Constraints Contributed by Chromatin Looping Limit Recombination Targeting during Ig Class Switch Recombination This information is current as of June 25, 2015.

Scott Feldman, Ikbel Achour, Robert Wuerffel, Satyendra Kumar, Tatiana Gerasimova, Ranjan Sen and Amy L. Kenter

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http://www.jimmunol.org/content/suppl/2015/01/23/jimmunol.140117 0.DCSupplemental.html This article cites 46 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/194/5/2380.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2015; 194:2380-2389; Prepublished online 26 January 2015; doi: 10.4049/jimmunol.1401170 http://www.jimmunol.org/content/194/5/2380

The Journal of Immunology

Constraints Contributed by Chromatin Looping Limit Recombination Targeting during Ig Class Switch Recombination Scott Feldman,*,1 Ikbel Achour,*,1 Robert Wuerffel,*,1 Satyendra Kumar,* Tatiana Gerasimova,† Ranjan Sen,† and Amy L. Kenter*

H

umoral immunity is mediated by Ig Ag receptors that are assembled from multiple V, D, and J segments during early B cell development (1). In mature B cells, Ig class switch recombination (CSR) promotes diversification of C region (CH) effector function while retaining the original V(D)J rearrangement (2). The mouse Igh locus spans 2.8 Mb within which a 220-kb genomic region contains eight CH genes, encoding m-, d-, g3-, g1-, g2b-, g2a-, ε-, and a-chains, each paired with repetitive switch (S) DNA (with the exception of Cd). CSR is focused on S regions and involves an intrachromosomal deletional rearrangement. Germline transcript (GLT) promoters (Prs), located upstream of I exon-S-CH regions, focus CSR to specific S *Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612; and †Gene Regulation Section, Laboratory of Cellular and Molecular Biology, National Institute on Aging/National Institutes of Health, Baltimore, MD 21224 1

S.F., I.A., and R.W. contributed equally to this work.

Received for publication May 7, 2014. Accepted for publication December 19, 2014. This work was supported by National Institutes of Health Grant AI052400 (to A.L.K.) and by the Intramural Research Program of the National Institute on Aging (to R.S.). Address correspondence and reprint requests to Dr. Amy L. Kenter, Department of Microbiology and Immunology, University of Illinois College of Medicine, 835 South Wolcott Avenue (M/C 790), Chicago, IL 60612-7344. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: AID, activation-induced cytidine deaminase; 3C, chromosome conformation capture; CSR, class switch recombination; CT, circle transcript; 3D, three-dimensional; DC-PCR, digestion circularization–PCR; FISH, fluorescence in situ hybridization; GLT, germline transcription; H3K9Ac, histone 3 lysine 9 acetylation; H3K4me3, trimethylated histone 3 lysine 4; hMT, human metallothionein IIA; hs, DNase hypersensitive site; Pol II, RNA polymerase II; Pr, promoter; qPCR, quantitative PCR; qRT-PCR, quantitative RT-PCR; S, switch; WT, wild-type. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401170

regions by differential transcription activation (2, 3). Activationinduced cytidine deaminase (AID) initiates a series of events culminating in formation of double strand breaks at donor Sm and a downstream acceptor S region to create S/S junctions and facilitate CSR. Gene expression is regulated by combinations of regulatory elements that interact over hundreds of kilobases. Use of chromosome conformation capture (3C) and its derivatives has demonstrated in numerous genetic loci that distant chromosomal elements associate to form chromatin loops, thereby providing a mechanism for Pr activation via long-range enhancer function (4). The I-S-CH region genes are embedded between the Em and 39Ea enhancers that are separated by 220 kb. Our 3C studies revealed that mature resting B cells engage in long-range Em and 39Ea chromatin interactions (5, 6). B cell activation leads to induced recruitment of the I-S-CH loci to the Em:39Ea complex that, in turn, facilitates GLT expression and S/S synapsis (6). Targeted deletion of DNase hypersensitive sites (hs) 3b,4, elements within 39Ea, leads to loss of all GLT expression except for g1 GLT, which is reduced, impairment of CSR (7), and abrogation of Em:39Ea and I-S-CH loci:39Ea looping interactions (6). Thus, CSR is dependent on three-dimensional (3D) chromatin architecture mediated by long-range intrachromosomal interactions between distantly located transcriptional elements. Given the importance of chromatin looping during CSR, several fundamental questions regarding the establishment and maintenance of DNA loop formation emerge. For example, what is the relationship of transcription, transcription factors, and specific transcriptional elements to the formation of DNA loops that promote or exclude GLT expression and S/S synapsis, preconditions for the CSR reaction? Additionally, it has been difficult to integrate the spatial relationships within the Igh locus with the preferential

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Engagement of promoters with distal elements in long-range looping interactions has been implicated in regulation of Ig class switch recombination (CSR). The principles determining the spatial and regulatory relationships among Igh transcriptional elements remain poorly defined. We examined the chromosome conformation of C region (CH) loci that are targeted for CSR in a cytokinedependent fashion in mature B lymphocytes. Germline transcription (GLT) of the g1 and « CH loci is controlled by two transcription factors, IL-4–inducible STAT6 and LPS-activated NF-kB. We showed that although STAT6 deficiency triggered loss of GLT, deletion of NF-kB p50 abolished both GLT and g1 locus:enhancer looping. Thus, chromatin looping between CH loci and Igh enhancers is independent of GLT production and STAT6, whereas the establishment and maintenance of these chromatin contacts requires NF-kB p50. Comparative analysis of the endogenous g1 locus and a knock-in heterologous promoter in mice identified the promoter per se as the interactive looping element and showed that transcription elongation is dispensable for promoter/enhancer interactions. Interposition of the LPS-responsive heterologous promoter between the LPS-inducible g3 and g2b loci altered GLT expression and essentially abolished direct IgG2b switching while maintaining a sequential m→g3→g2b format. Our study provides evidence that promoter/enhancer looping interactions can introduce negative constraints on distal promoters and affect their ability to engage in germline transcription and determine CSR targeting. The Journal of Immunology, 2015, 194: 2380–2389.

