of April 12, 2018.This information is current as

Gene ExpressionκIgRegulate and Ed, Differentially′Enhancers, E3

A New Hypersensitive Site, HS10, and the

T. GarrardXiaorong Zhou, Yougui Xiang, Xiaoling Ding and William

http://www.jimmunol.org/content/188/6/2722doi: 10.4049/jimmunol.1102758February 2012;

2012; 188:2722-2732; Prepublished online 8J Immunol 

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8.DC1http://www.jimmunol.org/content/suppl/2012/02/08/jimmunol.110275

Referenceshttp://www.jimmunol.org/content/188/6/2722.full#ref-list-1

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The Journal of Immunology

A New Hypersensitive Site, HS10, and the Enhancers, E39 andEd, Differentially Regulate Igk Gene Expression

Xiaorong Zhou,*,† Yougui Xiang,* Xiaoling Ding,* and William T. Garrard*

The mouse Igk gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 39 enhancer (E39), and a further

downstream enhancer (Ed). We previously discovered, using the chromosome conformation-capture technique, that Ei and E39

interact with a novel DNA sequence near the 39 end of the Igk locus, specifically in B cells. In the present investigation, we

examined the function of this far downstream element. The sequence is evolutionarily conserved and exhibits a plasmacytoma cell-

specific DNase I-hypersensitive site in chromatin, henceforth termed HS10 in the locus. HS10 acts as a coactivator of E39 in

transient transfection assays. Although HS102/2 mice exhibited normal patterns of B cell development, they were tested further

along with E392/2 and Ed2/2 mice for their Igk expression levels in plasma cells, as well as for both allelic and isotype exclusion in

splenic B cells. HS102/2 and Ed2/2, but not E392/2, mice exhibited 2.5-fold lower levels of Igk expression in antigenically

challenged plasma cells. E392/2 mice, but not HS102/2 mice, exhibited impaired IgL isotype and allelic exclusion in splenic

B cells. We have suggestive results that Ed may also weakly participate in these processes. In addition, HS102/2 mice no longer

exhibited regional chromosome interactions with E39, and they exhibited modestly reduced somatic hypermutation in the Jk-Ck

intronic region in germinal center B cells from Peyer’s patches. We conclude that the HS10, E39, and Ed differentially regulate Igk

gene dynamics. The Journal of Immunology, 2012, 188: 2722–2732.

The production of Ab molecules during B cell developmentoccurs by the stage-specific and sequential rearrangementof Ab genes in the bone marrow. The IgH gene locus

functionally rearranges first, by V(D)J joining events, leading tothe pre-B cell stage of differentiation (1). Next, the Igk locus ispoised for rearrangement, and in response to the appropriate en-vironmental cues, one of the 96 Vk genes is semi-randomly se-lected for recombination to a Jk region (2; reviewed in Refs. 3, 4).If Igk V-J joining is productively unsuccessful because of out-of-reading frame recombination junctions, then the Igl locus becomesactivated for rearrangement and expression, which, in wild-type(WT) mice, accounts for production of only ∼5% of the totalIgL chains. Interestingly, most l-producing B cells possess aber-rantly rearranged Igk genes or ones deleted because of Vk joiningto a recombining sequence near the 39-end of the locus (5–8).Research in our laboratory has focused on the Igk locus because

it offers the opportunity to visualize chromatin-remodeling eventsthat are linked to gene activation, as well as to identify changesin chromatin structure that may precede gene rearrangement and

transcriptional activation during B lymphocyte differentiation(Refs. 9 and refs. within). Rearrangement of the Igk locus resultsin its transcriptional activation because the process repositionsa Vk gene carrying its own promoter into a chromatin domaincontaining three powerful enhancers: an intronic enhancer (Ei)within the transcription unit (10), as well as two enhancersdownstream of the transcription termination region (11), a 39enhancer (E39) (12) and a further downstream enhancer (Ed) (9).Experimental evidence supports the proposal that these enhancersmediate long-range interactions to trigger transcriptional activa-tion via a regulated looping mechanism. In activated B cells, thethree enhancers exhibit all possible pair-wise interactions withrearranged Vk gene promoters and among themselves, with thelooping out of the intervening DNA sequences (13). However, inunstimulated B cells, rearranged Vk gene promoters only formcomplexes with either Ei or E39 but not with Ed. This results ina transcriptionally poised state in the locus (14).The functional significance of Ei, E39, and Ed in Igk gene

dynamics in B lymphocytes was previously studied by creatingindividual targeted deletions of each. The results of theseexperiments revealed that Ei and E39 each play quantitative rolesin gene rearrangement (15, 16), whereas E39 and Ed each playquantitative roles in rearranged gene transcription (16, 17). Ed,the farthest downstream enhancer, residing some 8 kb away fromE39, plays no role in gene rearrangement and maximally actsduring the latest stages of B cell differentiation (17). However,double deletions of pairs of enhancers has severe consequences onthe locus. Deletion of Ei and E39 eliminates Igk gene rearrange-ment (18), whereas deletion of E39 and Ed abolishes Igk genetranscription (19). These results reveal that these enhancers playredundant compensatory roles in this locus.Previously, we searched for new cis-acting elements that might

be involved in Igk gene regulation by using Ei or E39 as anchorsites with chromosome conformation-capture technology. Wefound that these enhancers exhibited B cell-specific interactionsspanning distances up to 40 kb with a DNA sequence near the 39end of the locus (13). In the current investigation, we determine

*Department of Molecular Biology, University of Texas Southwestern Medical Cen-ter, Dallas, TX 75390; and †Department of Microbiology and Immunology, MedicalSchool of Nantong University, Nantong, Jiangsu 226001, People’s Republic of China

Received for publication September 23, 2011. Accepted for publication January 4,2012.

This work was supported by Grants GM29935 and AI067906 from the NationalInstitutes of Health and Grant I-0823 from the Robert A. Welch Foundation (toW.T.G.).

Address correspondence and reprint requests to Dr. William T. Garrard, Departmentof Molecular Biology, University of Texas Southwestern Medical Center, 5323 HarryHines Boulevard, Dallas, TX 75390-9148. E-mail address: william.garrard@utsouthwestern.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: E39, 39 enhancer; Ed, downstream enhancer;Ei, intronic enhancer; ES, embryonic stem; GC, germinal center; HS10, DNase I-hypersensitive site number 10 in the region downstream of Vk21-1; KLH, keyholelimpet hemocyanin; MFI, mean fluorescence intensity; PNA, peanut agglutinin; TNP,2,4,6-trinitrophenyl; WT, wild-type.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102758

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the function of this new far downstream DNA element, whichproves to be evolutionarily conserved and forms a plasmacytomacell-specific DNase I-hypersensitive site in chromatin (HS10). Bycreating a targeted homozygous deletion of HS10 and characteriz-ing its resulting phenotype along with those resulting from homo-zygous deletions of either E39 or Ed, we demonstrate that bothHS10 and Ed, but not E39, play pivotal roles in high-level tran-scription in plasma cells, whereas E39 and Ed, but not HS10, sig-nificantly contribute to IgL isotype and Igk allelic exclusion insplenic B cells.

Materials and MethodsCell line culture

P815, EL-4, 3-1, and 38B9 cells were cultured in RPMI 1640 mediumsupplemented with 10% FBS, 1% penicillin-streptomycin, and 2 mML-glutamine. S194 and A20 cells were cultured in Iscove’s mediumcontaining 5% FBS, and MPC-11 cells were cultured in DMEM con-taining 20% horse serum. S194, A20, and MPC-11 cells were culturedat 37˚C with 10% CO2; all other lines were cultured at 37˚C with 5%CO2.

