The EMBO Journal vol.12 no.12 pp.4635-4645, 1993

Mutations of the intronic IgH enhancer and its flankingsequences differentially affect accessibility of the JHlocus

Jianzhu Chen, Faith Young, Andrea Bottaro,Valerie Stewart, Russell K.Smithand Frederick W.Alt"Howard Hughes Medical Institute, Children's Hospital and Departmentof Genetics, Harvard Medical School and Center for Blood Research,320 Longwood Avenue, Boston, MA 02115, USAICorresponding author

Communicated by K.RajewskyTo investigate the role of intronic immunoglobulin heavychain (gH) enhancer (Eit) in generating accessibility ofthe JH locus for VDJ recombination, we generated EScells in which EA or its flanking sequences were mutatedby replacement with or insertion of an expressed neorgene. Heterozygous mutant ES cells were used to generatechimeric mice from which pre-B cell lines were derivedby transformation of bone marrow cells with Abelsonmurine leukemia virus (A-MuLV). Comparison of therearrangement status of the normal and mutated allelesin individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bpregion immediately downstream from the core EIt [whichincludes part of the 3' matrix associated region (MAR)and the Iu exon] had no obvious effect on rearrangementof the targeted allele, indicating that insertion of atranscribed neor gene into the JH-CZ intron does notaffect JH accessibility. In contrast, replacement of anoverlapping 1 kb DNA fragment that contains the Elresulted in a dramatic cis-acting inhibition of rearrange-ment, demethylation and germline transcription of theassociated JH locus. Surprisingly, insertion of the neorgene into the 5' MAR sequence - 100 bp upstream of thecore Ey also dramatically decreased recombination of thelinked JH locus; but, in many lines, did not preventdemethylation of this locus. We conclude that integrityof the EA and upstream flankin sequences is requiredfor efficient rearrangement of the JH locus and thatdemethylation of this locus, per se, does not necessarilymake it a good substrate for VDJ recombination.Key words: EIt enhancer/gene targeting/methylation/re-arrangement/transcription

IntroductionDifferentiation of B and T lymphocytes requires the assemblyof immunoglobulin (Ig) and T cell receptor (TCR) variableregion genes, respectively, from individual V, D and J genesegments by a common site specific activity referred to asVDJ recombinase (Tonegawa, 1983; Davis and Bjorkman,1988; Blackwell and Alt, 1989). VDJ recombination occursin precursor B and T cells where its activity is dependenton the expression of the RAG-1 and RAG-2 gene products(Schatz et al., 1989; Oettinger et al., 1991; Mombaertset al., 1992; Shinkai et al., 1992). Witiin the pre-B and pre-

Oxford University Press

T lineages, VDJ recombination is highly regulated in anumber of different contexts including tissue-specificity,developmental stage-specificity and allelic exclusion(reviewed by Blackwell and Alt, 1989). Given a commonVDJ recombinase (Yancopoulos et al., 1986), the qualityof a particular locus as a substrate for the recombinase (e.g.accessibility) determines whether or not it will be rearrangedonce recombinase is expressed (Alt et al., 1992). Factorsthat confer an accessible character to endogenous V, D andJ gene segments are still poorly understood; althoughaccessibility has variously been correlated with 'open'chromatin structure, hypomethylation and transcription ofthe unrearranged gene segments (reviewed by Alt et al.,1992).Endogenous antigen receptor loci are often transcribed

before they are rearranged. For example, prior to D to JHrearrangement, at least one transcript (j0) appears to initiateupstream of DQ52 and run through the JH and CI constantregion gene segments (Alessandrini and Desiderio, 1991;Schlissel et al., 1991). VH to DJH rearrangement is alsousually preceded by active transcription of VH genesegments (Yancopoulos and Alt, 1985). However, despitethe strong correlation between transcription and accessibilityin these and other endogenous antigen receptor loci (Schlisseland Baltimore, 1989; Anderson et al., 1992), it has not beenpossible to test this relationship directly. Accessibility ofrecombination substrates and transgenes has also beencorrelated with hypomethylation. In cell lines, methylationdiminishes recombination potential of transiently introducedsubstrates (Hsieh and Lieber, 1992); in transgenic mice,recombination is restricted primarily to hypomethylatedtransgenic substrates (Engler et al., 1991). Becauseexpressed loci are generally hypomethylated (reviewed byCedar, 1988; Bird, 1992), the precise effect of transcriptionversus demethylation or other unknown factors in suchstudies is difficult to address.

Transcriptional enhancer elements can dominantly targetlinked gene segments for VDJ recombination both indeveloping lymphocytes of mice transgenic for particularrecombination substrates or in cell-based transfection systems(Ferrier et al., 1990; Rathbun et al., 1993). These studiesemployed a VDJ recombination substrate that consists ofgermline TCR V(3, Dol and Jo3 segments linked 5' to a portionof the IgH locus that includes most of the JH-C,U intron plusthe Cit exons. A version of this substrate that lacked anyknown transcriptional enhancer elements was neithertranscribed nor rearranged in transgenic mice or transfectedcell lines. However, insertion of a segment of DNA thatcontained the Elt element resulted in efficient rearrangementand expression of the substrate in developing lymphocytesand transfected lymphoid cell lines (Ferrier et al., 1990;Rathbun et al., 1993). Recent studies have shown that anumber of different lymphoid and non-lymphoid enhancerelements can target rearrangement of this substrate (Oltzet al., 1993).

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J.Chen et al.

To clarify the role of the E/s element in promotingdemethylation, transcription and rearrangement of theendogenous JH locus, we have introduced various mutationsinto ES cells, all based on insertion of an expressed neorgene, which replaces or inserts sequences in or around theendogenous Eli element. The heterozygous mutant ES cellswere used to generate somatic chimeric mice from whichES cell-derived A-MuLV transformed pre-B lines wereestablished. Analyses of these mutant cell lines have allowedus to demonstrate the importance of the integrity of theendogenous Eli element in promoting germline transcription,demethylation and rearrangement of the associated JH locus,and have provided some new insights into interrelationshipsbetween these processes.