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Materials and Methods Mice, cell culture, flow cytometry, and statistics C57BL/6 (wild-type [WT]), Stat62/2, and NF-kB p502/2 (Nfkb1) mice on the C57BL/6 background were purchased from The Jackson Laboratory. IgHhMT/hMT mice (9) were provided by C.O. Jacob (University of Southern California) on the C57BL/6 background. All procedures involving mice were approved by the Institutional Animal Care Committee of the University of Illinois College of Medicine or the National Institute of Aging. Splenic B cells were sorted for CD432 resting B cells using CD43 magnetic microbeads (MACS, Miltenyi Biotec) according to the manufacturer’s instructions and cultured in 50 mg/ml LPS (Salmonella typhimurium, phenol extract, Westphal; Sigma-Aldrich), 10 ng/ml rIL-4 (R&D Systems). To prepare for flow cytometry, B cells activated for 4 d were washed in HBSS plus 2% FCS and stained with Abs conjugated with FITC (FITC-IgG3 [553403] and FITC-IgG1 [553443], BD Pharmingen; FITC-IgG2b [406706], BioLegend) or with allophycocyanin–anti-mouse B220 (allophycocyanin-B220 [103211], BioLegend). The flow cytometry analyses represented 5,000–10,000 events and were gated for live lymphoid cells determined by forward and side scatter with CyAn ADP and Summit software (Becton Dickenson). The p values were calculated by using a two-tailed Student t test.

Real-time quantitative RT-PCR, circle transcript PCR, and 3D DNA fluorescence in situ hybridization Quantitative RT-PCR (qRT-PCR) assays were performed as described (6) except that primers for 18S rRNA were used (10) to normalize samples. Quantitative circle transcript (CT) RT-PCR assays were carried out as described (11) with modifications. Primers g2bF and g3R were used for Ig2b-Cg3 CT assays (Table I). Ig2b-Cm CTs were detected using the Ig2bF and CmR.1 primers (12) (Table I). Semiquantitative RT-PCR for Iε2Cm CTs was performed using primers IεF and CmR with Phire Hot Start II polymerase (Thermo Scientific) and an initial denaturation for 5 min at 98˚C followed by 34 cycles of 98˚C for 5 s, 60˚C for 5 s, 72˚C for 8 s, both in a 25 ml reaction on 5-fold serially diluted cDNA. 3D DNA fluorescence in situ hybridization (FISH) was carried out as described previously (13).

Chromosome conformation capture The 3C assay for the Igh locus was performed as described (6) and was optimized as follows. Cells were crosslinked with 1% formaldehyde for 8 min at room temperature and the reaction was quenched with glycine. All primers and probes for 3C product analyses were designed to avoid strain polymorphisms and are equally appropriate for both the C57BL/6 and 129 mouse strains (Table II). Several controls to confirm the efficacy of the 3C procedure were performed. Template concentration using the mb1 primers (Table II) was determined as previously described (6). The efficiency of HindIII restriction site digestion in chromatin was monitored by real-time PCR analysis using primers spanning the restriction sites under study (Supplemental Table I). To control for differences in amplification efficiency between primer sets we constructed a control template in which all

possible 3C ligation products are present in equimolar concentration (Supplemental Table II). DNA fragments that span restriction sites studied were PCR amplified and mixed in equimolar amounts, then digested with HindIII, ligated, and added to genomic DNA that had been digested and ligated to serve as the 3C control template and that is used in a standard curve in quantitative 3C PCR analyses. To permit sample-to-sample comparisons the data were normalized using the interaction frequency between two fragments within the nonexpressed Amylase 1 (Amy) gene, which are separated by 3.5 kb (Table II). The equation used to calculate the relative crosslinking frequency between two Igh restriction fragments is XIgh = [SIgh/SAmy] cell type/[SIgh/SAmy] control mix, where SIgh is the signal obtained using primer pairs for two different Igh restriction fragments and SAmy is the signal obtained with primer pairs for the Amy1 locus fragments. The crosslinking frequency for the two Amy1 fragments was arbitrarily set to a value of 1 to permit sample comparisons. A complete laboratory protocol for 3C is available upon request.

Quantitative PCR for 3C ligation products Quantitative PCR (qPCR) for 3C was used in combinations with 59FAMand 39BHQ1-modified probes (Integrated DNA Technologies) to detect 3C products (Table II). 3C primers were designed using Primer Express software (Applied Biosystems) (Table II). The qPCR protocol was run at 59˚C for 90 s to ensure maximum amplification, and these conditions were used for all optimization and assay reactions. Primer and probe optimization were carried out according to the manufacturer’s recommendations (http://www3.appliedbiosystems.com/cms/groups/mcb_support/ documents/generaldocuments/cms_042996.pdf). Dilutions of control mix were assayed under optimized conditions to determine the linear range of amplification (64 to 0.03 pg ligated mix/50 ng genomic DNA/ml). Finally, 3C chromatin template was analyzed in serial dilutions to determine the approximate minimum and maximum amounts of DNA that yield a constant crosslinking frequency for a sample (10–200 ng). For all qPCR 3C reactions, 100 ng chromatin were used. 3C assays for WT and NF-kB p502/2 B cells were analyzed with the H probe and T.H-H* primer whereas WT, Stat62/2, and IgHhMT/hMT B cells were analyzed with the H.1 probe and T.H-H.1* primer (Table II).