Mapping DNase I-hypersensitive sites

Cells were permeabilized with Nonidet P-40 and treated with increasingconcentrations (0–32 mg/ml) of DNase I (Roche Applied Science, Indi-anapolis, IN) for 3 min at room temperature (20). Cells were lysed, andDNase I was quenched in lysis buffer containing proteinase K. GenomicDNA was purified by phenol:chloroform extraction, and 10–15 mg wasdigested to completion with ScaI. Then DNA was transferred to z-probeGT genomic membrane (Bio-Rad Laboratories, Richmond, CA) afterelectrophoretic separation on agarose gels. Prehybridization and hybrid-ization were performed at 65˚C with ExpressHyb hybridization buffer(Clontech, Madison, WI). The membranes were hybridized with a-[32P]deoxy CTP-labeled probe. Membranes were exposed to Eastman Kodakfilm with intensifying screens at 270˚C or to PhosphorImager screens(Molecular Dynamics, Sunnyvale, CA). The probe was a 465-bp DNAfragment amplified by PCR, using primers that were Probe-forward: 59-TTCCCTTCTTCCTGCTAAGTCAA-39 and Probe-reverse: 59-GATGAT-GTGGGGTCTCAGATACC-39. PCR reactions were performed for 35 cy-cles of 30 s at 94˚C, 30 s at 56˚C, and 30 s at 72˚C.

Reporter gene constructions

Plasmids used in transient transfection luciferase assays were constructed asfollows: a 1.3-kb fragment encompassing HS10 was amplified from mousegenomic DNA. MluI or XhoI sites were introduced into the PCR fragmentby incorporating these restriction enzyme-recognition sequences into theprimers. PCR-amplified HS10 fragments were inserted into the MluI orXhoI site of the E39.kpLuc plasmid (21), designated in this article as HS10.E39.kpLuc and E39.HS10.kpLuc, respectively. The primer pairs for PCRamplification of HS10 fragments containing MluI or XhoI sites shown initalics were, respectively, HS10–MluI-forward: 59-CGTGACGCGTTGC-AAACAGCCATTCGCTAACA-39 and HS10–MluI-reverse: 59-CGTGAC-GCGTCATGCCTTTAATCACAGCCTAG-39 and HS10–XhoI-forward: 59-CGTGCTCGAGTGCAAACAGCCATTCGCTAACA-39 and HS10–XhoI-reverse: 59-CGTGCTCGAGCATGCCTTTAATCACAGCCTAG-39. ForHS10.kpLuc plasmid construction, an HS10 fragment was PCR amplifiedusing primers HS10–MluI-forward and HS10–XhoI-reverse and then thisfragment was cloned into MluI and XhoI sites of E39.kpLuc to replace theE39 enhancer. PCR reactions were performed for 35 cycles of 30 s at 94˚C,30 s at 56˚C, and 1 min at 72˚C.

Transient transfection luciferase assays

Cell lines were transiently transfected in triplicate using either ProFectionDEAE-dextran (Promega, San Luis Obispo, CA) for S194, as previouslydescribed (9), or Lipofectamine 2000 (Invitrogen, Carlsbad, CA) forothers. Typically, 106–107 cells and 1–2 mg DNA were used per trans-fection, adjusted for construct sizes to provide equimolarity with respectto the kpLuc reporter, along with 30 ng pRL-CMV Renilla luciferasereporter (Promega). Cells were harvested 24 h posttransfection, and cellextracts were assayed for luciferase activity using the Dual-Luciferasereporter assay system (Promega), following the manufacturer’s instruc-tions. The Renilla luciferase activity was used to normalize transfectionefficiencies.

Mouse strains

Mice possessing a 1.3-kb (1327-bp) deletion of the HS10 element in theendogenous Igk locus were generated by standard embryonic stem (ES)cell targeting technology; germline-transmissible mice were bred with Crerecombinase-expressing MORE mice to obtain neor deletion mice (22).Mice bearing a human Ck knocked-in gene were kindly provided byMichel C. Nussenzweig (The Rockefeller University, New York, NY) (23).E392/2 mice, originally produced by Fred Alt’s laboratory (16), werekindly provided by Mark Schlissel (University of California Berkeley,Berkeley, CA). Ed2/2 mice were previously established in our laboratory(17). All mice were extensively backcrossed onto a C57/BL6 geneticbackground. All mice strains were used in accordance with protocols ap-proved by the University of Texas Southwestern Medical Center Institu-tional Animal Care and Use Committee.

Flow cytometry

Single-cell suspensions were prepared from bone marrow and spleens of 6–14-wk-old mice. Single-cell suspensions were stained with Abs and ana-lyzed using a FACSCalibur with CellQuest software (BD Bioscience, SanDiego, CA) or FlowJo software (Tree Star, Ashland, OR). For IgL isotype-exclusion analysis, splenic cells were first stained with anti-FcgII/III (1 mg/ml; 553142; BD Bioscience) at 4˚C for 30 min. After washing, cells werestained with anti-mouse Igk-PE (0.4 mg/ml; 559940; BD Bioscience), anti-mouse Igl1-3–FITC (5 mg/ml; 553434; BD Bioscience), and anti-mouseB220-allophycocyanin (0.5 mg/ml; 553092; BD Bioscience) at 4˚C for 30min. For Igk allelic-exclusion analysis, splenic cells were first stained withanti-FcgII/III at 4˚C for 30 min. After washing, cells were stained withanti-mouse Igk-PE (0.4 mg/ml) at 4˚C for 30 min. After extensive furtherwashing, cells were stained with anti-human Igk-allophycocyanin (0.4 mg/ml; 9230-11; Southern Biotech, Birmingham, AL) and anti-mouse B220-FITC (1 mg/ml; 5530308; BD Bioscience) at 4˚C for 30 min. Intracellularstaining was performed with a BD Cytofix/Cytoperm Kit (BD Bioscience),and Igk levels were evaluated by mean fluorescence intensity (MFI) ofanti-mouse Igk-PE. A PE-isotype Ab (BD Bioscience) was used as neg-ative control for intracellular staining. Other reagents used were as follows:anti–IgM-allophycocyanin (Southern Biotech); anti–CD43-PE (BD Bio-science), anti–CD25-biotin (BD Bioscience), and streptavidin–PECy5.5(BD Bioscience).

Real-time PCR for expression of Igk and germline DNA levels

To examine Igk gene expression, total RNAwas extracted from 2–5 3 105

cells using TRIzol reagent (Invitrogen). RNA samples were treated withDNase I (Invitrogen) to remove genomic DNA contamination and werereverse transcribed into cDNA using a SuperScript VILO cDNA SynthesisKit (Invitrogen). Real-time PCR was performed to analyze Igk expressionlevels with the following primers: sense 59-GTATCCATCTTCCCAC-CATCCAGTGAGCAGTTAAC-39 and antisense 59-CTTGTGAGTGGC-CTCACAGGTATAGCTGTTATGTC-39. Transcript levels were calculatedusing the ΔCt method, according to the manufacturer’s instructions, andnormalized to the cDNA levels of the mouse HPRT gene.

Germline retention of Igk alleles was determined by real-time PCRassays, using forward and reverse primers that were complementary tosequences upstream (KGL-F) and downstream of Jk1 (KGL-R), as de-scribed previously (19). Germline levels were normalized to the levels ofa b-actin genomic region. The percentage of Igk germline alleles wascalculated by dividing the corresponding levels in WT or knockout miceB cells by those in ES cells.

Plasma cell purification

For isolating plasma cells from mice, splenic cells were first labeled withbiotinylated anti-mouse CD138 (BD Bioscience) Ab and then StreptavidinMicroBeads. Then CD138+ cells were isolated with MACS columns.Isolated CD138+ cells were labeled with anti-mouse B220-FITC and anti-mouse CD138-allophycocyanin. Plasma cells (B220low and negative

CD138high) were sorted on a MoFlo machine (Dako Cytomation, Carpin-teria, CA).

