (p values # 0.042after30minofpre-BCR to contribute to the allelic exclusion. The findings support that stimulation), with the exception of JH2(p = 0.054) and VHJ558 inhibition of binding of the complex to the key sites on Igh has (p = 0.074). The loss of inhibition after pre-BCR stimulation by the a dramatic inhibitory effect on further recombination. The pre- expression of CaMR E2A remained after 2 h (Fig. 6), including on BCR–induced reductions in binding of proteins in the complex to the JH2andVHJ558 sites, which supports the results after 30 min. individual key sites in the Igh locus were 2–10-fold in ChIP an- The reductions with CaM-sensitive E2A within 30 min, which is alyses (Fig. 6). However, reductions in binding of the complex to much before any significant changes in levels for proteins strongly important sites upon pre-BCR stimulation could be even larger for affected by AgR stimulation (41, 43), argue against the alternative several reasons: 1) overexpression of E2A in a part of the infected that the reductions are indirect through changed expression of an- cells could make inhibition of E2A by CaM appear less efficient, other unknown protein. Therefore, ChIP data strongly support the as indicated by the smaller effect of pre-BCR stimulation in in- notion that E12, RAG2, and PAX5 interact in vivo in a complex on fected cells than in noninfected cells in PLA (Figs. 3, 4, Sup- the Igh locus. The coimmunoprecipitation of the binding site of one plemental Fig. 4A–C); 2) reductions in the joining of the many protein with Ab against the Flag on the other protein implies binding sites relevant for recombination through the complex a physical interaction between the Flagged protein and the protein could be much larger than reductions in protein binding to indi- that binds to the site. Furthermore, mutation of E12 to CaM re- vidual sites; and 3) the total number of PAX5, E2A, RAG1, and sistance affects the level of this coimmunoprecipitation, even RAG2 sites in the locus is very high, and reductions in binding Downloaded from when RAG2 binding sites are analyzed with Flagged PAX5 and that are as large, or even larger, than those detected in this study when PAX5 binding sites are analyzed with the Flag on RAG2, might occur at some of the other sites. which strongly suggests that all three proteins are together in Li et al. (45) reported a cyclin-dependent kinase site in RAG2 a complex on the Igh locus. Therefore, the results further support and that accumulation of RAG2 is regulated during the cell cycle, that the effect of CaM is through E2A that is together with PAX5 which raises the question of whether increased degradation of andRAG2atPAX5,RAG,andE2AsitesoftheIgh locus. In RAG2 could be relevant for the reduction in RAG2 after pre-BCR http://www.jimmunol.org/ summary, ChIP analyses show that WT E12 allows the recombi- stimulation. However, the downregulation of proteins that we found nation complex to leave E2A, RAG, and PAX5 binding sites in the was not limited to RAG2 and was preceded by reductions in the Igh locus upon pre-BCR stimulation, whereas CaMR E12 sequesters mRNAs that occurred much more rapidly and were of approximately the complex on the Igh DNA. the same magnitude. Thus, the reductions are through reductions in the mRNA and are not through induction of degradation of the Discussion proteins. The reductions in the mRNAs after pre-BCR stimulation Feedback inhibition from AgRs has a well-established major role also occur too rapidly to be through a shift in the cell cycle from in allelic exclusion, but the molecular mechanism has remained G1 to S and G2/M, where degradation of RAG2 was reported to by guest on October 2, 2021 largely enigmatic (15–17). In this study, we show