Results The g1:39Ea looping requires NF-kB p50 and is independent of transcription Given the centrality of Em:39Ea looping to CSR (5, 6), we sought to better characterize the establishment and maintenance of Em:39Ea interactions with regard to a requirement for transcription, transcription factors, and specific transcriptional elements that promote or exclude GLT expression and S/S synapsis. To determine whether formation and/or maintenance of GLT Pr:enhancer contacts are dependent on transcription, we analyzed Stat6or NF-kB p50–deficient B cells for AID and GLT expression, CSR frequency, and Igh looping interactions. The IL-4–inducible STAT6 transcription factor, in collaboration with LPS–inducible NF-kB, binds to the Prs of GLT g1 and ε, mediates their expression (14–16), and enhances AID transcription (17). As a control for B cell activation, AID transcripts are highly expressed upon induction with LPS alone or LPS plus IL-4 in both WT and Stat62/2 B cells (Fig. 1A, Table I). Typically, GLT g3 and g2b are stimulated by LPS, whereas GLT g1 and ε also require IL-4 (Fig. 1A). Although GLT g3 and g2b expression is intact in Stat6-deficient B cells, the GLTs g1 and ε fail to express, as expected (Fig. 1A). LPS plus IL-4 represses g3 and g2b GLTs in WT but not in Stat6-deficient B cells, demonstrating that STAT6 negatively regulates the expression of these GLTs either directly or indirectly (Fig. 1A). We observe that Stat6 deficiency leads to impaired IgG1 switching in response to LPS plus IL-4 relative to WT, as expected. The findings are shown in a representative FACS analysis and summarized for multiple ex vivo B cell cultures from independent mice (Fig. 1B, 1C). In contrast, IgG3 switching was comparable in WT and Stat6 knockout B cells stimulated with LPS alone (Fig. 1B, 1C). Thus, the pattern of IgG1 and IgG3 switching in WT and Stat62/2 B cells is paralleled by the g1 and g3 GLT expression pattern (Fig. 1A–C).

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expression of some isotypes. Notably, IgG1 and IgE are both induced by CD40L and IL-4 and require STAT6 and NF-kB, but the g1 locus is highly favored for CSR (8). We have addressed these questions by characterizing Igh chromatin topologies, GLT expression, and CSR in the context of specific transcription factor deficiencies and GLT Pr substitutions in mice. In this study, we report that long range interactions between I-S-CH loci and Igh enhancers are independent of GLT production and STAT6, whereas the establishment and maintenance of these chromatin contacts require NF-kB p50. Replacement of the g1 GLT Pr with the LPS-responsive human metallothionein IIA (hMT) Pr (9) shows that the GLT Pr directly contacts the Igh enhancers and this looping is independent of productive transcription elongation. Strikingly, intercalation of the hMT Pr between the LPS-inducible g3 and g2b loci constrains g2b GLT expression and essentially abolishes direct m→g2b CSR whereas sequential m→g3→g2b switching is retained, albeit at a reduced frequency. These findings demonstrate that specific long-range contacts contribute spatial constraints that functionally impinge on gene expression, determine CSR targets, and provide a mechanistic basis for direct IgG1 and sequential IgE switching.

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B cell activation with LPS and LPS plus IL-4 leads to long-range chromatin looping between I-S-CH loci and the Em:39Ea complex that in turn facilitates GLT expression and S/S synapsis (6) (Fig. 1D). The differential expression of g1 GLTs in STAT6deficient and -sufficient B cells allowed us to assess the requirement for transcription in establishing and maintaining g1:39Ea looping interactions using highly sensitive TaqMan 3C assays. 39Ea is composed of DNase hypersensitivity sites, termed hs3a, hs1,2, and hs3b,4 (18). In the present study, we evaluated the association of g3, g2b, and g1 loci with Em, 39Ea hs1,2, and hs3b,4 in resting and activated WT B cells. LPS induction significantly increased Em:g3 and Em:g2b interactions, whereas LPS plus IL-4 treatment modestly reduced these interactions relative to LPS (Fig. 1E, Table II), as previously observed (6). This reduction

did not achieve statistical significance. Conversely, LPS plus IL-4 treatment significantly induced Em:g1 (A–C) looping as compared with unstimulated B cells (Fig. 1E). A similar trend of LPS and LPS plus IL-4–inducible contacts between I-S-CH loci and hs1,2 and hs3,b4 was also found. However, the frequency of induced contacts between hs3b,4 with g3, g1, or g2b was 4- to 8-fold higher than the equivalent interactions with Em (in all cases p , 0.0005) and 1.5- to 3-fold higher than the equivalent interactions with hs1,2 (fragment G) (in all cases p , 0. 01), indicating preferential association of I-S-CH loci with hs3b,4 (Fig. 1E). We conclude that the hs3b,4 element within 39Ea is the primary point of contact with I-S-CH loci, consistent with the critical requirement for hs3b,4 in mediating GLT expression (7). Based on the foregoing observations, 3C assays were focused to looping