Mouse immunization and ELISA analysis

WT, HS102/2, E392/2, and Ed2/2 mice, 10–16 wk of age, were eachimmunized i.p. with 2 3 108 SRBCs (Sigma-Aldrich, Carlsbad, CA)emulsified in CFA (Sigma-Aldrich). Spleens were harvested at the indi-cated times for analysis of Igk expression in plasma cells. To inducea T cell-dependent Ab response, WT, HS102/2, and Ed2/2 mice were eachimmunized i.p. with 100 mg keyhole limpet hemocyanin (KLH; Sigma-Aldrich) in emulsified CFA. To induce a T cell-independent Ab response,

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mice were each immunized with 10 mg 2,4,6-trinitrophenyl-(TNP)-Ficoll(BioSearch Technologies, Novato, CA) i.p. in PBS. Sera were collected 7and 14 d later. Anti-KLH–specific IgM or IgG Ab concentrations wereanalyzed with mouse anti–KLH-IgM or anti–KLH-IgG ELISA kits (LifeDiagnostics, West Chester, PA). Anti–TNP-specific IgM was analyzed byELISA. Briefly, polyvinyl chloride microtiter plates (DYNEX Technolo-gies, Chantilly, VA) were coated with TNP-BSA (25 mg/ml) and incubatedovernight at 4˚C. After washing with PBS, diluted sera were added andincubated at 4˚C overnight. Plates were subsequently washed three timesand incubated with goat anti-mouse IgM-HRP (Southern Biotech) for 2 h.After washing three times, plates were developed with the ABTS substrate(Southern Biotech), following the manufacturer’s instructions. Mouse anti-TNP Ab (BD Bioscience) was used to quantitate ELISA data.

Splenic cell culture

Single-cell suspensions of spleens from WT and HS102/2 mice were in-cubated with an optimal concentration of biotinylated anti-CD43 (Ly-48;BD Bioscience) Ab and biotinylated anti–Igl1-3 (R26-24; BD Bioscience)Ab. Then MACS MS separation columns (Miltenyi Biotec, Auburn, CA)were used to deplete CD43+ and l+ B cells. k+-Enriched B cells werecultured in RPMI 1640 medium supplemented with 10% FBS, penicillin/streptomycin, L-glutamine, and 50 mM 2-ME at 106 cells/ml and then were

stimulated with 20 mg/ml LPS (Sigma-Aldrich) and 1.0 mg/ml anti-CD40Ab (1C10; eBioscience, San Diego, CA) for 3 d at 37˚C.

Chromosome conformation capture

Chromosome conformation-capture assays were performed, as described,using the previously reported PCR primers, with minor modifications (13,14). These previous studies established the validity of this technique in ourhands by performing many controls, including the demonstration, by ge-nomic Southern assays, that restriction enzyme digestion goes to com-pletion at efficiencies ranging from 65 to 89% within cross-linkedchromatin DNA for both transcriptionally active and inactive Igk loci. Atotal of 107 cells was cross-linked and lysed, and nuclei were digested with1000 IU AseI at 37˚C for 16 h. After ligation and subsequent DNA puri-fication, the cross-linking frequencies between the anchor and test frag-ments, as measured by the amount of corresponding ligation product, wereestimated by duplicate SYBR Green real-time PCR reactions relative tostandards. The standards were used to normalize for the PCR primer ef-ficiencies. BAC clone RP24-387E13, which contains all of the relevantregions studied, was mixed at equal molar ratios with the PCR productspanning the housekeeping gene (PBGD) AseI cutting site. The mixture

FIGURE 1. Mapping and sequence analysis of DNase I-hypersensitive

site HS10. (A) Schematic diagram depicting a rearranged Igk locus. The

coordinates of Ei, E39, Ed, and HS10 in the NCDI37/mm9 mouse chro-

mosome 6 sequence are 70,675,570–70,676,084, 70,685,250–70,686,058,

70,693,704–70,694,944, and 70,713,758– 70,715,084, respectively. (B)

Upper panel, Schematic diagram of the positions of the ScaI and NspI

restriction sites, HS10, and the hybridization probe. Lower panel, HS10

was mapped by indirect end-labeling after DNase I digestion of the various

indicated permeabilized cell lines and ScaI digestion of the resulting pu-

rified DNA, followed by Southern blotting. The arrow indicates the posi-

tion of HS10, which is plasmacytoma cell specific. Cell lines correspond to

plasmacytomas S194 and MPC-11 (25, 26), mastocytoma P815 (27), pro-B

38B9 (28), pre-B 3-1 (28), mature B A20 (29), and mature T EL-4 cells

(30). (C) HS10 sequences are conserved among mouse, human, and rat.

White spaces indicate no match, and gray and black boxes indicate two

or three matches among species, respectively. Two conserved E-boxes

(CANNTG) are shown above the sequences.

FIGURE 2. DNA sequences encompassing HS10 specify transcriptional

enhancer coactivator activity. (A) Schematic diagram of luciferase reporter

gene constructs (labeled 1–5). (B) The luciferase activities of the above

constructs were measured after transient transfection into the indicated

cell lines. Data (mean 6 SD) are representative of several independent

experiments and are plotted as the activity of each construct relative to the

activity of kpLuc after correction for the relative transfection efficiencies

by monitoring the activity of a cotransfected pRL-CMV Renilla luciferase

reporter gene. Statistical significance was determined by a paired Student t

test. *p , 0.01, **p , 0.05, relative to the activity exhibited by the kpLuc

construct.

2724 Igk GENE REGULATION BY DOWNSTREAM HYPERSENSITIVE SITES

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was digested with AseI to completion and ligated at high concentration togenerate equimolar mixtures of all possible ligation products. The cross-linking and ligation efficiencies between different samples and differentexperiments were also normalized to the cross-linking frequencies of twoadjacent AseI fragments of the PBGD gene. The relative cross-linkingfrequency was then calculated using the following formula: (PCR signalof a given Igk gene-ligation product from chromatin/PCR signal of thatIgk gene-ligation product from naked DNA standard)/(PCR signal ofa given PBGD gene-ligation product from chromatin/PCR signal of thatPBGD gene-ligation product from naked DNA standard). The error barsrepresent the SD from the means from at least three independent experi-ments.

SHM analyses

Single-cell suspensions prepared from Peyer’s patches were stained withPE–anti-B220 and FITC-peanut agglutinin (PNA) (Vector Laboratories,Burlingame, CA). B200+ PNAhigh germinal center (GC) cells were sortedon a MoFlo machine (Dako Cytomation). For the Jk-Ck intronic regionSHM analysis, genomic DNA was purified from sorted WT and HS102/2

GC B cells. The Jk-Ck intronic regions from rearranged genes were PCRamplified using Phusion Hot Start II High-Fidelity DNA Polymerase(Finnzymes; Thermo Scientific/Dharmacon, Lafayette, CO) with a degen-erate Vk primer and a reverse primer located ∼600 bp downstream of theJk5, as described elsewhere (17). Gel-purified Vk-Jk5 PCR products werecloned into the StrataClone Blunt Vector Mix amp/kan (Agilent Tech-nologies, La Jolla, CA). Vk-Jk5 clones were identified and sequenced byuse of a T7 primer. Sequences were aligned with the mouse Jk5 down-stream sequence using the Vector NT I (Invitrogen) AlignX program, andmismatches were scored as mutations in the 500-bp region downstream ofJk5.

ResultsIdentification of HS10, a new DNase I-hypersensitive site in theIgk gene locus with E39 coactivator activity

We identified a DNA sequence that exhibited B cell-specific, long-range interactions with Ei and E39 using chromosome con-formation-capture technology (13). This sequence resides some40 kb downstream of Ei, within 2 kb of the neighboring house-keeping gene, Rpia, at the 39 end of the Igk gene locus (Fig. 1A,HS10). Because functionally active cis-acting elements exhibitDNase I-hypersensitive sites in chromatin (24), we first mappedthe chromatin structure of the element as a function of B celldifferentiation using a panel of cell lines. The element formeda DNase I-hypersensitive site, termed HS10, in the terminallydifferentiated plasmacytoma cell lines S194 and MPC-11 but notin other B cell lines arrested at earlier developmental stages (3.1,A20, and 38B9) or in non-B cells (P815 and EL-4) (Fig. 1B) (25–30). Sequence analysis revealed that HS10 contains a core region110 bp in length that is highly conserved among multiple species,which exhibits 76% identity with human and 87% identity with ratIgk gene sequences (Fig. 1C). Two E-boxes are present in the coreregion, which are known to bind E2A proteins crucial for B celldevelopment (31). Importantly, our previous studies showed thatE-boxes with associated E2A proteins are present in Ei, E39, andEd in mouse B cells (13).To determine whether HS10 corresponded to a new transcrip-

tional enhancer in the locus, we performed transient-transfectionassays with a luciferase reporter gene containing a Vk gene pro-moter (Fig. 2A, construct 1). As a positive control, we found thatwhen E39 alone was inserted into this reporter, it exhibited sig-nificant enhancer activities in both S194 and MPC-11 cells, as