ResultsTargeted mutations of the E,u region in ES cellsThe IgH intronic enhancer (Eli) is within the 1 kbXbaI-XbaI fragment (Baneri et al., 1983; Gillies et al.,

1983). It includes the core enhancer region, the 220 bpHinfl -Hinfl fragment, which contains most defined factor-binding motifs (solid box in Figure IA; reviewed by Staudtand Lenardo, 1991; Nelsen and Sen, 1992), as well as twoflanking matrix associated regions (MARs: hatched boxesin Figure IA) which contain topoisomerase II and nuclearmatrix binding sites (Cockerill et al., 1987; Dickinson et al.,1992). Three different targeted mutations were generatedin ES cells (Figure LA). The first mutation, termed Elkreplacement, replaced the entire 1 kb XbaI-XbaI fragment,including the core EA and flanking MARs, with a neorgene. The neor gene was driven by the phosphoglyceratekinase promoter (PGK-1; Tybulewicz et al., 1991), which,although ubiquitously expressed, does not have any knownenhancer activity (Adra et al., 1987). The second mutation,termed 3' replacement, replaced the 0.7 kb EcoRI-HindHlfragment 3' of the core E/t with the PGK-neor gene. Thisreplaced region contains part of the 3' MAR and the IA exon(Lennon and Perry, 1985; Su and Kadesch, 1990). The thirdmutation, termed 5' insertion, inserted the PGK-neor gene

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Fig. 1. Targeted mutations of the Eu region. (A) Genomic configuration of the targeted loci after homologous recombination. The Ey region includesthe core enhancer (220 bp Hinfl-Hinfl fragment, Nelsen and Sen, 1992) as indicated by the solid box and the flanking matrix associated regions(MARs, Cockerill et al., 1987) as shown by the hatched boxes. DQ52, AHand Cy gene segments are represented by solid or open boxes. Threeindependent mutations were introduced in the Ept region. The EA replacement mutation replaces the 1.0 kb XbaI -XbaI fragment that includes thecore E1s and flanking MARs with a PGK-neor gene. The 5' insertional mutation inserts the same PGK-neor gene at the ScaI site 5' of the coreenhancer but within the MAR. The 3' replacement mutation replaces the 0.7 kb EcoRl-HindiH fragment 3' of the core Eu with the PGK-neor gene.The region replaced in the latter mutation includes part of the 3' MAR and the IA exon (Lennon and Perry, 1985). Restriction sites are: E, EcoRI;H, HindIm; S, SacI; A, ApaI; N, NaeI; X, XbaI; and Sc, ScaI. (B) Probes used in Southern and Northern analyses. The intron probe is the 380 bpSacI-ApaI fragment between DQ52 and JH1 The JH probe is the 440 bp NaeI-XbaI fragment between JH4 and Eu. The neor probe is the 0.9 kbPstI-PstI fragment from the neor gene. (C) Expected hybridization patterns of the normal and the targeted JH alleles before and after rearrangementby SacI digestion and hybridization with the neor, JH and intron probes. (D) An example of Southern blotting analyses that verify homologousrecombination at the EA region. DNA from STO feeder cells, normal ES cells and potentially targeted ES cells was digested with SacI and assayedfor hybridization with the JH probe. DNA samples assayed are as follows: lane 1, STO; lane 2, CCE; lanes 3 and 4, heterozygous mutant ES cellswith Ea replacement; lanes 5 and 6, heterozygous mutant ES cells with 5' insertion; and lane 7, heterozygous mutant ES cells with 3' replacement.The normal allele generates a hybridizing band at the expected size of 5.0 kb. The targeted alleles generate hybridizing bands at the expected sizes of5.8, 6.8 and 6.2 kb for Eu replacement, 5' insertion and 3' replacement, respectively. The normal and targeted bands are indicated. X-HindJHmarkers are shown.4636

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at the Scal site 5' of the core EA but within the 5' MAR.Homologous recombination was achieved by transfection

of the three independent targeting constructs (see Materialsand methods for details) into CCE ES cells and selectionfor doubly (G418 and gancyclovir) resistant clones.Homologous recombinants at the EIL region were confirmedby Southern blotting analyses with multiple digests andprobes (diagrammed in Figure 1). For example, digestionof DNA with SacI and hybridization with the 440 bpNaeI-XbaI JH probe (Figure lA and B) gave a 5.0 kbband from the normal allele and 5.8, 6.8 and 6.2 kb bandsof the targeted alleles, corresponding to the E,u replacement,5' insertion and 3' replacement, respectively (Figure IC andD). The DQ52-JHl intron and the neor gene probes also gavethe expected hybridization patterns (Figure 1 and Figure 2,lanes 1, 12 and 23), confirming that intended mutations hadbeen achieved.

Generation of chimeric mice and A-MuLV transformedpre-B cell linesTo assay effects of introduced mutations on the associatedJH locus, we injected the heterozygous mutant ES cellclones into C57BL/6J blastocysts which were then allowedto develop into chimeric mice. Because A-MuLVtransformed pre-B cell lines can be generated more efficientlyfrom mice of the 129 strain than from C57BL/6J mice(Rosenberg and Baltimore, 1976), the different backgroundsof the ES cells and blastocysts were expected to facilitatederivation of pre-B lines from precursor lymphocytes of EScell origin. Resulting chimeras (recognized by their agouticoat color) were sacrificed at 1 month of age and the bonemarrow cells were infected with A-MuLV to derive pre-Bcell lines. Splenocytes were analyzed by flow cytometry withanti-allotpe antibodies to assess contribution of injected EScells (IgMa) versus blastocyst (IgMb) to the B cellcompartment. As anticipated, over 90% of A-MuLVtransformants were of ES cell origin as demonstrated by thepresence of the integrated neor gene (Table I, Figure 2Aand data not shown), even though ES-derived B cells rangedfrom only 20% to 50% of total splenic B cells. A-MuLVtransformants derived from chimeras were B220+ andindependent as determined by viral integration patterns (datanot shown). We established 53, 34 and 33 independent A-MuLV transformants from pre-B cells that, respectively,harbored the 3' replacement, EA replacement, and 5'insertion (Table 1). In addition, for each experiment, severalA-MuLV transformants were obtained from pre-B cells thatwere derived from the host blastocyst (Table I).