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FIGURE 1. NF-kB but not transcription is required for g1:39Ea and Em:39Ea looping. Resting B cells from WT, Stat62/2, or NF-kB p502/2 mice were stimulated with LPS or LPS and IL-4lo (10 ng/ml) for 40 h (RT-PCR, 3C assays) or 4 d (FACS). (A) qRT-PCR assays for GLT g3, g2b, and g1 and AID were normalized to the 18S rRNA gene transcript using two to four samples from two to four independent experiments. (B) FACS analyses of B cells stained with anti-IgG1 or anti-IgG3 in combination with anti-B220. Numbers indicate percentage of switched cells. (C) Average percentage of IgG3 (top) or IgG1 (bottom) from FACS analyses of LPS- or LPS plus IL-4lo–activated B cells. Each symbol represents a single mouse and the line indicates the average. (D) Schematic for long-range chromatin looping interactions for LPS-activated WT B cells in which Em:39Ea interacts with g3 (a) or g2b (b) loci. (E) HindIII restriction fragments used in the 3C analysis are indicated (fragments A–D, G, and H). 3C assays were anchored at Em (fragment A), hs1,2 (fragment G), or hs3b,4 (fragment H) and were analyzed for interaction with I-S-CH loci (fragments B–D). (F) 3C assays anchored at hs3b,4 (fragment H) interrogated interactions with the g1 (C fragment) or Em (A fragment) as indicated. (G) qRT-PCR assays for GLT g3 and g1 and AID from WT or NF-kB p502/2 B cells activated with LPS or LPS plus IL-4 as indicated. qRT-PCR assays were normalized to the 18S rRNA gene transcript using two to four samples from two to four independent experiments. *p # 0.05, **p # 0.001 by two-tailed Student t test.

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Table I. Primers for qRT-PCR and CT analyses Primera

RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR DC-PCR DC-PCR DC-PCR DC-PCR CT CT CT CT

HprtF HprtR 18SR 18SR g1F g1R g3Fb g3R g2bF g2bR eF eR AIDF AIDR DC-εF1 DC-εF2 DC-mR1 DC-mR2 Ig2bFc CmR.1c IεFd CmRd

Sequence

59-GTTGGATACAGGCCAGACTTTGTTG-39 59-TACTAGGCAGATGGCCACAGGACTA-39 59-TTGACGGAAGGGCACCACCAG-39 59-GCACCACCACCCACGGAATCG-39 59-AGGAATGTGTTTGGCATGGAC-39 59-CACTGTCACTGGCTCAGGGAA-39 59-TGTCTGGAAGCTGGCAGGA-39 59-GGCTCAGGGAAGTAGCCTTTG-39 59-TAAGCTGCGCACACCTACAGACAA-39 59-AGGGATCCAGAGTTCCAAGTCACA-39 59-CAGAAGATGGCTTCGAATAAGAACA-39 59-CGTTGAATGATGGAGGATGTGT-39 59-CCATTTCAAAAATGTCCGCT-39 59-CAGGTGACGCGGTAACACC-39 59-CCGATTTGACCTACCAGATGCT-39 59-TCTATGGGCATCAGGACCACTCC-39 59-TGAAGCCGTTTTGACCAGAAT-39 59-GGAGACCAATAATCAGAGGGAAGAA-39 59-CACTGGGCCTTTCCAGACCTA-39 59-TGGTGCTGGGCAGGAAGT-39 59-GCGGCCCCTAGGTACTACCA-39 59-AATGGTGCTGGGCAGGAAGT-39

References

(6) (6) (10) (10) (43) (43) (44) (45) (45) (44) (46) (46) (6) (6) (45) (45) (45) (45) This study (12) (45) (11)

a

For primers, F and R indicate forward and reverse primers, respectively. g3F and Ig2bF are used for Ig2b-Cg3 CT. Ig2bF and CmR.1 are used to detect Ig2b-Cm CT. d IεF4 and CmR are used to assess Iε-Cm CT. b c

between hs3b,4 (fragment H), Em (fragment A), and g1 (fragment C) loci. Next we evaluated the frequency of the g1:hs3b4 and Em:hs3b,4 looping interactions in Stat6-proficient and -deficient B cells to assess the contribution of GLT expression to these chromatin contacts. In WT and Stat62/2 B cells, hs3b,4:g1 (H–C) and hs3b,4:Em (H–A) contacts are comparably induced by LPS plus IL-4, indicating that these long-range interactions are independent of both STAT6 and g1 GLT expression (Fig. 1F). Induction of hs3b,4:g1 interactions by LPS plus IL-4 in Stat6-deficient B cells may occur through a STAT6-independent pathway involving the insulin receptor substrate family (19). In contrast, NF-kB p50–deficient B cells do not express GLTs or AID, and they fail to support

hs3b,4:Em (H–A) (Fig. 1F, 1G) (17, 20). Although hs3b,4:g1 (H– C) looping contacts are present in NF-kB p50–deficient B cells, these contacts are not IL-4 inducible as compared with WT (Fig. 1F). Evidence indicates that NF-kB p65 (RelA) is required for inducible transcription elongation in some genes, whereas the role of NF-kB p50 has been less clear (21). NF-kB binding has also been detected in 39Ea hs4 (22) and could be directly involved in long-range looping. Taken together, these studies show that 1) long-range looping interactions between 39Ea:g1 and Em:39Ea occur independent of GLT expression as observed in Stat6deficient B cells, and 2) that NF-kB p50 is a major contributor to inducible g1 GLT expression and hs3b,4:g1 looping as well as the basic integrity of the Em:39Ea complex as found in p502/2

Table II. Primer and probe sequences used in the quantitative 3C assay Probea

Primerb

Sequence

References

mb1F mb1R

59-CCACGCACTAGAGAGAGACTCAA-39 59-CCGCCTCACTTCCTGTTCAGCCG-39 59-TTGAATATGTACCGAGTACACATGGATGGTGCAT-39 59-GAGATCTTACGTAGGCACTTAGTGGTATAA-39 59-GCTTCCATGATACTCTATGTTCTTCCT-39 59-TGGCTTACCATTTGCGGTGCCTGGTTT-39 59-TCCACACAAAGACTCTGGACCTCT-39 59-CTGACCCAGGAGCTG-39 59-CAGATCACAGGGTCCCAGGTT-39 59-TGACTCATCCACATCACCTTGCCTGTG-39 59-CTCATCCACATCACCTTGCCTGTGTATTGTC-39 59-CTCCCACCAGCCAAGACAAT-39 59-ATAGGCCCTCCTCCCACCA-39 59-GAAACCAGGCACCGCAAATG-39 59-AGTAGATAGGACAGATGGAGCAGTTACA-39 59-GTGATAATGAACTGAATCCCACATGTAC-39 59-AGTACCCAGCATGTTCACATC-39 59-AGGACCAAGGTTCACAGCCA-39 59-GCCCCTAAGACCCTACTCTGCTA-39