FIGURE 3. Flow cytometric analysis of cell surface Ig expression and B

cell development in spleen and bone marrow. (A) FACS analysis of cell

surface Ig expression in WT and HS102/2 mice. Single-cell suspensions

from spleen were simultaneously stained with anti-B220 and anti-Igk Abs

(upper panels) or anti-B220 and anti-Igl Abs (lower panels). Stained cells

were analyzed by FACS. Only cells residing in the lymphocyte gate were

analyzed. Percentages of cells residing in various windows are shown. Data

are representative of independent FACS analyses from at least six mice of

each genotype. The percentages of Igk+ B cells between HS102/2 and WT

were not significantly different (n = 6, p = 0.88, Student t test), nor were

the percentages of Igl+ B cells (n = 6, p = 0.54, Student t test). (B) FACS

analysis of cell surface Ig expression in bone marrow of WT and HS102/2

mice. Single-cell suspensions from bone marrow were stained and ana-

lyzed, as described in (A). The percentages of Igk+ B cells between

HS102/2 and WT mice were not significantly different (n = 6, p = 0.73,

Student t test), nor were the percentages of Igl+ B cells (n = 6, p = 078,

Student t test). (C) Four-color FACS analysis of B cell development in

bone marrow of WT and HS102/2 mice. Single-cell suspensions from

bone marrow were stained with anti-B220 and anti-IgM Abs to assay for

mature B cells (B220highIgM+) and immature B cells (B220+IgM+) (upper

right subpanels). There were no significant differences between these cell

populations (n = 6, p = 0.63, Student t test; n = 6, p = 0.95, Student t test;

n = 6, p = 0.47, Student t test). B220+IgM2 cells were gated, and their

expression of CD25 and CD43 was analyzed (lower panels). Data are

representative of independent FACS analyses from at least six mice of each

genotype. There were no significant differences between these cell pop-

ulations (n = 6, p = 0.71, Student t test; n = 6, p = 0.87, Student t test;

n = 6, p = 0.72, Student t test).

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expected, but not in 38B9 pro-B cells or P815 mastocytoma cells(Fig. 2A, 2B, construct 2). Insertion of HS10 alone resulted inonly mild enhancer activity in MPC-11 cells (Fig. 2A, 2B, con-struct 3). By contrast, HS10 significantly stimulated transcriptionin constructs possessing E39 in the plasmacytoma cell lines S194and MPC-11, and it showed this trend, but to a lesser extent, in38B9 pro-B cells; HS10 was not active in P815 mastocytoma cells(Fig. 2A, 2B, constructs 4 and 5). We conclude that HS10 exhibitstranscriptional synergism with E39 in plasmacytoma cells, a resultobserved earlier with Ei and Ed (9). However, HS10 does notstimulate transcription when combined with Ei in plasmacytomacells (Supplemental Fig. 1A). We suspect that this is because Eialone does not act as an enhancer in transient transfectionexperiments with these cells (Supplemental Fig. 1A) (9).

HS102/2 mice exhibit normal surface Ig expression and B celldevelopment in spleen and bone marrow

The observations that HS10 sequences are evolutionarily con-served, exhibit a plasmacytoma-specific DNase I-hypersensitivesite in chromatin, and possess enhancer coactivation activity

prompted us to examine the function of HS10 in the endogenousIgk locus. We generated germline-transmissible mice with a tar-geted 1.3-kb deletion of HS10, leaving only a single loxP site inits place, through standard ES cell-targeting technology. The tar-geted and deleted loci were confirmed by Southern blotting andPCR assays (Supplemental Fig. 2). We found that HS102/2 miceexhibited no significant differences in bone marrow and spleen cellnumbers or spleen weight compared with their WT littermates orage-matched WT mice controls (bone marrow cell numbers [twofemurs and two tibias]: 50.96 4.23 106 versus 53.06 4.63 106,n = 6, p = 0.75, Student t test; spleen cell numbers: 82.2 6 15.8 3106 versus 88.3 6 17.6 3 106, n = 8, p = 0.54, Student t test;spleen weight: 89.4 6 13.6 mg versus 93.8 6 13.4 mg, n = 6, p =0.57, Student t test). Furthermore, HS102/2 mice exhibited similarlevels of Igk+ and Igl+ B cells in spleen compared with WT mice(Fig. 3A). We further investigated the effect of HS10 deletion onthe development of B cell subpopulations in bone marrow byFACS. The percentages of Igk+ and Igl+ cells in bone marrowwere not significantly different between HS102/2 and WT mice(Fig. 3B). Finally, we evaluated early B cell compartments in bone

FIGURE 4. HS10 and Ed, but not E39, are re-

quired for maximal Igk expression in plasma cells.

(A) FACS analysis of splenic plasma cell levels

(B220low and negative CD138high, boxed or circled

regions) from WT, HS102/2, E392/2, and Ed2/2

mice, before (upper panels) and 5 d after (lower

panels) immunization with SRBCs. Data are repre-

sentative of independent FACS analyses from at least

six mice of each genotype. (B) FACS analysis of Igk

expression in permeabilized splenic plasma cells from

WT, HS102/2, E392/2, and Ed2/2 mice, before (up-

per panels) and 5 d after (lower panels) immunization

with SRBCs. Data are representative of independent

FACS analyses from at least six mice of each geno-

type. (C) Real-time PCR assays of Igk transcript levels

in FACS-sorted splenic plasma cells from WT,

HS102/2, E392/2, and Ed2/2 mice before and 5 d

after SRBC immunization. **p , 0.01.

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marrow by FACS. We found no significant differences in thepercentages of immature B cells (B220+IgM+) and circulatingmature B cells (B220highIgM+) (Fig. 3C). No significant differ-ences were observed in the percentage of bone marrow B220+

IgM2 B cells, which include both pro-B and pre-B cells, fromHS102/2 and WT mice (Fig. 3C). These B220+IgM2 B cells weregated and further analyzed for their surface expression of CD43and CD25. The results revealed that HS102/2 and WT mice havecomparable levels of pro-B cells (B220+IgM2CD43+CD252),large cycling pre-B cells (B220+IgM2CD43+CD25+), and smallpre-B cells (B220+IgM2CD432CD25+) (Fig. 3C). In addition, wethoroughly analyzed FACS-sorted pre-B cells and found no dif-ferences in the levels of Igk gene rearrangement or Jk regionusage between HS102/2 and WT mice (Supplemental Fig. 3).Therefore, we conclude that deletion of HS10 causes no signifi-cant defects in B cell development or in cell surface Ig expressionin spleen and bone marrow cells.