The integrity of the Eu region is required for efficientDJH rearrangementTo analyze the effect of the three targeted mutations on JHrearrangement, genomic DNA from individual pre-B cell

lines was digested with Sacl, which cuts 5' between DQ52and JH1 and 3' before the SA region (Figure IA), andassayed for hybridization with probes specific for the neorgene, the JH region and the DQ52-JHl intervening sequences(Figure iB). In the context of this analysis, the neor probewill hybridize to and detect potential rearrangements of onlythe targeted allele; the JH probe will hybridize to and detectpotential rearrangements of both the targeted and the normalalleles; and the intron probe will hybridize only to fragmentsderived from alleles that have not undergone DJH or VHDJHrearrangement (summarized in Figure IC). A-MuLV-transformed pre-B cell lines from normal mice almost alwayshave rearrangements of both JH loci at the time of isolation;these may be either DJH or VHDJH rearrangements (Altet al., 1984). In addition, many such lines actively makeD to JH replacements or VH to DJH rearrangements duringpropagation; these are evidenced by multiple JH-hybridizingbands of varying intensity (Alt et al., 1984; Reth et al.,1986). When genomic DNA was assayed as described above,lines that represented normal, blastocyst-derived pre-B cellswere identified by lack of neor-hybridizing fragments, lackof germline JH-hybridizing fragments accompanied by thepresence of two or more non-germline-sized JH-hybridizingfragments, and lack of any fragments that hybridized to theDQ52-JH1 intron probe (e.g. Figure 2, lane 18).When assayed for hybridization to the neor probe, DNA

from A-MuLV transformants derived from ES cellsharboring the 3' replacement gave hybridizing fragmentswhich differed from that of the parental ES cells (Figure 2A,lane 1) and which were generally different from each other(Figure 2A, lanes 2-1 1; representative data are shown). Inaddition, fragments of multiple sizes were observed in DNAfrom a single line, indicating ongoing rearrangement of thetargeted allele. When assayed for hybridization with the JHprobe, all the neor-hybridizing fragments were identifiedand at least one additional novel fragment appeared for eachline. The latter fragments represent rearrangements of thenon-targeted allele (Figure 2B, lanes 1-11). Finally, theDQ52-JH intron probe hybridized to DNA fragments fromboth the normal and targeted alleles of mutant ES cells butfailed to hybridize to any DNA fragments from mutant pre-B cell lines (Figure 2C, lanes 1-11), confirming thecomplete rearrangement of both the targeted and normalallele in the pre-B cells. Of 53 independent pre-B cell linesharboring the 3' replacement mutation, all rearranged boththe normal and the targeted JH alleles (Table I).Furthermore, ES cells homozygous for the 3' replacementmutation generated sIgM-positive B cells in chimeric mice(A.Bottaro, J.Chen and F.Alt, unpublished data). Thus,insertion of an expressed neor transcription unit (see below)into the JH-C!t intron at a position just 3' of the core E/t hasno obvious effect on the ability of the mutated allele toproduce and express VH(D)JH rearrangements.

Table I. Summary

Mutations % ES-derived Abelson lines Rearrangementa Transcripts Methylationbsplenic B cells ES-derived Blastocyst-derived DQ52-JH neor JH neor

3' replacement 22-28 53 2 53/53 0/20 20/20 - -EA replacement 20 34 3 4/34 0/19 19/19 22/23 0/125' insertion 49 33 4 6/33 0/17 17/17 11/22 0/11

aThe normal alleles are rearranged in all mutant Abelson lines and both alleles are rearranged in Abelson lines derived from blastocysts.bThe HpaII site within the 2.3 kb Hindm JH fragment are not methylated in nine RAG-2 and two RAG-1 deficient Abelson lines analyzed.

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Fig. 2. Rearrangement status of the normal and the targeted JH alleles in heterozygous mutant A-MuLV transformants. DNA from heterozygousmutant ES cells and A-MuLV transformants was digested with SacI and electrophoresed on 0.8% agarose gels. (A) Filters were first hybridized withthe neor probe (Figure IB) to determine the rearrangement status of the targeted alleles. (B) The neor probe was removed from the filters shown inpanel A and they were assayed for hybridization to the JH probe (Figure iB) to determine the rearrangement status of the normal alleles in the samelines. (C) The JH probe was removed from the filters shown in panel B and they were assayed for hybridization with the intron probe (Figure IB) toconfirm the rearrangement status of both the normal and the targeted alleles. DNA from A-MuLV transformants with the 3' replacement tends togenerate similarly migrating bands following SacI digestion and hybridization to the neor probe (lanes 3 and 6). When the same DNA was digestedwith EcoRV and hybridized with the neor probe, different sized bands were generated (data not shown), suggesting different rearrangements. Inaddition, these A-MuLV trasnformants were known to be independent because of their different endogenous rearrangements (panel B, lanes 3 and 6)and different viral integrations (data not shown). Band position of the normal and targeted alleles from the heterozygous mutant ES cells areindicated. X-HindIII markers are shown.

Based on the finding that replacements of sequences, perse, in the JH-C,U intron in the vicinity of the core E/t do notblock rearrangement of the locus, we assayed for the effectof replacing the actual E,u element and obtained a

dramatically different result. Assay for hybridization to theneor probe demonstrated that only four of 33 pre-B celllines analyzed had rearranged their E,u-replaced allele;although faint hybridizing fragments of novel size werepresent in DNAs of some lines (Table I; Figure 2A, lanes12-22; representative data are shown), suggesting a lowlevel of ongoing rearrangement. We confirmed thatrearrangement of the mutant allele was inhibited specificallyby assaying for hybridization to the JH probe. All of themutant 'germline-sized' fragments that hybridized to theneor probe, also hybridized to the JH probe. In addition, allof these lines lacked a wild-type germline-sized fragmentthat hybridized to the JH probe but did have at least oneother non-germline size, JH-hybridizing fragment thatrepresented the rearranged normal allele (Figure 2B, lanes

13 -22). Finally, nearly all of the ES-cell derived pre-B linesmaintained a mutant 'germline-sized' fragment thathybridized to the intron probe (Figure 2C, lanes 12-22;Table I). Clearly, these lines were derived from pre-B cellsthat underwent the normal differentiation process in whichthe JH alleles were exposed to physiological levels of VDJrecombinase as evidenced by the rearranged normal alleles(Figure 2B and C; Table I). Therefore, these resultsdemonstrate that the Elt replacement resulted in a cis-actingblock in JH rearrangement.We next determined the effects of inserting the neor gene

in the MAR just 5' of the core Eli. Remarkably, this mutationyielded results quite similar to those obtained with A-MuLV-transformants in which the entire E,u region was replaced.Thus, DNA from the majority of pre-B cell lines with 5'insertion maintained an unrearranged neor-hybridizingfragment that also hybridized to the JH and the intronprobes (Figure 2, lanes 23-33). In addition, none of thelines had wild-type germline-sized JH-hybridizing fragments