(8) (8) This study This study This study (8) (8) (8) (8) This study (8) (8) (8) (8) (8) (8) (8) (8) (8)

Amy T.AmyF T.AmyR A (Em) T.A-A* B (g3) T.B-B* H (hs3b,4) H.1 (hs3b,4) T.H-H* T.H-H.1* T.A T.B T.C T.D T.F T.H

a 3C probes specific for unique HindIII fragments are denoted by a capital letter referring to the 3C fragments. The Amy probe is in the Amylase1 gene. The probes and anchor primers can be used in combination with any other 3C primers. b F and R indicate forward and reverse primers, respectively. Anchor primers marked with an asterisk are used in combination with anchor probes. 3C assay primers specific for each HindIII fragment are denoted by a capital letter referring to the 3C fragments.

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Assay

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FIGURE 2. The g1hMT/hMT locus perturbs g2b and ε GLT expression. Resting B cells from WT or IgHhMT/ hMT mice were stimulated with LPS or LPS and IL-4lo (10 ng/ml) or IL-4hi (20 ng/ml) for 40 h or as indicated. (A) GLT g3, g2b, g1, and ε and AID expression were analyzed by qRTPCR and signals were normalized to the 18S rRNA gene transcript using two to five samples from two to three independent experiments. (B) FACS analyses of B cells stained with anti-IgG1 in combination with anti-B220. Numbers indicate percentage of switched cells. (C) Average percentage of IgG3 (top) or IgG1 (bottom) from FACS analyses of LPS- or LPS plus IL-4–activated B cells, as indicated. Each symbol represents a single mouse and the line indicates the average. *p # 0.05, **p # 0.001 by two-tailed Student t test.

GLT Pr identity determines chromatin contacts with Igh enhancers Earlier studies showed that the enhancer elements Em:39Ea are in close proximity in resting splenic B cells and this interaction is increased upon activation of CSR (6). Furthermore, downstream S regions gain proximity to the universal donor, Sm, in a cytokinedependent fashion (6). However, it remained unclear whether the GLT Pr is responsible for bringing its associated S region into proximity with Sm. To address this question, we investigated whether the GLT Pr per se interacts with Em and 39Ea. We studied IgHhMT/hMT mice in which the LPS plus IL-4–reactive g1 GLT Pr is replaced by the LPS-responsive hMT Pr, referred to in this study as the g1hMT/hMT Pr (9). The g1hMT/hMT Pr lacks the Ig1 splice donor (9) that is required for cotranscriptional pre–mRNA processing (24, 25). Unspliced or aberrantly spliced mRNA is prevented from leaving the nucleus by nuclear surveillance pathways, primarily mediated by the exosome (24). Despite impaired expression of g1 GLTs, the g1hMT/hMT Pr is responsive to LPS as assessed in nuclear run-on assays (9), which detect induced transcription and are an indicator of elongation-competent RNA polymerases (26). Activation of WT and IgHhMT/hMT B cells with CSR inducers is confirmed by induction of AID transcripts and LPS plus IL-4 stimulation of ε GLTs (Fig. 2A). It is unknown why AID expression is elevated in activated IgHhMT/hMT B cells relative to WT cells. IgHhMT/hMT B cells fail to express g1 GLTs whereas WT B cells are competent in this regard (Fig. 2A) (9). FACS analyses confirm an impaired IgG1 switching pattern in LPS plus IL-4–induced IgHhMT/hMT B cells as compared with WT (Fig. 2B, 2C). Furthermore, IgG3 switching in response to LPS activation is significantly reduced in IgHhMT/hMT versus WT B cells

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B cells. NF-kB has also been implicated in chromatin looping associated with Igk gene expression (23). Previous studies suggested AID deficiency led to a severe reduction of I-S-CH:Em and Em:39Ea interactions in stimulated B cells (6), raising questions regarding the relative contribution of transcriptional elements versus DNA damage to long-range chromatin contacts. Owing to breeding issues, the AID2/2 mouse was rederived by four backcrosses to C57BL/6. We now find a smaller yet reproducible reduction of Em:hs3b,4 (A–H) interactions (p , 0.026) and hs3b,4:Em (H–A) (Supplemental Fig. 1). We also find that the frequency of Em:g2b (A–D) interactions is reduced and that hs3b,4:g2b (H–D) contacts display a similar trend (Supplemental Fig. 1). We conclude that induced Em:39Ea and I-S-CH:39Ea chromatin interactions are modestly influenced by AID-mediated DNA lesions. The earliest detectable switching occurs at 2.5 d after B cell activation under our culture conditions (6). We isolated chromatin at 40 h of B cell activation to capture B cell chromatin in a germline configuration prior to CSR. It is formally possible that low-level m→g2b CSR occurs shortly after B cell activation. These early m→g2b CSR events would give the appearance of Em:g2b (A–D) chromatin looping in 3C assays. However, this is not the case for Em:39Ea (A–H and H–A) and 39Ea:g2b (H–D) interactions because these sites do not become contiguous postswitching and therefore are not prone to create false-positives in the 3C assay. Therefore, the greatest contribution to Igh looping and S/S synapsis is via chromatin looping of GLT Prs and Igh enhancers with AID providing a more modest contribution. These findings also indicate that in p502/2 B cells the severe loss of Em:39Ea looping, described above, is unlikely to be due to impaired AID expression.