HS10 and Ed, but not E39, are required for maximal Igkexpression in plasma cells

Because HS10 forms a plasmacytoma cell-specific DNase I-hypersensitive site, we decided to focus our further immediateattention on its potential function in splenic plasma cells before andafter immunological challenge. We also evaluated the contributionsof E39 and Ed using knockout mice, because their roles in plasmacells have not been previously addressed. Splenic cells from un-immunized mice were stained with anti-CD138 and anti-B220Abs, and the percentage of plasma cells (B220low and negative

CD138high) were evaluated by FACS. We found no significantdifferences in the percentages of plasma cells from HS102/2,E392/2, and Ed2/2 mice compared with those of WT mice (Fig. 4A,upper panels) (HS102/2 versus WT: 0.25 6 0.08% versus 0.26 60.10%, n = 4, p = 0.88, Student t test; E392/2 versus Ed2/2: 0.28 60.08% versus 0.256 0.07%, n = 4, p = 0.59, Student t test; E392/2

and Ed2/2 versus HS102/2 and WT: n = 4, p = 0.94, one-wayANOVA). To trigger plasma cell expansion, we immunized WT,HS102/2, E392/2, and Ed2/2 mice with SRBCs in CFA andmonitored splenic plasma cell levels by FACS, as before. Wefound that the plasma cell populations dramatically increased5 d after immunization (Fig. 4A, lower panels) but that HS102/2,E392/2, and Ed2/2 mice exhibited similar increases in the numberof plasma cells compared with their WT littermates (WT versusHS102/2: 2.01 6 0.27% versus 2.05 6 0.3%, n = 4, p = 0.85,Student t test; E392/2 versus Ed2/2: 1.95 6 0.21% versus 1.88 60.32%, n = 4, p = 0.72, Student t test).We also analyzed Igk expression levels in plasma cells from

WT, HS102/2, E392/2, and Ed2/2 mice by intracellular Igkstaining, both before and after immunological challenge. FACSanalysis revealed that before immunization, the plasma cells fromWT, HS102/2, E392/2, and Ed2/2 mice expressed comparablelevels of Igk, which was indicated by the similar levels of Igkmean MFI: WT: (1084 6 69), HS102/2 (1105 6 64), E392/2

(1031 6 75), and Ed2/2 (1071 6 63) (Fig. 4B, upper panels,n = 4, p = 0.48, one-way ANOVA). It also should be noted thata significant fraction of plasma cells from E392/2 mice are Igkminus because they are Igl+; this observation but to a lesser extent

FIGURE 5. Both HS10 and Ed are required for

maximal Igk expression during a T cell-dependent Ab

response. (A–C) Production of anti-KLH and anti-TNP

Abs after immunization of WT, HS102/2, and Ed2/2

mice. The results show sera titers measured by ELISA

assays 7 and 14 d after immunization. Sera titers were

only at background levels at day 0 (6 0.2). The p

values are shown at the top of panels (compared with

WT levels, determined by one-way ANOVA using

SPSS 11.5 software).

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also occurs for Ed2/2 mice (Fig. 4B). However, 5 d after immu-nization, the plasma cells from HS102/2 and Ed2/2 mice, but notthose from E392/2 mice, expressed significantly lower levels of Igk(MFI) compared with those from WT cells (Fig. 4B, lower panels):HS102/2 versus WT: 1140 6 72 versus 1587 6 78, p = 0.01, n =4, Student t test; Ed2/2 versus E392/2 versus WT: 1124 6 64 and1598 6 71 and 1613 6 79, n = 4, p , 0.01, one-way ANOVA;Ed2/2 versus WT or Ed2/2 versus E392/2: p , 0.01, one-wayANOVA). These results indicate that both HS10 and Ed, but notE39, are required for maximal Igk expression in plasma cells.Finally, to confirm the above results we performed real-time PCR

assays to quantitate Igk gene transcript levels in plasma cellpopulations before and 5 d after immunization. In agreement withthe above results, ∼2.5-fold lower Igk transcript levels were ob-served in isolated plasma cells from HS102/2 and Ed2/2 micesplenic cells after immunization compared with levels in WT mice(Fig. 4C) (p , 0.01, n = 4, Student t test). We conclude that HS10and Ed, but not E39, are required for maximal Igk expression inplasma cells under conditions of strong Ag-induced immune re-sponse challenge.

HS10 and Ed are required for maximal T cell-dependent, butnot T cell-independent, Igk Ab responses

Because the above results revealed that HS10 and Ed are importantelements in maximizing Ab production in plasma cells, we decidedto evaluate their roles further in response to either T cell-dependentor T cell-independent Ag challenges. For this purpose, we im-munized WT, HS102/2, and Ed2/2 mice with the T cell-dependent Ag, KLH, as well as the T cell-independent Ag,TNP-Ficoll, and assayed the resulting Ab titers in sera by ELISA.As shown in Fig. 5A, 7 d after immunization, high levels of anti-KLH (IgM) were detected and remained at similar levels after2 wk. However, we found that the levels of anti–KLH-IgM inHS102/2 and Ed2/2 mice sera were significantly lower than thosein WT mice. In addition, although the serum levels of anti-KLH

(IgG) were quite low 7 d after immunization, they dramaticallyincreased after 2 wk. HS102/2 and Ed2/2 mice produced signif-icantly less anti–KLH-IgG 2 wk after immunization comparedwith WT mice (Fig. 5B, right panel). By contrast, no significantdifferences in anti–TNP-IgM were observed among WT, HS102/2,and Ed2/2 mice 7 or 14 d after immunization (Fig. 5C). Weconclude that HS10 and Ed are important elements in maximizingthe T cell-dependent immune response, presumably through sig-naling by CD40, but not in the T cell-independent process.

Effect of HS10 on chromosome conformation

To approach understanding the mechanism of HS10 function, weused the chromosome conformation-capture technique to addressthe consequences of deleting HS10 on various pair-wise inter-actions between the enhancers and a DNA restriction fragment thatwould bear HS10 in WT mice (Fig. 6). Unfortunately, because thisanalysis requires biochemical amounts of cells, plasma cells arepresent in insufficient amounts for this experiment to be practicalor feasible. Therefore, as an alternative, we generated biochemicalamounts of activated splenic cells for this analysis by culturingsplenic cells for 3 d with anti-CD40 Abs and LPS to trigger themex vivo to differentiate toward the plasma cell phenotype. We usedsamples from CD3ε+ T cells as a negative control. The results ofthe chromosome conformation-capture assays revealed that B cell-specific pair-wise interactions between Ei and E39 or Ei and Edwere similar between WT and HS102/2 mice samples (Fig. 6B,6C). However, it is noteworthy that the interactions between Eiand HS10 previously seen in plasmacytoma cells (13) were notobserved in stimulated splenic cells from WT mice (Fig. 6D).However, these culturing conditions do not achieve the level ofIgk gene expression exhibited by in vivo Ag-stimulated CD138+

plasma cells. Nevertheless, upon HS10 deletion, regional inter-actions with E39 were significantly diminished (Fig. 6E). Weconclude that HS10 itself is largely responsible for the previouslyobserved chromosome conformation-capture results.

FIGURE 6. Importance of HS10 in pair-wise

interactions between selected cis-acting sequences in

the Igk locus in stimulated splenic B cells from WT

and HS102/2 mice or in T cells from WT mice. (A)

Map of the 39 region of the mouse Igk locus. Cis-

acting sequences are indicated by rectangles. Num-

bered arrows indicate the primers used in selected

chromosome conformation-capture assays. Vertical

lines depict AseI enzyme cut sites. The region of the

HS10 deletion is demarcated by brackets. (B) Inter-

actions between Ei and E39 were assayed using PCR

primers 1 and 2 (n = 3, p , 0.01, WT versus T; p =

0.73, WT versus HS102/2). (C) Interactions between

Ei and Ed were assayed using PCR primers 1 and 3

(n = 3, p , 0.01, WT versus T; p = 0.96, WT versus

HS102/2). (D) Interactions between Ei and HS10

were assayed using PCR primers 1 and 4 (n = 3, p =

0.54, WT versus T; p = 0.71, WT versus HS102/2; p =

0.83, HS102/2 versus T). (E) Interactions between E39

and HS10 were assayed using PCR primers 2 and 4

(n $ 3, p , 0.01, WT versus T; p , 0.01, WT versus

HS102/2; p = 0.09, HS102/2 versus T). Data are

mean 6 SD. The p values were determined by one-

way ANOVA using SPSS 11.5 software. **p , 0.01.

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E39 and Ed, but not HS10, are involved in IgL isotype and Igkallelic exclusion

We next assayed for the role of HS10 in specifying IgL isotype andIgk allelic exclusion. We also evaluated how E392/2 and Ed2/2

mice would behave in these assays, because the functions of these

enhancers in these processes have not been directly addressed. We

first examined the population of Igk+Igl+ cells in the spleen from

WT, HS102/2, E392/2, and Ed2/2 mice by FACS analysis. As

shown in Fig. 7A, like WT mice, a normal very low level of

isotype-coexpressing B cells (B220+Igk+Igl+) was observed in

HS102/2 mice (WT: 0.68 6 0.05%; HS102/2: 0.73 6 0.04%).