that CaM resis- occur. Li et al. (45) showed that coupling of V(D)J recombination tance of E2A dramatically increases the normally very low fre- to the cell cycle is not essential for enforcement of allelic exclu- quency of double expression of the Igh alleles at the cell surface to sion. However, our data showing that CaM sensitivity of E2A is 5–7%. This is close to the theoretical maximum in the absence of essential for allelic exclusion does not exclude the participation of feedback inhibition that was estimated at ∼6.4%, because most other mechanisms. After the Ca2+ signal from the pre-BCR has VDJ recombinations do not lead to a functional IgH (15–17). At terminated the VDJ recombination of Igh, something also enables the genomic level, CaM resistance of E2A resulted in a remarkable initiation of the recombination of Igk or Igl. It should be noted ∼7-fold increase in the ratio between B cells VDJ recombined on that a Ca2+ signal is time limited, which is suitable for an inactivation both alleles and B cells that had one allele VDJ recombined and the that should be temporary. Because E2A also is needed in recombi- other allele only DJ recombined. Thus, the feedback inhibition is al- nation of L chains (3, 5, 6), it is likely that the retargeting to a L chain most completely lost in B cells with E2A to which CaM cannot bind. gene happens after the Ca2+ signal from the pre-BCR has ended. RAG1, RAG2, PAX5, and E2A are all indispensable for VDJ When Igh is accessible to the recombination machinery, chromo- recombination at Igh (2–4, 10–14). In this study, signaling from somal contraction and DNA looping enable recombination, whereas the pre-BCR was shown to downregulate the expression of several chromosomal expansion inhibits the recombination (46, 47). During components of the recombination machinery, including RAG1, VDJ recombination of Igh, the chromatin of the locus is organized RAG2, and PAX5, through inhibition of E2A by CaM. Data also in compartments containing clusters of loops separated by linkers, were provided supporting the notion that E2A, PAX5, and RAG and the entire repertoire of V regions merges and juxtaposes to D are in a complex bound together to E2A, PAX5, RAG1, and RAG2 elements (48, 49). Productive V-DJ recombination at the Igh locus sites on the Igh gene before pre-BCR activation. Upon activation leads to reversal of locus compaction, and this decontraction was of the pre-BCR, this complex instead bound to CaM and left these postulated to enforce allelic exclusion (47). The large-scale chromatin Igh sequences when E2A was CaM sensitive. What is the relative structures and the changes in them upon productive V–DJ recombi- role in the allelic exclusion for the two negative effects on VDJ nation indicate the existence of protein complexes binding to multiple recombination? One argument that the latter is more important is places in Igh and, thereby, enabling looping between the critical DNA that downregulation of expression of PAX5, RAG1, and RAG2 elements during the VDJ recombination but not after feedback takes several hours, whereas the inhibition of the binding of the inhibition from the pre-BCR. The results presented in this study are complex to the Igh gene was complete after 30 min, and Ca2+/ consistent with PAX5, E2A, RAG1, and RAG2 being key participants CaM inhibits DNA binding of E2A in vitro in ,5 min (44). Thus, in such protein complexes. The complexes also might contain CTCF, CaM inhibition of the binding of the recombination complex cohesin, YYI, and/or Ikaros, because studies (46, 50–52) support could inhibit recombination of the second Igh allele much faster that these proteins contribute to the shaping of the conformation of than the reductions in protein levels could. In addition, the re- the Igh locus. The Journal of Immunology 11