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FIGURE 3. Igh enhancers contact the g1 GLT Pr in the absence of GLT production. Resting WT or IgHhMT/ hMT B cells were activated with LPS or LPS plus IL-4lo for 40 h and then analyzed in 3C, 3D DNA FISH, or ChIP assays as indicated. (A) Schematic for long-range looping interactions in LPSactivated WT or IgHhMT/hMT B cells in which Em:39Ea interacts with g3 (a), g2b (b), or ghMT/hMT (c) loci. (B–D) In 3C assays, two to three 3C chromatin samples from at least two independent experiments were analyzed. (B) Schematic showing HindIII restriction fragments (B–D) analyzed with anchor hs3b,4 (fragment H) (upper panel). 3C assays are shown in the lower panel. (C) 3C HindIII restriction fragments (C and H) analyzed with anchor g3 (fragment B) or Em (fragment A) (upper panel). 3C assays are shown in the lower panel. (D) 3C HindIII restriction fragments (A and H) analyzed with anchor Em (fragment A) or hs3b,4Em (fragment H) (upper panel). 3C assays are shown in the lower panel. (E) A 220-kb segment of the Igh locus (top). 3D DNA FISH probes h4 (green) (27), Em5 (red) (13), and BAC210H14 (blue) (top panel) are shown. High-resolution three-color 3D DNA FISH with WT splenic B cells. Probes were labeled with Alexa Fluor 594 (red) and 488 (green) and BAC RP23-201H14 was labeled with Alexa Fluor 697 (blue) and hybridized with fixed splenic B cells. Signals were visualized by epifluorescence microscopy (original magnification 3100) and distances between probes were determined as described (13, 27). B cells were purified from two mice (middle panel). Quantitation of FISH data are shown (bottom panel). (Figure legend continues)

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decreased to ,0.2 mm in ∼80% of Igh alleles originating in LPS plus IL-4–activated B cells as compared with ∼20% of resting B cells (Fig. 3E). In ∼80% of resting B cells, the signals were separated by 0.2–0.5 mm as compared with 20% in activated B cells. However, the separation distances detected for LPS plus IL-4–activated IgHhMT/hMT B cells were similar to WT (0.1483 6 0.0546 mm). Thus, unlike our 3C assays, the 3D FISH studies did not detect the deficit of Em:39Ea looping in IgHhMT/hMT B cells. One explanation for this discrepancy is that the considerably higher formaldehyde concentration used in 3D FISH (4%) as compared with 3C assays (1%) may obscure the partial Em:39Ea looping deficiency in IgHhMT/hMT B cells, as suggested (28). Another explanation for these findings is that 3D FISH reports more global chromatin interactions whereas 3C assays are capable of detecting chromatin contacts at the molecular level. Consequently, 3D FISH detects a similar LPS plus IL-4–induced Igh locus conformation in both WT and IgHhMT/hMT B cells, whereas 3C assays indicate that Em and 39Ea are less frequently in molecular proximity in IgHhMT/hMT B cells. These studies independently confirm that Em:39Ea looping interactions in resting B cells are significantly induced by CSR stimuli. Bromo- and chromodomain factor binding to activating histone modifications at Pr-proximal positions and at enhancers could in principle mediate Pr:enhancer looping (29). In transcribed I-S-CH regions, trimethylated histone 3 lysine 4 (H3K4me3) and acetylated histone 3 lysine 9 (H3K9Ac) marks accumulate in S regions as a result of R-loop formation and RNA polymerase II (Pol II) pausing (30). H3K4me3 and H3K9Ac are enriched at the Sm locus upon LPS and LPS plus IL-4 activation in WT and IgHhMT/hMT B cells, whereas these marks were severely depleted at the Sg1hMT/hMT locus relative to WT (Fig. 3F). We conclude that factor binding to S region epigenetic marks is dispensable for the formation and maintenance of g1hMT/hMT Pr:Igh enhancer looping. Chromatin topology constrains the order of isotype switching Intercalation of the g1hMT/hMT Pr between the g3 and g2b loci offers an opportunity to assess the contribution of locus topology to differential isotype choice during CSR (Figs. 3A, 4A). We then asked whether interposition of the g1hMT/hMT Pr between the LPSresponsive g3 and g2b loci will create a new topological constraint by means of LPS-induced g1hMT/hMT Pr:39Ea looping that now inhibits direct m→g2b CSR. Such a constraint may underpin sequential CSR, which occurs when the Sm donor recombines with a proximal downstream I-S-CH locus to form hybrid S/S regions (Fig. 4A). The hybrid S/S regions can in turn serve as a donor substrate in a second round of CSR events (31) (Fig. 4A). Thus, Sg2b, located downstream of Sg1, may become available for CSR only following the removal of the constraints contributed by g3:39Ea:Em and g1hMT/hMT:39Ea:Em looping (Fig. 3A). Because the relative strength of g3 and g2b GLT expression in WT and IgHhMT/hMT B cells could be an indicator of constraints on I-S-CH looping with 39Ea, we assessed GLT expression levels using qRT-PCR. The g3 GLT was expressed similarly in WT and IgHhMT/hMT B cells upon treatment with LPS (Fig. 2A). Furthermore, g3 and g2b GLT expression levels were reduced in response to LPS plus IL-4lo as compared with LPS alone in both strains of

Distances between red and green FISH signals were divided into two categories (,0.2 and 0.2–0.5 mm) for ∼100 nuclei. The probes h4 (green) and Em5 (red) correspond to hs3b,4 and Em, respectively. The percentage of Igh alleles in each group (y-axis) was determined for each activation condition (x-axis) and are displayed in different colors. *p # 0.05, **p # 0.001, ***p # 0.0001. (F) ChIP assays were performed on nuclei from four to six samples derived from two independent experiments using anti-H3K9Ac or anti-H3K4me3 Abs. All samples were analyzed in duplicate then averaged, and SEM are shown. Histone modifications and primer pairs tested are indicated. The qPCR product concentration is relative to 10% input and indicates the enrichment of a sequence following immunoprecipitation.