Surprisingly, relative to levels in WT mice, a dramatic increase in

isotype-coexpressing cells was detected in E392/2 mice (4.46 6

0.35%), and a less striking, but significant, ∼2-fold increase was

also observed in Ed2/2 mice (1.926 0.20%). Because the number

of splenic Igk+ B cells in E392/2 and Ed2/2 mice was less than in

WT mice, we evaluated isotype exclusion by comparing the ratios

of the number of double producers (B220+Igk+Igl+) to the total

number of B cells that express Igk (B220+Igk+Igl+ plus B220+

Igk+Igl2). As shown in Fig. 7B, isotype coexpression was in-

FIGURE 7. E39 and Ed, but not HS10, are involved in IgL isotype and Igk allelic exclusion. (A) Analysis of Igk and Igl isotype exclusion. Single-cell

suspensions from spleen were simultaneously stained with anti-B220, anti-Igk, and anti-Igl Abs. The expression of Igk and Igl in WT, HS2/2, E392/2, and

Ed2/2 mice splenic B220+ cells was analyzed by FACS. Data are representative of independent FACS analyses from at least four mice of each genotype.

(B) Percentage of Igk+ cells that also express Igl. Data are mean6 SD. (C) Analysis of Igk allelic exclusion. Single-cell suspensions from spleen of Igkm/h,

IgkΔHS10m/h, IgkΔE39m/h, and IgkΔEdm/h mice were simultaneously stained with anti-B220, anti–mouse-Igk (mCk), and anti–human-Igk (hCk). The ex-

pression of mCk and hCk was analyzed by FACS. Percentages of cells residing in various windows are shown. Data are representative of independent FACS

analyses from at least four mice of each genotype. (D) Percentage of mCk+ cells that also express hCk. Data are mean6 SD. (E) Results of a real-time PCR

assay used to detect germline retention of Igk sequences in splenic B220+ Igk+(hi) B cells from the indicated lines of mice. The percentages of germline

retention were calculated by dividing the germline sequence levels of B cells by those of ES cells. Data are mean 6 SD (n = 3). (F) The levels of Igk gene

expression in B220+Igk+ splenic B cells between the indicated pairs of homo- and heterozygotic genetic lines were assayed for by determining MFI of Igk

in B220+ gated cells. (G) Biallelic expression was evaluated by measuring the relative MFI between homozygotes and the heterozygotes from each pair of

groups. *p , 0.05, **p , 0.01.

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creased significantly in both E392/2 and Ed2/2 mice (WT versusE392/2 or WT versus Ed2/2, n = 4, p , 0.01, one-way ANOVA)but not in HS102/2 mice.As one approach to assay for allelic exclusion, we bred our

mouse lines with human Ck knockin mice (23) to generate het-

erozygotes (Igkm/h). As shown in Fig 7C, HS10 deficiency ap-

parently did not alter the pattern of allelic exclusion, because, like

WT m/h heterozygotes, most cells producing molecules with

mouse Ck exons from DHS10 alleles did not produce counterparts

also possessing human Ck exons. In marked contrast, because of

competition between DE39 mouse Ck alleles and WT knockin

human Ck alleles, DE39 alleles were only able to contribute to

surface Igk expression in ∼10% of the total splenic B cells. Al-

though much less striking, heterozygotes possessing DEd alleles

also exhibited more human Ck+ B cells than mouse Ck+ B cells,

indicating that Ed may also play a role in allelic choice. In the

same way that we quantitated isotype exclusion, we evaluated

biallelic expression by comparing the ratios of the number of

double producers (B220+mCk+hCk+) to the number of total

B cells that expressed mouse Igk (B220+mCk+hCk+ plus B220+

mCk+hCk2). These results suggest that there is a severe impair-

ment of allelic exclusion in heterozygotes possessing DE39 alleles

compared with those possessing WT or DHS10 alleles and that

heterozygotes possessing DEd alleles may also exhibit a modest,

but significant, increase in biallelic expression (Fig. 7D, WT

versus E392/2 or WT versus Ed2/2, n = 4, p , 0.01, one-way

ANOVA).As another approach to assay for allelic exclusion, we measured

the extent of rearrangement of the locus in Igk+ splenic B cells using

real-time PCR to quantitate the level of germline Igk sequences

from HS102/2, E392/2, Ed2/2, and WT mice. As shown in Fig.

7E, the levels of such germline sequences in HS102/2, Ed2/2, and

WT mice samples were each ∼30% of those of ES cell controls,

whereas these levels were only ∼20% in E392/2 mice, a statisti-

cally significantly lower value than the other controls (p , 0.05).

This result further supports increased biallelic expression in E392/2

mice.As a final approach to assay for biallelic expression, we bred

our mouse lines with E392/2Ed2/2 mice, which are inactive for

Igk gene expression (19), to generate heterozygotes. We then

compared the level of Igk gene expression between these het-

erozygotic lines and their homozygous counterparts in B220+

Igk+ splenic cells by measuring their respective MFI of Igk. We

reasoned that if significant biallelic expression occurs in the

homozygotes, then it should be detectably higher than the level

of monoallelic expression in the heterozygotes. As shown in

Fig. 7F, HS10 or Ed deficiency did not alter the levels of Igk,

because HS102E39+Ed+/HS102E39+Ed+ homozygotes and HS102

E3+Ed+/HS10+E32Ed2 heterozygotes, or HS10+E39+Ed2/HS10+

E39+Ed2 homozygotes and HS10+E39+Ed2/HS10+E392Ed2 het-

erozygotes, expressed comparable levels of Igk, just like WT

control mice after being crossed with E392/2Ed2/2 mice. In

marked contrast, HS10+E392Ed+/HS10+E392Ed+ homozygotes

expressed significantly higher levels of Igk compared with HS10+

E392Ed+/HS10+E392Ed2 heterozygotes, consistent with signifi-

cant biallelic expression. We quantitated these results by mea-

suring the relative increase in MFI of Igk in B220+ Igk+ B cells

between homozygotes and the heterozygotes for each pair of

groups, an approach that revealed a statistically significant dif-

ference for the DE39 pair (Fig. 7G; WT versus E39, n = 4, p ,0.05, one-way ANOVA). In conclusion, each of these three ap-

proaches yield results that are strongly suggestive that allelic ex-

clusion is hampered in mice deficient in E39.

SHM is only modestly decreased in the Jk-Ck intronic regionof HS102/2 GC B cells

The E-box motif was reported to enhance SHM (32, 33). In ad-dition, Ig transcription efficiency has also been correlated withlevels of SHM (34). Because HS10 contains two E-box sites, andwe also observed reduced Igk expression in HS102/2-stimulatedplasma B cells along a reduced T cell-dependent Ab response, weinvestigated the possibility that SHM might be decreased inHS102/2 GC B cells. We purified B220+PNAhigh GC B cells fromPeyer’s patches of HS102/2 and WT mice by flow cytometry.Genomic DNA was isolated from these GC B cells, and VkJkrearrangements were amplified using high-fidelity PCR. VkJkrearrangement products were gel purified, cloned, and sequenced.We found that the mutation frequency within a 500-bp intronicregion downstream of Jk5 was 14.5 3 1023 mutations per base inWT GC B cells. In HS102/2 GC B cells, the mutation frequencyin the corresponding region was 11.6 3 1023 mutations per base(Fig. 8). Thus, the overall mutation frequency in HS102/2 GCB cells was reduced by only 20% compared with that of WTGC B cells, which, although small, was statistically significant(p , 0.01). Among clones bearing mutations, the mutation fre-quency was 20.1 3 1023 mutations per base in WT GC B cellsand 17.1 3 1023 mutations per base in HS102/2 GC B cells,a 15% reduction (Fig. 8). Control experiments revealed that theerror frequency of the high-fidelity PCR was only ∼0.2 mutations/kb. Furthermore, we found that the percentage of clones with nomutations was 33% in HS102/2 GC B cells compared with 25%in WT GC B cells (Fig. 8). In summary, these data indicate thatIgk SHM is modestly decreased in HS102/2 GC B cells fromPeyer’s patches.