The RAG proteins are essential for V(D)J recombination of all 24. Hughes, K., A˚ . Antonsson, and T. Grundstrøm. 1998. Calmodulin dependence of Ig and TCR loci. Also, E2A and E-boxes control many aspects of NFkappaB activation. FEBS Lett. 441: 132–136. 25. Hauser, J., A. Wallenius, N. Sveshnikova, J. Saarikettu, and T. Grundstro¨m. V(D)J recombination of Ig L chain and the TCR loci (3). Therefore, 2010. Calmodulin inhibition of E2A stops expression of surrogate light chains of related protein complexes, as at the Igh locus, containing at least E2A, the pre-B-cell receptor and CD19. Mol. Immunol. 47: 1031–1038. 26. Krutzik, P. O., and G. P. Nolan. 2003. Intracellular phospho-protein staining RAG1 and RAG2 might play corresponding roles on other AgR loci. techniques for flow cytometry: monitoring single cell signaling events. Cytom- Elucidation of this will be an interesting subject for future research. etry A 55: 61–70. 27. Bhaumik, D., B. Yang, T. Trangas, J. S. Bartlett, M. S. Coleman, and D. H. Sorscher. 1994. Identification of a tripartite basal promoter which regulates Acknowledgments human terminal deoxynucleotidyl transferase gene expression. J. Biol. Chem. We thank Dr. Per-Arne Oldenborg (Umea˚ University) for tail-vein injections. 269: 15861–15867. PAX52/2 pro-B cells were a kind gift of Drs. A. Ebert and M. Busslinger 28. Fong, I. C., A. A. Zarrin, G. E. Wu, and N. L. Berinstein. 2000. Functional analysis of the human RAG 2 promoter. Mol. Immunol. 37: 391–402. (Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria). 29. Hsu, L. Y., J. Lauring, H. E. Liang, S. Greenbaum, D. Cado, Y. Zhuang, and M. S. Schlissel. 2003. A conserved transcriptional enhancer regulates RAG gene expression in developing B cells. Immunity 19: 105–117. Disclosures 30. Zarrin, A. A., I. Fong, L. Malkin, P. A. Marsden, and N. L. Berinstein. 1997. The authors have no financial conflicts of interest. Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions. J. Immunol. 159: 4382–4394. 31. Hauser, J., J. Verma-Gaur, A. Wallenius, and T. Grundstro¨m. 2009. Initiation of References antigen receptor-dependent differentiation into plasma cells by calmodulin in- hibition of E2A. J. Immunol. 183: 1179–1187. 1. Bergman, Y., and H. Cedar. 2010. Epigenetic control of recombination in the 32. Jarvius, M., J. Paulsson, I. Weibrecht, K. J. Leuchowius, A. C. Andersson, immune system. Semin. Immunol. 22: 323–329. C. Wa¨hlby, M. Gullberg, J. Botling, T. Sjo¨blom, B. Markova, et al. 2007. In situ 2. Matthews, A. G., and M. A. Oettinger. 2009. RAG: a recombinase diversified. detection of phosphorylated platelet-derived growth factor receptor beta using Nat. Immunol. 10: 817–821. Downloaded from a generalized proximity ligation method. Mol. Cell. Proteomics 6: 1500–1509. 3. Osipovich, O., and E. M. Oltz. 2010. Regulation of antigen receptor gene as- 33. So¨derberg, O., M. Gullberg, M. Jarvius, K. Ridderstra˚le, K. J. Leuchowius, sembly by genetic-epigenetic crosstalk. Semin. Immunol. 22: 313–322. J. Jarvius, K. Wester, P. Hydbring, F. Bahram, L. G. Larsson, and U. Landegren. 4. Subrahmanyam, R., and R. Sen. 2010. RAGs’ eye view of the immunoglobulin 2006. Direct observation of individual endogenous protein complexes in situ by heavy chain gene locus. Semin. Immunol. 22: 337–345. proximity ligation. Nat. Methods 3: 995–1000. 5. Inlay, M. A., H. Tian, T. Lin, and Y. Xu. 2004. Important roles for E protein 34. So¨derberg,O.,K.J.Leuchowius,M.Gullberg,M.Jarvius,I.Weibrecht,L.G.Larsson, binding sites within the immunoglobulin kappa chain intronic enhancer in ac- and U. Landegren. 