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Fig. 3. Analyses of germline transcripts of the JH locus. Approximately 10 itg of total RNA from a RAG-2-deficient A-MuLV transformant,heterozygous mutant ES cells and A-MuLV transfornants with targeted mutations in the EA region was fractionated on 1 % formaldehyde-agarosegels. Filters were first hybridized to the intron probe (Figure iB) to assay for transcripts that initiate from the DQ52 region on the unrearranged,targeted alleles (panel A). Then, the intron probe was removed from the filters and they were assayed for hybridization to the neor probe(Figure IB). Lanes 1, 8 and 15 are RNA from a RAG-2-deficient A-MuLV transformant that expresses the it' transcript from the DQ52 region butfew neo transcripts. Lanes 2, 9 and 16 are RNA from the three types of mutant ES cells which do not express the it transcript but expressabundant neo transcripts. The rest of the lanes are RNA from Abelson lines with either 3' replacement, Eli replacement or 5' insertion which do notexpress the y° transcript but express abundant neor transcripts. 28S and 18S ribosomal RNAs are indicated.

that hybridized to the intron probe and all had rearrangedJH-hybridizing fragments that did not hybridize to the neoror intron probes, indicating complete rearrangement of thenormal allele (Figure 2, lanes 23-33; Table I). Therefore,insertion of an expressed neor gene into the MARsequences just 5' of the core EM resulted in a major cis-actingblock in JH-rearrangement.

E# replacement abolishes transcription initiated fromthe D052 regionThe transcriptional enhancer activity of the EM region wasdefined by its ability to augment transcriptional activity ofeither a heterologous promoter or a VH promoter intransfected or transgenic expression constructs (reviewed byStaudt and Lenardo, 1991; Nelsen and Sen, 1992). We havenow tested this function with respect to the endogenous locus.In developing B cell precursors that have not yet undergonea DJH rearrangement, the A' primary transcript appears toinitiate upstream of DQ52, terminate downstream of the CMexons and is processed to generate an -2.3 kb transcript(Alessandrini and Desiderio, 1991; Schlissel et al., 1991;J.Gorman and F.W.Alt unpublished data). To assay for theexpression of y° transcripts, total RNA from various A-MuLV pre-B cell lines was assayed for hybridization to theintron probe (Figure 3A). The A' transcripts were readilydetected in total RNA from RAG-2-/- A-MuLVtransformants, which have germline JH loci (Figure 3A,lanes 1, 8 and 15, Shinkai et al., 1992). As expected, no

A' transcripts were observed in RNA from mutant ES cells(Figure 3A, lanes 2, 9 and 16) and pre-B cell lines harboringthe 3' replacement, as these cells had undergone DJHrearrangement and, therefore, deleted the intron probe-hybridizing sequences from their genome (Figure 3A, lanes3-7; representative data are shown; Table I). However, nointron probe-hybridizing transcripts were detected in totalRNA from either the pre-B cell lines harboring the EMreplacement or the 5' insertion (Figure 3A, lanes 10-14and 17-21; representative data are shown; Table I). Thesepre-B lines all had a non-rearranged JH allele; therefore, thetwo mutations resulted in a substantially reducedaccumulation of germline transcripts.To assay for expression of the inserted neor gene in these

lines, the intron probe was removed from the filters describedabove and they were re-hybridized to the neor probe(Figure 3B). Abundant neor gene transcripts, at theexpected size of 1.5 kb, were detected in all of the variousEl mutant A-MuLV transformants analyzed (Figure 3B;Table I) and also in the heterozygous mutant ES cells(Figure 3B). A much less abundant and slightly smallerneor transcript was detected in RAG-2-deficient A-MuLVtransformants. The difference in expression level of theneor gene between RAG-2-deficient and EM-mutant A-MuLV transformants was probably due to the differenttranscriptional regulatory elements used to drive neor geneexpression; the PGK-1 promoter was used (PGK-neo') forthe various targeted mutations of the EM region and the

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Fig. 4. Nuclear run-on assays for transcription initiation from the DQ52 region in A-MuLV transformants. (A) A schematic map of the JH locus. Thegennline 1° transcription is postulated to initiate upstream of DQ52; however, the precise initiation site(s) has not been defined (Alessandrini andDesiderio, 1991). B represents BamHI and the rest of the map is the same as in Figure 1A. (B) Plasmid fragment patterns after digestion withvarious emzymes. The pJH plasmid, which contains the 6.2 kb EcoRI fragment of the JH locus subcloned into pUC18, generates 0.8, 2.3, 1.0 and2.0 kb JH fragments and a 2.7 kb vector fragment after EcoRl and BamHI digestion. EcoRI plus Hindm digestion of the PGK-neor plasmidgenerates a 1.9 kb neor insert fragment and a 3.5 kb vector fragment. PstI digestion of the pGAPDH plasmid generates a 1.4 kb insert fragment and3.2 kb vector fragment. (C) Hybridization patterns of duplicate filters with run-on RNA probes. Ethidium bromide-stained gel patterns of variousdigested plasmid DNAs are shown in the left panel. As indicated on the figure, other panels (from left to right) represent filters assayed forhybridization to run-on probes from two independent RAG-2-deficient A-MuLV transformants; a normal A-MuLV transformant and three independentA-MuLV transformants derived from pre-B cells that carried the heterozygous Eti replacement.