DiscussionThe DNA sequence corresponding to HS10 was first identifiedthrough exploratory experiments using chromosome conformation-capture assays to search for sequences that interacted with the Igkgene enhancers (13). In the present investigation, we found thatHS10 is a plasmacytoma cell-specific DNase I-hypersensitive sitein chromatin and that its underlying sequence is evolutionarilyconserved. These observations provided the impetus for uncov-ering the function of HS10, because every B cell-specific hyper-

FIGURE 8. Analysis of Igk SHM in GC B cells. B220+PNAhigh GC B

cells were sorted from 4–6-mo-old WT (left panel) and HS102/2 (right

panel) mice, and SHM in a 500-bp Jk-Ck region that is immediately 39 of

Vk-Jk5 recombination products was analyzed. Each pie slice represents the

proportions of sequences having the indicated numbers of mutations. The

numbers of plasmid clones that were sequenced are shown in the center of

the pie. The mutation frequencies from the total or mutated clones are

shown at the bottom.

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sensitive site that has been identified previously in the mouse Igklocus has proven to have a significant function in B cells. HS102/2

mice exhibited normal B cell development, surface Igk expres-sion, and Vk-Jk rearrangement (Fig. 3, Supplemental Fig. 3). Inplasma cells, instead, we found that HS10 was required formaximal Igk expression but only after immunological challenges,which were T cell-dependent. This was also true for Ed but not forE39. In the case of T cell-dependent responses, B cells receivea primary stimulation that is transduced through BCR cross-linking, as well as costimulatory signaling from CD40 or TLRligands (35–38). Thus, our results imply that HS10 and Ed may beactivated by costimulatory signaling and mediate a special geneconformation that facilitates maximal Igk gene expression duringT cell-dependent Ab responses.It is not without precedent that the order of sequences along

amulti-gene family correlates with the developmental order of theirsequence usage (39). Interestingly, the locations of the cis-actingsequences 59 to 39 along the Igk locus correlate with the stages oftheir functional usage during B cell development: Ei and E39 arerequired for Vk-Jk joining (18), E39 and Ed are essential forrearranged gene transcription (19), and Ed and HS10 are neces-sary for maximal Igk expression in differentiating plasma cells(Figs. 4, 5). Our previous chromosome conformation-captureexperiments revealed that these cis-acting elements form pair-wise interactions with each other (13, 14), and work by us andother investigators revealed that strong phenotypes only resultwhen pairs of these cis-acting sequences are deleted together (18,19). These observations suggest that Ed and HS10 may haveoverlapping functions in plasma cells. This view is consistent withthe observation that HS10 exhibits pair-wise interactions with Eiand E39, but not with Ed, in plasmacytoma cells (13). Our currentchromosome conformation-capture experiments reveal that HS10is not required for Ei–E39 or Ei–Ed interactions but is required formaximal regional interactions with E39 in stimulated splenic cells.Our results also revealed that E39, but not HS10, contribute to

IgL isotype and Igk allelic exclusion. We have suggestive resultsthat Ed may also weakly participate in these processes. In thesecases, the level of surface Igk is detectably lower than that ob-served in WT cells, which would lead to less tonic BCR signalingand a less stringent suppression of continued RAG expression (40–43), which presumably accounts for the observed increases inisotype coexpression and biallelic expression due to continuedgene rearrangements.One of our experimental designs used to assay for allelic ex-

clusion put two Igk alleles in competition in the same cells: anallele carrying entirely WT cis-acting sequences marked by a hu-man Ck exon and another allele, with or without selected deletionsin HS10, E39, or Ed, possessing a mouse Ck exon. This approachnot only revealed the percentage of cells that are double Igkproducers but also allowed measurement of the allelic preferencesfor expression. The most striking skewed result in expression wasobserved in IgkΔE39m/h heterozygotes, where ,10% of the splenicB cells expressed the E39-deleted allele. This test also revealedthat the HS10 deletion had no effect on splenic cell Igk expres-sion, in agreement with our earlier results from FACS analyses ofHS102/2 cells.Previous studies of SHM in mice possessing targeted Igk gene-

enhancer deletions in the endogenous locus revealed that E39 andEd are each necessary to achieve WT levels of SHM in the 59region of the Igk gene’s J-C intron (17, 44). They further showedthat Ei in the native locus plays no role in determining the level ofSHM (44). We found that HS102/2 mice exhibited a modest 20%reduction in SHM over WT levels. We conclude that HS10 playsonly a very minor role in contributing to SHM under the con-

ditions studied, where, like the previous studies (17, 44), there wasno specific Ag challenge. Notably, both E39 and Ed are enhancersthat contribute maximally to Igk gene transcription levels, andeach plays a more significant role in SHM (17, 19, 44).HS10 resides near the 39 end of the Igk locus, ∼2 kb away from

the downstream Rpia1 housekeeping gene. Rpia is expressed atthe same low level in B cells as in fibroblasts (45), indicating thatthis housekeeping gene’s expression is totally buffered from theextraordinarily high level of expression exhibited by the Igk genein B cells. Moreover, we found that HS102/2 mice still expressthe Rpia gene at the same low constitutive level in B cells atdifferent developmental stages and in non-B cells (SupplementalFig. 4). Furthermore, the results of transient-transfection andstable-integration experiments proved that HS10 lacked silenceror insulator activities (Fig. 2, Supplemental Fig. 1), but theyrevealed that HS10 is a transcriptional coactivator of E39 but notof Ei (Fig. 2, Supplemental Fig. 1A).In conclusion, the results of the current study, together with our

previously published studies on the mouse Igk locus, revealedmultiple levels of communication between distal cis-acting regu-latory sequences through long-range interactions (9, 13, 14, 17,19). In the future, it will be germane to determine how theseelements contribute to the three-dimensional organization of thelocus in B cell nuclei and to the determine what other genes sharetranscription factories and common regulatory circuits with theIgk locus transcriptional interactome.

AcknowledgmentsWe thank Mark Schlissel for providing mice and Jose Cabrera for graphic

illustrations. We are indebted to Nicolai van Oers for insightful comments

and suggestions.

DisclosuresThe authors have no financial conflicts of interest.

References1. Yancopoulos, G. D., and F. W. Alt. 1985. Developmentally controlled and tissue-

specific expression of unrearranged VH gene segments. Cell 40: 271–281.2. Brekke, K. M., and W. T. Garrard. 2004. Assembly and analysis of the mouse

immunoglobulin kappa gene sequence. Immunogenetics 56: 490–505.3. Schatz, D. G., and Y. Ji. 2011. Recombination centres and the orchestration of

V(D)J recombination. Nat. Rev. Immunol. 11: 251–263.4. Schlissel, M. S. 2004. Regulation of activation and recombination of the murine

Igkappa locus. Immunol. Rev. 200: 215–223.5. Durdik, J. M., M. W. Moore, and E. Selsing. 1984. Novel k light-chain gene

rearrangements in mouse l light chain-producing B lymphocytes. Nature 307:749–752.

6. Nadel, B., P. A. Cazenave, and P. Sanchez. 1990. Murine lambda gene rear-rangements: the stochastic model prevails over the ordered model. EMBO J. 9:435–440.

7. Shimizu, T., T. Iwasato, and H. Yamagishi. 1991. Deletions of immunoglobulinC k region characterized by the circular excision products in mouse splenocytes.J. Exp. Med. 173: 1065–1072.

8. Vela, J. L., D. Aı̈t-Azzouzene, B. H. Duong, T. Ota, and D. Nemazee. 2008.Rearrangement of mouse immunoglobulin kappa deleting element recombiningsequence promotes immune tolerance and lambda B cell production. Immunity28: 161–170.

9. Liu, Z. M., J. B. George-Raizen, S. Li, K. C. Meyers, M. Y. Chang, andW. T. Garrard. 2002. Chromatin structural analyses of the mouse Igkappa genelocus reveal new hypersensitive sites specifying a transcriptional silencer andenhancer. J. Biol. Chem. 277: 32640–32649.