2008. Characterizing proteins and their interactions in cells and tivating Vkappa Jkappa rearrangement. J. Exp. Med. 200: 1205–1211. tissues using the in situ proximity ligation assay. Methods 45: 227–232. 6. Beck, K., M. M. Peak, T. Ota, D. Nemazee, and C. Murre. 2009. Distinct roles 35. So¨derberg, O., K. J. Leuchowius, M. Kamali-Moghaddam, M. Jarvius, http://www.jimmunol.org/ for E12 and E47 in B cell specification and the sequential rearrangement of S. Gustafsdottir, E. Schallmeiner, M. Gullberg, J. Jarvius, and U. Landegren. immunoglobulin light chain loci. J. Exp. Med. 206: 2271–2284. 2007. Proximity ligation: a specific and versatile tool for the proteomic era. 7. Romanow, W. J., A. W. Langerak, P. Goebel, I. L. Wolvers-Tettero, J. J. van Genet. Eng. (N. Y.) Dongen, A. J. Feeney, and C. Murre. 2000. E2A and EBF act in synergy with the 28: 85–93. V(D)J recombinase to generate a diverse immunoglobulin repertoire in non- 36. Greenbaum, S., and Y. Zhuang. 2002. Identification of E2A target genes in lymphoid cells. Mol. Cell 5: 343–353. B lymphocyte development by using a gene tagging-based chromatin immuno- 8. Ebert, A., S. McManus, H. Tagoh, J. Medvedovic, G. Salvagiotto, M. Novatchkova, precipitation system. Proc. Natl. Acad. Sci. USA 99: 15030–15035. I. Tamir, A. Sommer, M. Jaritz, and M. Busslinger. 2011. The distal V(H) gene cluster 37. Chowdhury, D., and R. Sen. 2001. Stepwise activation of the immunoglobulin of the Igh locus contains distinct regulatory elements with Pax5 transcription mu heavy chain gene locus. EMBO J. 20: 6394–6403. factor-dependent activity in pro-B cells. Immunity 34: 175–187. 38. Matthews, A. G., A. J. Kuo, S. Ramo´n-Maiques, S. Han, K. S. Champagne, 9. Johnston, C. M., A. L. Wood, D. J. Bolland, and A. E. Corcoran. 2006. Complete D. Ivanov, M. Gallardo, D. Carney, P. Cheung, D. N. Ciccone, et al. 2007. RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombina-
sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V by guest on October 2, 2021 region. J. Immunol. 176: 4221–4234. tion. Nature 450: 1106–1110. 10. Fuxa, M., J. Skok, A. Souabni, G. Salvagiotto, E. Roldan, and M. Busslinger. 39. ten Boekel, E., F. Melchers, and A. Rolink. 1995. The status of Ig loci rear- 2004. Pax5 induces V-to-DJ rearrangements and locus contraction of the im- rangements in single cells from different stages of B cell development. Int. munoglobulin heavy-chain gene. Genes Dev. 18: 411–422. Immunol. 7: 1013–1019. 11. Hesslein, D. G., D. L. Pflugh, D. Chowdhury, A. L. Bothwell, R. Sen, and 40. Barreto, V., and A. Cumano. 2000. Frequency and characterization of phenotypic D. G. Schatz. 2003. Pax5 is required for recombination of transcribed, acety- Ig heavy chain allelically included IgM-expressing B cells in mice. J. Immunol. lated, 59 IgH V gene segments. Genes Dev. 17: 37–42. 164: 893–899. 12. Johnson, K., D. L. Pflugh, D. Yu, D. G. Hesslein, K. I. Lin, A. L. Bothwell, A. Thomas- 41. Hauser, J., J. Verma-Gaur, and T. Grundstro¨m. 2013. Broad feedback inhibition Tikhonenko, D. G. Schatz, and K. Calame. 2004. B cell-specific loss of histone 3 lysine of pre-B-cell receptor signaling components. Mol. Immunol. 54: 247–253. 9 methylation in the V(H) locus depends on Pax5. Nat. Immunol. 5: 853–861. 42. Lin, Y. C., S. Jhunjhunwala, C. Benner, S. Heinz, E. Welinder, R. Mansson, 13. Johnson, K., K. L. Reddy, and H. Singh. 2009. Molecular pathways and M. Sigvardsson, J. Hagman, C. A. Espinoza, J. Dutkowski, et al. 2010. A global mechanisms regulating the recombination of immunoglobulin genes during B- network of transcription factors, involving E2A, EBF1 and Foxo1, that orches- lymphocyte development. Adv. Exp. Med. Biol. 650: 133–147. trates B cell fate. Nat. Immunol. 11: 635–643. 