thymidine kinase promoter and polyoma enhancer(pMClneo, Stratagene) was used for the RAG-2 targeting(Shinkai et al., 1992).The absence of it' transcripts in RNA from the Elt-

replaced or 5' insertion A-MuLV transformants could bedue to lack of transcription from the germline promoter orto events subsequent to transcription that lead to decreasedaccumulation of the steady-state A' transcripts. To distinguishbetween these possibilities, we employed nuclear run-ontranscription methods (Smith et al., 1992) to assay fortranscription through the DQ52-JH region. For thisexperiment, nuclei were incubated with [32P]dUTP andRNA was isolated. Southern blotting procedures were usedto assay these labeled RNA probes for hybridization tovarious restriction fragments generated by BamHI plusEcoRI digestion of the pJH plasmid (Alt et al., 1981) thatcontains the germline 6.2 kb JH-containing EcoRI fragment(Figure 4A). BamHI plus EcoRI digestion yields four JHfragments of 0.8, 2.3, 1.0 and 2.0 kb plus a 2.7 kb fragmentthat represents the pUC18 vector (Figure 4A, B and C). TheAO transcripts probably initiate just upstream of DQ52(Alessandrini and Desiderio, 1991); therefore thesetranscripts should hybridize to the 2.3, 1.0 and 2.0 kb JHfragments (Figure 4A and B). In contrast, transcripts ofDJH or VHDJH rearrangements would not contain sequences

upstream of DQ52 (except DQ52-JH rearrangements) orsequences between DQ52 and JH1. They also could lacksome additional downstream sequences depending on whichJH is rearranged (Figure 4B). Therefore, such transcriptswould hybridize either weakly or not at all to the 2.3 and1.0 kb fragments, but would hybridize to the downstream2.0 kb fragment. As controls, we assayed for hybridizationto inserts from neor and GAPDH-containing plasmids(Figure 4B).Run-on probes prepared from the RAG-2-deficient pre-B

cell nuclei, which clearly had detectable steady-state levelsof ,°t transcripts from their germline alleles (Figure 3A),strongly hybridized to the 1.0 and 2.0 kb JH fragments andweakly hybridized to the 2.3 kb JH fragment (Figure 4C,panels 2 and 3), consistent with transcription initiation fromthe upstream of DQ52. In contrast, run-on probes preparedfrom the EA-replaced pre-B cell nuclei did not showhybridization to the 1.0 and 2.3 kb JH fragments, indicatinga lack of transcription through the germline DQ52-JHl regionon the targeted allele in these lines (Figure 4C, panels 5 -7).As expected, hybridization of run-on transcripts to the 2.0kb JH fragment was detected from these lines; thisrepresents transcription through that region of the rearrangednormal allele in these cells. Similarly, hybridization to the2.0 kb fragment was the only predominant signal detected

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Fig. 5. Methylation patterns of mutant JH loci in A-MuLV transformants. (A) A schematic map of the Eg replaced allele. Two MspI (Hpal) siteswithin the 2.3 kb Hindm fragment were suggested from previous sequencing analyses, one 300 bp upstream of DQ52 segment and the other one inbetween DQ52 and JHl (Early et al., 1980; Sakano et al., 1981). However, we found only the MspI (Hpal) site between DQ52 and JH1 by Southernblotting analyses with the intron probe following digestion with HindHI and MspI (panel C). Nucleotide sequencing analyses confirmed that there isno MspI (HpaIl) site 300 bp upstream of DQ52 segment. The previous sequences of TTCCCGGAGGC which contains an MspI (HpalI) site (CCGG)corresponds to our sequences of TTCCCAGGGAGGGGC which does not contain any MspI (Hpall) sites. The MspI (M, or Hpal) sites in the JHlocus are shown. The rest of the map is the same as in Figure IA. (B) This panel depicts the possible intron-probe hybridizing fragments aftergenomic DNA is digested with HindfI, HindIlI+MspI, or HindIl+Hpall. (C) Southern blotting analysis of DNA from heterozygous mutant EScells, RAG-2-deficient A-MuLV transformants and A-MuLV transformants from pre-B lines heterozygous for the Eg replacement or 5' insertion.Lanes 1-3 contain DNA from heterozygous mutant ES cells harboring the Elt replacement mutation; lanes 4-10 contain DNA from threeindependent RAG-2-deficient A-MuLV transformants; lanes 11-18 contain DNA from four independent Eli replaced A-MuLV transformants; andlanes 19-26 contain DNA from four independent A-MuLV transformants harboring 5' insertion. The digestions employed are as follows: lanes 1

and 4, HindU; lanes 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25, HindIH and MspI; lanes 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26,Hindi and HpaH. The probe used was the DQ52-JHI intron probe (panel A). X-HindEI markers are indicated.

from normal A-MuLV transfonnants that had rearrangedboth JH alleles and had deleted the upstream sequences

(Figure 4C, panel 4). These interpretations were confirmedby quantifying the various bands using a PhosphorImager(data not shown). Furthermore, a pattern of hybridizationtotally consistent with the results shown was obtained whenrun-on transcripts were assayed for hybridization to EcoRI-and HindlI-digested pJH plasmid which generates a differentseries of restriction fragments spanning the JH locus

(Figure 4A and data not shown).As expected, we found substantial transcription of the

GAPDH gene in all assayed cells (1.4 kb GAPDH insert;Figure 4C). The different level of steady-state of neor genetranscripts observed by Northern analysis was found tocorrelate to similar levels of neor gene transcription by thenuclear run-on assay (Figure 3B; Figure 4C). The insertedneor gene was actively transcribed in EA replaced A-MuLVtransformants (1.9 kb neor gene insert; Figure 4C).However, transcription from the inserted neor gene inRAG-2-deficient Abelson nuclei was barely detectable(Figure 4C).

Effect of the Eu mutations on demethylation of the JHlocusAccessibility of gene segments for rearrangement has alsobeen correlated with hypomethylation of the unrearrangedloci (Engler et al., 1991; Hsieh and Lieber, 1992). Thereis one methylation sensitive Hpall site in between DQ52 andJH1 of the 2.3 kb Hindm fragment (Figure 5A). The effectof EA replacement and 5' insertion mutations on methylationstatus of this site was analyzed by digesting genomic DNAfrom the various lines with either HindI, HindllI+MspIor HindHI+Hpall and assaying for hybridization to theDQ52-JHl intron probe. This probe was used because it onlyhybridizes to unrearranged, mutated JH loci in these pre-Bcells. When assayed as described above, DNA fromheterozygous mutant ES cells gave the expected 2.3 kbfragment when digested by HindIH alone (Figure 5C, lane 1

and data not shown) and the expected fragments of -0.6and 1.7 kb when digested with HindIH and MspI (Figure 5C,lane 2 and data not shown). When digested with HindIl andHpaH, DNA from heterozygous ES cells had a majorhybridizing fragment of 2.3 kb (Figure SC, lane 3 and data