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(Fig. 2B, 2C). These findings indicate that substitution of the g1 GLT Pr with a heterologous Pr has unanticipated collateral effects on the expression of adjacent I-S-CH regions. This observation is explored in the sections below. We tested the proposition that the GLT Pr is the primary regulatory element engaged in looping interactions with the Igh enhancers. In the WT, the LPS plus IL-4–reactive g1 GLT Pr interacts with the Igh enhancers, whereas this contact profile is predicted to shift to LPS-inducible looping for the g1hMT/hMT Pr (Fig. 3A) (6). 3C studies for WT B cells show that LPS plus IL-4 induces abundant hs3b,4:g1 (H–C) interactions (5.3-fold; p , 0.007) relative to resting B cells whereas LPS stimulates a more muted induction (2.3-fold; p , 0.016) (Figs. 1E, 3B). A similar pattern, albeit with lower frequency, is found in WT for Em:g1 (A-C), as expected (Figs. 1E, 3C). In contrast, in IgHhMT/hMT B cells, LPS preferentially increases hs3b,4:g1hMT/hMT (H-C) interactions (2.6-fold, p = 0.02), but LPS plus IL-4 has less impact on longrange contacts (p , 0.013) relative to resting B cells (Fig. 3B). A similar trend for Em:g1hMT/hMT (A–C) looping was observed but did not achieve statistical significance (Fig. 3C). Importantly, equivalent LPS-induced crosslinking frequencies were detected for hs3b,4:g3 (H–B and B–H) and hs3b,4:g2b (H–D) in WT and IgHhMT/hMT B cells, indicating that looping to flanking isotypes is unperturbed (Fig. 3B, 3C). Thus, the shift from LPS plus IL-4 to LPS-inducible looping is limited to the LPS responsive g1hMT/hMT Pr, thereby identifying the Pr as the primary element engaging with the Igh enhancers. We further conclude that GLT Pr:enhancer interactions are modulated by different stimuli (LPS versus LPS plus IL-4), require specific transcription factors (NF-kB p50), and that looping is independent of GLT expression. It is pertinent to ask whether the identity of the GLT Pr influences the abundance of Em:39Ea contacts. This is because CSR requires a tripartite Em:39Ea:GLT Pr interaction complex to provide synapsis between Sm and the targeted downstream S region (Fig. 3A) (6). In WT B cells, hs3b4:Em (H–A and A–H) interactions are of approximately equivalent frequency in response to LPS plus IL-4 and LPS (Fig. 3D). In contrast, in IgHhMT/hMT B cells, hs3b4:Em contacts are significantly (H–A, p , 0.05) or modestly (A–H, p , 0.067) impaired in response to LPS plus IL-4 versus LPS (Fig. 3D). These findings strongly suggest that g1 GLT Pr interactions with Em and 39Ea contribute to the juxtaposition of tripartite Em:39Ea:GLT Pr complex, leading to synapsis of donor Sm and downstream acceptor S regions (Fig. 3A). To independently assess the frequency of the prominent longrange Em:39Ea looping interactions in WT and IgHhMT/hMT B cells, we carried out high-resolution 3D FISH with 10-kb probes. The H14 BAC probe hybridizes outside the Igh locus and independently identifies chromosome 12. Previously described probes h4 (27) and Em5 (13), separated by ∼200 kb, were employed to mark sequences close to Em and within 39Ea, respectively (Fig. 3E, top). This probe combination produced closely juxtaposed signals in LPS plus IL-4–activated B cells (0.1529 6 0.0621 mm) but not in resting B cells (0.2719 6 0.0987 mm) (Fig. 3E). We examined the proportion of Igh alleles in which two FISH signals are separated by different interprobe distances. The separation distances between FISH signals were

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mice, indicating appropriate IL-4–dependent repression (Fig. 2A). In contrast, the g2b GLT was reduced (2.8-fold; p , 0.021) in IgHhMT/hMT B cells relative to WT, consistent with the notion that the g1hMT/hMT Pr element or its chromatin conformation interferes with g2b GLT expression (Fig. 2A). Next, the impact of the LPS-reactive g1hMT/hMT Pr on IgG3 and IgG2b switching was directly tested. Following LPS induction, the frequency of IgG3+ IgHhMT/hMT B cells is modestly though significantly lower (1.51-fold; p , 0.002) relative to WT (Fig. 2B, 2C). Similarly, FACS analyses show a reduced incidence (1.7-fold; p . 0.016) of IgG2b+ IgHhMT/hMT B cells relative to WT in response to LPS activation (Fig. 4B, 4C). The elevated levels of AID in IgHhMT/hMT B cells appear not to compensate for the diminished expression of g2b GLTs (Fig. 2A). Taken together, these findings indicate that the g1hMT/hMT Pr identity impairs switching at both I-S-CH loci flanking the g1hMT/hMT Pr. It was of interest to determine whether direct or sequential IgG2b switching was differentially impacted by the presence of the g1hMT/hMT

Pr. CT assays measure hybrid Ig2b-Cm transcripts, which arise from excised circular DNA generated by direct m→g2b CSR. These CTs are detected using a forward primer in Ig2b together with a reverse primer in Cm in qRT-PCR assays (Fig. 4D, Table I). Sequential IgG2b switching through the g3 locus involves m→g3 (step 1) followed by g3→g2b (step 2) CSR and is detected in Ig2b-Cg3 CT assays (Fig. 4E). Quantitative Ig2b-Cm CT assays show that direct m→g2b CSR was severely diminished 12.5-fold (p . 0.02) in LPS activated IgHhMT/hMT B cells relative to WT (Fig. 4D). In contrast, sequential CSR through the g3 locus is reduced ∼3.1-fold (p . 0.018) and is generally in accord with the lower incidence of IgG3 and IgG2b switching relative to WT (Figs. 2B, 4B, 4C, 4E). We conclude that insertion of the g1hMT/hMT Pr between the g3 and g2b loci substantially impairs direct m→g2b CSR whereas the sequential m→g3→g2b format remains largely intact. Thus, LPS-inducible g1hMT/hMT Pr:39Ea contacts appear to create a chromatin architecture that interferes with the ability of the g2b locus to directly access the Sm donor substrate.