10. Queen, C., and D. Baltimore. 1983. Immunoglobulin gene transcription is ac-tivated by downstream sequence elements. Cell 33: 741–748.

11. Xu, M., M. B. Barnard, S. M. Rose, P. N. Cockerill, S.-Y. Huang, andW. T. Garrard. 1986. Transcription termination and chromatin structure of theactive immunoglobulin k gene locus. J. Biol. Chem. 261: 3838–3845.

12. Meyer, K. B., and M. S. Neuberger. 1989. The immunoglobulin k locus containsa second, stronger B-cell-specific enhancer which is located downstream of theconstant region. EMBO J. 8: 1959–1964.

13. Liu, Z., and W. T. Garrard. 2005. Long-range interactions between three tran-scriptional enhancers, active Vkappa gene promoters, and a 39 boundary se-quence spanning 46 kilobases. Mol. Cell. Biol. 25: 3220–3231.

The Journal of Immunology 2731

by guest on April 12, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

14. Liu, Z., Z. Ma, L. S. Terada, and W. T. Garrard. 2009. Divergent roles of RelAand c-Rel in establishing chromosomal loops upon activation of the Igkappagene. J. Immunol. 183: 3819–3830.

15. Xu, Y., L. Davidson, F. W. Alt, and D. Baltimore. 1996. Deletion of the Ig k lightchain intronic enhancer/matrix attachment region impairs but does not abolishV k J k rearrangement. Immunity 4: 377–385.

16. Gorman, J. R., N. van der Stoep, R. Monroe, M. Cogne, L. Davidson, andF. W. Alt. 1996. The Ig(kappa) enhancer influences the ratio of Ig(kappa) versusIg(lambda) B lymphocytes. Immunity 5: 241–252.

17. Xiang, Y., and W. T. Garrard. 2008. The downstream transcriptional enhancer,Ed, positively regulates mouse Ig kappa gene expression and somatic hyper-mutation. J. Immunol. 180: 6725–6732.

18. Inlay, M., F. W. Alt, D. Baltimore, and Y. Xu. 2002. Essential roles of the k lightchain intronic enhancer and 39 enhancer in k rearrangement and demethylation.Nat. Immunol. 3: 463–468.

19. Zhou, X., Y. Xiang, and W. T. Garrard. 2010. The Igk gene enhancers, E39 andEd, are essential for triggering transcription. J. Immunol. 185: 7544–7552.

20. Aasland, R., and A. F. Stewart. 1999. Analysis of DNaseI hypersensitive sites inchromatin by cleavage in permeabilized cells. Methods Mol. Biol. 119: 355–362.

21. Fulton, R., and B. G. Van Ness. 1993. Kappa immunoglobulin promoters andenhancers display developmentally controlled interactions. Nucleic Acids Res.21: 4941–4947.

22. Tallquist, M. D., and P. Soriano. 2000. Epiblast-restricted Cre expression inMORE mice: a tool to distinguish embryonic vs. extra-embryonic gene function.Genesis 26: 113–115.

23. Casellas, R., T.-A. Y. Shih, M. Kleinewietfeld, J. Rakonjac, D. Nemazee,K. Rajewsky, and M. C. Nussenzweig. 2001. Contribution of receptor editing tothe antibody repertoire. Science 291: 1541–1544.

24. Gross, D. S., and W. T. Garrard. 1988. Nuclease hypersensitive sites in chro-matin. Annu. Rev. Biochem. 57: 159–197.

25. Horibata, K., and A. W. Harris. 1970. Mouse myelomas and lymphomas inculture. Exp. Cell Res. 60: 61–77.

26. Seidman, J. G., and P. Leder. 1980. A mutant immunoglobulin light chain isformed by aberrant DNA- and RNA-splicing events. Nature 286: 779–783.

27. Lundak, R. L., and D. J. Raidt. 1973. Cellular immune response against tumorcells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cellsuspensions against DBA mastocytoma cells. Cell. Immunol. 9: 60–66.

28. Lieber, M. R., J. E. Hesse, K. Mizuuchi, and M. Gellert. 1987. Developmentalstage specificity of the lymphoid V(D)J recombination activity. Genes Dev. 1:751–761.

29. Kim, K. J., C. Kanellopoulos-Langevin, R. M. Merwin, D. H. Sachs, andR. Asofsky. 1979. Establishment and characterization of BALB/c lymphomalines with B cell properties. J. Immunol. 122: 549–554.

30. Shevach, E. M., J. D. Stobo, and I. Green. 1972. Immunoglobulin and u-bearingmurine leukemias and lymphomas. J. Immunol. 108: 1146–1151.

31. Quong, M. W., W. J. Romanow, and C. Murre. 2002. E protein functionin lymphocyte development. Annu. Rev. Immunol. 20: 301–322.

32. Michael, N., H. M. Shen, S. Longerich, N. Kim, A. Longacre, and U. Storb.2003. The E box motif CAGGTG enhances somatic hypermutation withoutenhancing transcription. Immunity 19: 235–242.

33. Schoetz, U., M. Cervelli, Y. D. Wang, P. Fiedler, and J. M. Buerstedde. 2006.E2A expression stimulates Ig hypermutation. J. Immunol. 177: 395–400.

34. Odegard, V. H., and D. G. Schatz. 2006. Targeting of somatic hypermutation.Nat. Rev. Immunol. 6: 573–583.

35. Haxhinasto, S. A., and G. A. Bishop. 2004. Synergistic B cell activation byCD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factorregulation. J. Biol. Chem. 279: 2575–2582.

36. Meyer-Bahlburg, A., S. Khim, and D. J. Rawlings. 2007. B cell intrinsic TLRsignals amplify but are not required for humoral immunity. J. Exp. Med. 204:3095–3101.

37. Rubtsov, A. V., C. L. Swanson, S. Troy, P. Strauch, R. Pelanda, and R. M. Torres.2008. TLR agonists promote marginal zone B cell activation and facilitateT-dependent IgM responses. J. Immunol. 180: 3882–3888.

38. Jain, S., S. B. Chodisetti, and J. N. Agrewala. 2011. CD40 signaling synergizeswith TLR-2 in the BCR independent activation of resting B cells. PLoS ONE 6:e20651.

39. Pearson, J. C., D. Lemons, and W. McGinnis. 2005. Modulating Hox genefunctions during animal body patterning. Nat. Rev. Genet. 6: 893–904.

40. Monroe, J. G. 2006. ITAM-mediated tonic signalling through pre-BCR and BCRcomplexes. Nat. Rev. Immunol. 6: 283–294.

41. Tze, L. E., B. R. Schram, K. P. Lam, K. A. Hogquist, K. L. Hippen, J. Liu,S. A. Shinton, K. L. Otipoby, P. R. Rodine, A. L. Vegoe, et al. 2005. Basalimmunoglobulin signaling actively maintains developmental stage in immatureB cells. PLoS Biol. 3: e82.

42. Keren, Z., E. Diamant, O. Ostrovsky, E. Bengal, and D. Melamed. 2004.Modification of ligand-independent B cell receptor tonic signals activatesreceptor editing in immature B lymphocytes. J. Biol. Chem. 279: 13418–13424.

43. Verkoczy, L., B. Duong, P. Skog, D. Aı̈t-Azzouzene, K. Puri, J. L. Vela, andD. Nemazee. 2007. Basal B cell receptor-directed phosphatidylinositol 3-kinasesignaling turns off RAGs and promotes B cell-positive selection. J. Immunol.178: 6332–6341.

44. Inlay, M. A., H. H. Gao, V. H. Odegard, T. Lin, D. G. Schatz, and Y. Xu. 2006.Roles of the Ig kappa light chain intronic and 39 enhancers in Igk somatichypermutation. J. Immunol. 177: 1146–1151.

45. Apel, T. W., A. Scherer, T. Adachi, D. Auch, M. Ayane, and M. Reth. 1995.The ribose 5-phosphate isomerase-encoding gene is located immediatelydownstream from that encoding murine immunoglobulin k. Gene 156: 191–197.

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