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not shown), indicating that this HpaII site was methylatedon both the wild-type and mutated alleles in most ES cells.In contrast, DNA from A-MuLV transformants derived fromeither RAG-1 or RAG-2-deficient mice yielded the 2.3 kbhybridizing fragment following HindIII digestion(Figure SC, lane 4), but yielded the 0.6 and 1.7 kbhybridizing fragments following digestion with eitherHindIl +MspI or HindIll+HpalI (Figure 5C, lanes 5-10,representative data are shown; Table I) indicating that theHpaH site is demethylated in the lymphoid lineage beforeVHDJH recombination occurs at the JH locus.The hybridization patterns ofDNA from the EAt-replaced

A-MuLV transformants were similar to those obtained withDNA from the parental ES cells (Figure SC, lanes 11-18and data not shown), indicating that the bulk of the mutated(unrearranged) JH loci in these lines remained methylated.Of 23 independent and unrearranged E,u-replaced pre-B celllines assayed, the HpaH site was methylated in 22 lines andpartially demethylated in one line (Table I). In contrast,DNA from -50% of the A-MuLV transformants thatharbored the 5' insertional mutation gave 0.6 and 1.7 kbhybridizing fragments following digestion with eitherHindIII+MspI or HindIII+HpaII [Figure 5C (lanes19-26), Table I and data not shown], indicating that theHpaHl site was demethylated in these lines. Because theselines did not rearrange the mutated allele but did rearrangethe normal allele, these results suggest that demethylationof the JH locus alone is not sufficient to target efficientrearrangement. Finally, the various restriction endonulceasedigested DNAs described above were also assayed forhybridization to the neor probe. The patterns ofhybridization observed indicated that the neor gene wasdemethylated in both parental mutant ES cells and their A-MuLV derivatives (data not shown), consistent with theobservation that the neor gene was transcribed in theselines.

DiscussionA novel system to assay the effects of targetedmutations on recombination accessibilityPrevious studies have clearly demonstrated that E,u canconfer accessibility to associated TCR V,B, D,B and Josegments in a transgenic VDJ recombination substrate(Ferrier et al., 1990). We now have used gene targetedmutation in ES cells to test further the role of EA and itsflanking sequences in targeting rearrangement of theendogenous JH locus during normal development. For thispurpose, we developed a novel procedure in whichheterozygous mutant ES cells from a mouse strain (129)highly permissive to pre-B cell transformation by A-MuLVwere injected into blastocysts from a less permissive mousestrain (C57BL/6J; Rosenberg and Baltimore, 1976).Subsequently, A-MuLV transformed pre-B cell lines wereestablished from bone marrow of resulting chimeras; thechoice of ES cell and blastocyst background ensured thatover 90% of A-MuLV transformants were ES cell derived(Table I). The availability of large numbers of independentpre-B cell lines derived from normal pre-B cells heterozygousfor the various mutations allowed us to compareunequivocally the rearrangement potential of mutant andnormal JH alleles.

Mutations in the E, region differentially affect VDJrecombinationA-MuLV transformants derived from normal pre-B cellshave rearrangements of both JH loci and often undergoadditional JH-associated rearrangements during propagationin culture (Alt et al., 1984; Reth et al., 1986). A-MuLVtransformants harboring the 3' replacement, like wild-typecells, had rearranged both of their JH alleles. Thus,replacement of a segment of DNA within the JH-C,U intron,at the 3' of the core Eli (Nelsen and Sen, 1992) and includingpart of the 3' MAR (Cockerill et al., 1987), with anexpressed neor transcription unit does not grossly affect theability of the upstream JH locus to undergo VDJrecombination. In addition, the 3' replacement deletes mostof the Ipt exon that is part of a separate type of germlineyt transcript (Ilt; Lennon and Perry, 1985); this transcriptionunit was also postulated to play a role in targeting the JHlocus for rearrangement (Lennon and Perry, 1985; Su andKadesch, 1990). We now can conclude that the I/i transcriptsare not required to target the JH locus for recombination.However, we cannot rule out the possibility that transcriptionwithin this region is important, because the I/,^ exon wasreplaced by an expressed neor gene.A-MuLV transformants harboring the E,u replacement

mutation all rearranged their wild-type allele but fewrearranged the mutant allele. This finding, coupled with theresults of the 3' replacement study, strongly suggests thatintegrity of the Eli region is required for efficient JHrearrangement. However, the residual level of observedrearrangements demonstrates that the linked E/A is notabsolutely required for the occurrence of JH rearrange-ments. Perhaps the most surprising finding of our study wasthat the 5' insertional mutation also substantially inhibitedrearrangement of the linked JH segments. Extensiverestriction mapping of the targeted JH -C/ locus in the EScell clone used for these experiments revealed no obviousalterations outside of the inserted neor gene. Thus, althoughwe cannot unequivocally preclude an unknown alteration ofthe targeted allele that affects recombination, the presentresults indicate that the presence of the core E,u near the JHsegments is not sufficient to ensure their efficient rearrange-ment. Furthermore, the complete rearrangement of the wild-type alleles in the El-replaced and 5' insertion mutantsconfirms that the effect of the introduced mutations werecis-acting and not due to a more generalized defect indifferentiation or VDJ recombinase expression. Finally,because the 3' replacement mutation does not interfere withD to JH or VH to DJH rearrangements, it is unlikely that theinhibition observed with the upstream mutations resultedfrom interference with potential downstream cis-actingelements. Therefore, we conclude that the El replacementand 5' insertional mutations inhibit JH rearrangement byinterfering with E/z function and/or activity of potentiallyinvolved elements upstream of the core enhancer.There are several possible mechanisms by which the 5'

insertion blocks rearrangement of the upstream JH locus.One is disruption of an unknown element that is importantfor efficient JH rearrangement. In this regard, the insertiondisrupts the 5' matrix associated region (Cockerill et al.,1987; Dickinson et al., 1992) and the EA associated originof replication (Ariizumi et al., 1993; K.Ariizumi,M.R.Ghosh and P.W.Tucker, submitted). Although theprecise function of these sequences is unknown, there are

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several scenarios in which one could speculate a role in therearrangement process. Another, not mutually exclusivepossibility is that insertion of the transcribed neor unitupstream of the EA region somehow interferes with its abilityto function as a recombination enhancer for the JH locus;for example, by disrupting DNA -protein complexesnecessary for recombination enhancement. Because the 3'replacement does not have any apparent effect on rearrange-ment, such putative interference must be position-dependent.