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FIGURE 4. GLT g1hMT/hMT Pr alters the balance between direct and sequential CSR. Resting B cells from three WT and three IgHhMT/ hMT mice were independently stimulated with LPS or LPS and IL-4lo for 42 h (DC-PCR and CT assays) or 4 d (FACS). (A) Diagram depicting direct m→g2b (above the line) or sequential m→g3→g2b (below the line) CSR. (B) FACS analyses of B cells stained with anti-IgG2b in combination with anti-B220. Numbers indicate percentage of switched cells. (C) Average percentage of IgG2b from FACS analyses of LPS- or LPS plus IL-4lo–activated B cells, as indicated. Each symbol represents a single mouse and the line indicates the average. (D) CT assays measure hybrid Ig2b-Cm transcripts that arise from excised circular DNA generated by direct m→g2b CSR and are detected using a forward primer in Ig2b together with a reverse primer in Cm in qRT-PCR and normalized to the 18S rRNA gene transcript using six samples from three independent experiments. (E) CT Ig2b-Cg3 switching is detected using a forward primer in Ig2b together with a reverse primer in Cg3 indicating sequential CSR by qRT-PCR, and signals were normalized to the 18S rRNA gene transcript using six samples from three independent experiments. (F) Schematic of the DC-PCR assay with nested PCR primers and EcoRI (RI) sites indicated by the arrows and filled circles, respectively (upper panel), are compared with the nAChR gene used as a loading control. Representative gel images for the semiquantitative DC-PCR assays are shown in the lower panel. PCR products were harvested in the second round at 26 (lanes 1, 4, 7, and 10), 29 (lanes 2, 5, 8, and 11), and 32 cycles (lanes 3, 6, 9, and 12). (G) CT assays measure hybrid Iε-Cm transcripts, which are detected using a forward primer in Iε with a reverse primer in Cm using semiquantitative RT-PCR and are compared with the Hprt loading control. The p values are # 0.05 (*) and 0.001 (**).

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remain intact when transcription elongation is inhibited but they are disrupted when transcription machines are disrupted (40). Accordingly, Pr-centered chromatin looping provides a topological infrastructure for transcription regulation (41). Taken together, these findings suggest that some features of the transcription apparatus contribute to the spatial organization of genetic elements in chromatin. However, it remains unclear precisely what those features are and how they may vary with the transcriptional requirements of specific loci. We show in the present study that DNA looping in the Igh locus is driven by association of transcriptional elements and has the functional property of influencing partner selection during CSR. Emerging evidence suggests extensive Pr:Pr and Pr:enhancer interactions exist in multigene complexes and these interactions can cooperatively regulate the transcriptional activity (41). Therefore, it might be expected that coregulated GLT Prs would be cooperatively expressed. Although IgG1 and IgE are induced by identical stimuli and require STAT6 and NF-kB, the g1 locus is highly favored for CSR (42). Our studies indicate that the LPS-inducible hMT Pr engages in GLT Pr:enhancer looping and thereby creating chromatin constraints that affect the downstream LPS-responsive g2b locus by reducing GLT expression and impairing direct IgG2b switching. We find that the potentially coregulated GLT Prs are constrained by the 3D architecture of the multigenic Igh locus. These findings represent an example of signal-induced Pr:enhancer interactions that constrain a similarly induced downstream locus, limit gene expression, and determine recombination outcomes. Thus, chromatin looping per se contributes positively and negatively to the efficacy of gene expression.

Acknowledgments We thank Dr. A. Radbruch for the IgHhMT/hMT mouse.

Disclosures Discussion Our studies demonstrate that the establishment and maintenance of induced GLT Pr:enhancer chromatin interactions are contingent upon the integrity of the GLT Pr as well as NF-kB activation by CSR stimuli. I-S-CH:Igh enhancer interactions form in the absence of GLT production and are independent of activating chromatin modifications associated with productive transcription elongation or STAT6. The dispensability of productive transcription elongation for GLT Pr:enhancer looping focuses attention to the early stages of transcription. Pol II transcription is comprised of two phases, that is, initiation and elongation. Following recruitment of Pol II to Prs, the C-terminal domain heptapeptides become phosphorylated at Ser5, permitting transcription initiation (26). During initiation, Pol II pauses ∼40 bp downstream of the transcription start site prior to elongation (26). Following phosphorylation of C-terminal domain Ser2, the paused Pol II is released for elongation (26). Although the paradigm for inducible gene expression has been signal-dependent Pol II recruitment and transcription initiation, several studies suggest that some genes (37) are regulated after transcription initiation, in accord with genome-wide studies of Pol II occupancy (26). In some cases, ongoing transcription elongation is dispensable for sustaining preformed chromatin loops (38, 39). Our studies indicate that GLT Pr:hs3b,4 looping can form in the absence of transcription elongation but they do not discriminate whether signal-induced Pol II engagement with the GLT Pr is necessary and sufficient for Pr:enhancer engagement or transcription initiation is also required. Notably, long-range Igh locus looping interactions involving transcriptionally active Em and Pax5-activated intergenic repeat sites

The authors have no financial conflicts of interest.

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