14. Zhang, Z., C. R. Espinoza, Z. Yu, R. Stephan, T. He, G. S. Williams, 43. Verma-Gaur, J., J. Hauser, and T. Grundstro¨m. 2012. Negative feedback regu- P. D. Burrows, J. Hagman, A. J. Feeney, and M. D. Cooper. 2006. Transcription lation of antigen receptors through calmodulin inhibition of E2A. J. Immunol. factor Pax5 (BSAP) transactivates the RAG-mediated V(H)-to-DJ(H) rear- 188: 6175–6183. rangement of immunoglobulin genes. Nat. Immunol. 7: 616–624. 44. Onions, J., S. Hermann, and T. Grundstro¨m. 2000. A novel type of calmodulin 15. Brady, B. L., N. C. Steinel, and C. H. Bassing. 2010. Antigen receptor allelic interaction in the inhibition of basic helix-loop-helix transcription factors. Bio- exclusion: an update and reappraisal. J. Immunol. 185: 3801–3808. chemistry 39: 4366–4374. 16. Cedar, H., and Y. Bergman. 2008. Choreography of Ig allelic exclusion. Curr. 45. Li, Z., D. I. Dordai, J. Lee, and S. Desiderio. 1996. A conserved degradation Opin. Immunol. 20: 308–317. signal regulates RAG-2 accumulation during cell division and links V(D)J re- 17. Vettermann, C., and M. S. Schlissel. 2010. Allelic exclusion of immunoglobulin combination to the cell cycle. Immunity 5: 575–589. genes: models and mechanisms. Immunol. Rev. 237: 22–42. 46. Bolland, D. J., A. L. Wood, and A. E. Corcoran. 2009. Large-scale chromatin 18. Grawunder, U., T. M. Leu, D. G. Schatz, A. Werner, A. G. Rolink, F. Melchers, remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene and T. H. Winkler. 1995. Down-regulation of RAG1 and RAG2 gene expression regulation. Adv. Exp. Med. Biol. 650: 59–72. in preB cells after functional immunoglobulin heavy chain rearrangement. Im- 47. Rolda´n,E.,M.Fuxa,W.Chong,D.Martinez,M.Novatchkova,M.Busslinger,and munity 3: 601–608. J. A. Skok. 2005. Locus ‘decontraction’ and centromeric recruitment contribute to 19. Corneliussen, B., M. Holm, Y. Waltersson, J. Onions, B. Hallberg, A. Thornell, allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6: 31–41. and T. Grundstro¨m. 1994. Calcium/calmodulin inhibition of basic-helix-loop- 48. Jhunjhunwala, S., M. C. van Zelm, M. M. Peak, S. Cutchin, R. Riblet, J. J. van helix transcription factor domains. Nature 368: 760–764. Dongen, F. G. Grosveld, T. A. Knoch, and C. Murre. 2008. The 3D structure of 20. Onions, J., S. Hermann, and T. Grundstro¨m. 1997. Basic helix-loop-helix protein the immunoglobulin heavy-chain locus: implications for long-range genomic sequences determining differential inhibition by calmodulin and S-100 proteins. interactions. Cell 133: 265–279. J. Biol. Chem. 272: 23930–23937. 49. Jhunjhunwala, S., M. C. van Zelm, M. M. Peak, and C. Murre. 2009. Chromatin 21. Saarikettu, J., N. Sveshnikova, and T. Grundstro¨m. 2004. Calcium/calmodulin architecture and the generation of antigen receptor diversity. Cell 138: 435–448. inhibition of transcriptional activity of E-proteins by prevention of their binding 50. Chaumeil, J., and J. A. Skok. 2012. The role of CTCF in regulating V(D)J re- to DNA. J. Biol. Chem. 279: 41004–41011. combination. Curr. Opin. Immunol. 24: 153–159. 22. Zhuang, Y., P. Soriano, and H. Weintraub. 1994. The helix-loop-helix gene E2A 51. Ribeiro de Almeida, C., R. Stadhouders, S. Thongjuea, E. Soler, and R. W. Hendriks. is required for B cell formation. Cell 79: 875–884. 2012. DNA-binding factor CTCF and long-range gene interactions in V(D)J recom- 23. Hauser, J., N. Sveshnikova, A. Wallenius, S. Baradaran, J. Saarikettu, and bination and oncogene activation. Blood 119: 6209–6218. T. Grundstro¨m. 2008. B-cell receptor activation inhibits AID expression through 52. Seitan, V. C., M. S. Krangel, and M. Merkenschlager. 2012. Cohesin, CTCF and calmodulin inhibition of E-proteins. Proc. Natl. Acad. Sci. USA 105: 1267–1272. lymphocyte antigen receptor locus rearrangement. Trends Immunol. 33: 153–159.