Comparison of various targeted mutational studies ofE,u and Ex functionA parallel study has demonstrated that deletion of the sameE,u-containing fragment targeted by our El replacement alsoaffects recombination of the associated JH segments (Serweand Sablitzky, 1993). Qualitatively, the results of the twostudies are consistent; however, there are quantitativedifferences that may provide important insights. In the otherstudy, PCR assays of purified heterozygous mutant splenicB cells indicated that 70-85% of the mutated alleles hadundergone DJH rearrangement as compared with thecomplete rearrangement of the normal alleles; however, VHto DJH rearrangement on the targeted allele was moreseverely inhibited. That study, like ours, concluded that theEy region is important for efficient V(D)J rearrangementbut is not absolutely required. The rearrangements observedon E/ deleted chromosomes also implied the existence ofadditional elements within the JH locus that contribute to JHaccessibility (Serwe and Sablitzky, 1993). Our finding ofa residual level of JH rearrangements in the E,u-replacedmutant pre-B cells is consistent with this interpretation.However, it remains possible that the residualrearrangements we observed may be facilitated by theinsertion of the transcribed neor gene.

There are several possible explanations for the quantitativedifferences in the two analyses of EA function. One is thatthe PCR amplification assay from heterogeneous B cellpopulations may be subject to more quantitative variationthan our approach. However, it seems unlikely that this couldlead to the magnitude of the observed difference in D to JHrearrangements (70-85 % versus - 15 %). Anotherpossibility is that the presence of an expressed neor gene inour studies interfered with rearrangement. Although thispossibility cannot be ruled out, it seems unlikely becauseinsertion of the neor gene just downstream of the EA hadno major effect. Another difference between the two studiesis the stage ofB lineage cells analyzed: pre-B versus B cells.In this regard, the DJH rearrangements observed in splenicB cells with the EA deletion may have occurred at a laterstage than normal. For example, it has been suggested thatVDJ recombination activity is activated in bone marrow Bcells whose receptor is engaged, leading to ongoing VDJrearrangement and 'receptor editing' (Gay et al., 1993; Tiegset al., 1993). In such a case, re-exposure of mutant germlinealleles to VDJ recombinase may lead to increasedaccumulation of D to JH rearrangements due to theactivation of additional control elements, even though VHto DJH rearrangement remains inefficient.Recent gene targeted mutational analyses have similarly

implicated the Ig x intronic enhancer (Ex) in the controlof x light chain gene rearrangement (Takeda et al., 1993).Replacement of the Ex with an apparently unexpressed neor

gene almost completely blocks the rearrangement of the xlocus as determined by exclusive X light chain expressionin homozygous mutant splenic B cells. As a control, insertionof the same neor gene 3' of the Ex had only a minimaleffect on Jx rearrangements. However, it is unknownwhether insertional mutations 5' of the Ex will have anadverse effect on Jx rearrangements.

Effects of mutations in the Eu region on germlinetranscription and demethylation of the associated JHlocusTranscription and hypomethylation are closely associatedprocesses (Cedar, 1988; Bird, 1992) that have been linkedto recombinational accessibility (Engler et al., 1991; Hsiehand Lieber, 1992). RAG-2-deficient pre-B cell lines produceAO germline transcripts that probably initiate upstream ofDQ52 and proceed through the JH and CA regions (J.Gormanand F.W.Alt, unpublished data; Alessandrini and Desiderio,1990; Schlissel et al., 1991). We failed to detect theaccumulation of steady-state y° transcripts from unrear-ranged mutant alleles of numerous analyzed pre-B lines thatharbored either the E/t replacement or 5' insertion mutations.In the case of Ey replacement, the failure to accumulate thesetranscripts cannot be attributed simply to decreased stabilityor downstream transcriptional blockage; because we failedto detect run-on transcription through the DQ52-JH intronregion. Thus, the Eit replacement mutation eliminates orsubstantially decreases either the rate of y°0 transcriptioninitiation or the elongation of these transcripts through theimmediate downstream DQ52-JHl intron sequences. Thisfinding strongly implicates the E,t element as an enhancerof germline transcription of the endogenous JH locus.The JH loci are methylated in ES cells but are

demethlyated in RAG-deficient A-MuLV transformants.However, the unrearranged JH loci from A-MuLV trans-formants harboring the Ept replacement remained methylated.These results indicate that the JH locus is demethylatedduring normal development at a point prior to VDJrecombination and that replacement of the EIt regioninterferes with the occurrence of both of these processes.Notably, the transcribed neor gene which replaces the Eyremains demethylated, indicating the methylation status ofthe closely linked JH and neor genomic DNA segments isdifferentially controlled. Finally, many of the unrearrangedJH loci of pre-B lines harboring the 5' insertional mutationwere demethylated, indicating that the 5' insertion and theE/A replacement mutations either affect different elements orsomehow differentially affect EA function. Together theseresults also suggest that sequences in or very near EI maycontain cis-acting elements important for demethylation ofthe JH locus during development.

Function of cis-acting elements in controlling VDJrecombinationThe El region contains multiple motifs known to interactwith various factors that have been implicated intranscriptional regulation of Ig expression as assayed inexpression constructs (reviewed by Staudt and Lenardo,1991; Nelsen and Sen, 1992). The JH loci in RAG-2-deficient A-MuLV transformants are actively transcribed andare demethylated. Introduction of RAG-2 expression vectorsinto these lines leads to efficient rearrangement of the

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endogenous JH loci, indicating that they are accessible(G.Rathbun and F.Alt, unpublished data). On the other hand,the relatively inaccessible mutant alleles in A-MuLV trans-formants that harbor the Eli replacement have diminishedtranscription through the DQ52-JH region and remainmethylated, once again linking these three phenomena.However, the results from the 5' insertional mutation clearlysuggest that demethylation, per se, is not sufficient to renderthe locus fully accessible for VDJ recombination, althoughwe note that these results do not eliminate the possibility thatdemethylation is a requirement for efficient JH rearrange-ment. Therefore, the ability of E,u to promote transcriptionfrom the IA° promoter remains the most tightly linkedcorrelate of its ability to function as a VDJ recombinationalenhancer (Alessandrini and Desiderio, 1991; Schlissel et al.,1991).A role for germline transcription in VDJ recombinational

targeting has been suggested by the finding that DJHrearrangement is usually preceded by germline transcriptionof the JH locus (Alessandrini and Desiderio, 1991; Schlisselet al., 1991). One possible function for this process is thatthe generated RNA transcripts facilitate VDJ recombination.This notion, in its simplest form, seems obviated by ourfinding that the heterozygous E,t replacement and 5' insertionmutations prevent rearrangement of only the targeted alleles.We also note that the presence of the transcribed neor gene,just 500 bp downstream of JH4, is not sufficient to driveefficient rearrangement of adjacent JH segments. Thus, wesuggest that activation of it' promoter may help generaterecombinational accessibility by promoting transcriptionthrough the JH locus and/or by co-activating binding sitesfor transcription and VDJ recombination factors. Wepreviously demonstrated that the E,t region efficiently targetsan associated TCR,3 mini-locus for rearrangement; deletionof the Ey region from the construct completely eliminatedrearrangements of the construct (Ferrier et al., 1990).Mutations of the endogenous EA element do not indicate asstrict a dependence on its presence for rearrangement (Serweand Sablitzky, 1993; this study). Thus, it seems likely thatthe TCR(3 mini-locus may lack additional elements presentin the endogenous JH locus that allow targeting of D to JHrearrangements at some stage of B cell development.

Materials and methodsGene targeted mutation of the E# region in ES cellsThe El replacement construct was made by replacing the 1 kb XbaI-XbaIfragment containing the E/s enhancer in a 7.8 kb EcoRI-AseI DNA fragmentfrom the JH locus with a neor gene. The neor gene is under thetranscriptional control of the phosphoglycerate kinase (PGK-1) promoter(Tybulewicz et al., 1991), which does not have any known enhancer activity(Adra et al., 1987). The 5' insertion construct was made by inserting thesame PGK-neor in the ScaI site in the 7.8 kb EcoRI-AseI fragment. The3' replacement construct was made by replacing the 0.7 kb EcoRIl-HindImfragment 3' of the core enhancer with the same PGK-neor gene in a 3.1kb HindlI-HindI genomic DNA fragment that spans the Ey region. Theherpes simplex virus thymidine kinase (HSV-tk) gene, also driven by thePGK-1 promoter, was added to the 3' end of the targeting vectors forselection against random integration events (Mansour et al. 1988).Approximately 20 jig of linearized construct DNA was electroporated

into 2 x 107 CCE ES cells at 300 V and 70 AF, after which the cells werediluted in culture medium, plated into five 60mm plates containing mitoticallyinactivated STO feeder cells, and placed under selection 24 h later in eitherG418 (400 Ag/ml powder, Gibco) or G418 and gancyclovir (1 AM, Sigma).Doubly resistant clones were picked and expanded for freezing and DNAisolation. Potential homologous recombinants were identified by Southernblot analyses with probes that represent sequences adjacent to those used

in the targeting constructs. Candidates were verified for homologousrecombination with various restriction digests and multiple probes.

Generation of chimeric mice and A-MuLV transformation ofpre-B cellsHeterozygous mutant ES cells were injected into C57BL/6J blastocysts togenerate chimeric mice. Chimeric mice, as determined by agouti coat color,were sacrificed at about 1 month of age for analysis. To generate A-MuLVtransformants from pre-B cells (Rosenberg and Baltimore, 1976), singlecell suspensions prepared from bone marrow of the various chimeric micewere incubated with A-MuLV for 4 h in the presence of polybrene. 1 mlof the cell mixture was plated in a final concentration of 0.3% BactoAgar;an additional 1 ml of0.3% BactoAgar in RPMI and 20% FCS was overlaid5 days later. Foci were picked between days 8 and 10 and individual lineswere established for analysis.

Southern and Northern blot analysesFor Southern blot analyses, 10 yg of genomic DNA was digested with theindicated restriction enzymes, electrophoresed through a 0.85% agarose gel,transferred to Zetaprobe membrane (Bio-Rad) and hybridized with the 32p-radiolabeled probes as described previously (Shinkai et al., 1992). ForNorthern blot analyses, 10 g of total RNA from ES cells or pre-B celllines was electrophoresed through a 1% formaldehyde-agarose gel,transferred to Zetaprobe membrane and hybridized with the 32p-radiolabeled probes as described previously (Shinkai et al, 1992). Hybridizedprobes were removed from filters by submersion in boiling water for 2-5min.

Nuclear run-on assaysThe assay was performed as previously described (Smith et al., 1992).Briefly, nuclei from RAG-2-deficient, normal and EA-replaced A-MuLVtranformants were isolated by NP-40 lysis of cytoplasmic membranes andstored at -800C. Run-on RNA transcripts were labeled by incubating-2 x 107 nuclei in elongation reaction buffer in the presence of[32P]UTP. The extended transcripts were purified by removing DNA withRNase-free DNase (Boehringer), proteins with protease K treatment followedby phenol-chloroform extraction and passage through a Sephadex G-50spin column. The labeled RNA was subjected to limited alkaline hydrolysisprior to being used as a probe. 5 Ag of each plasmid was digested withappropriate restriction enzymes and Southern blotted on to nitrocellulosemembranes. Duplicate filters were hybridized to the 32P-labeled run-ontranscripts from each cell line. Filters were washed, autoradiographed onX-ray film and quantified using a Phosphorlmager.

AcknowledgementsWe thank Drs Eugene Oltz, Jean-Christophe Bories, Michel Cogne, AmiOkada, Paschalis Sideras and James Gorman for critically reading thismanuscript and for helpful discussions. We also thank Drs F.Sablitzky andK.Rajewsky for communication of results prior to publication. This workwas supported by Howard Hughes Medical Institute and by NIH grantsAI20047 and U01 AI31541 (to F.W.A.). J.C. is a fellow of the CancerResearch Institute, F.Y. is a fellow of the Robert Wood Johnson Foundationand A.B. is a fellow of the European Molecular Biology Organization.

ReferencesAdra,C.N., Boer,P.H. and McBurney,M.W. (1987) Gene, 60, 65-74.Alessakirini,A. and Desiderio,S.V. (1991) Mol. Cell. Biol., 11, 2096-2107.Alt,F.W., Rosenberg,N., Lewis,S., Thomas,E. and Baltimore,D. (1981)

Cell, 27, 381-390.Alt,F.W., Yancopoulos,G.D., Blackwell,T.K., Wood,C., Thomas,E.,

Boss,M., Coffnan,R., Rosenberg,N., Tonegawa,S. and Baltimore,D.(1984) EMBO J., 3, 1209-1219.

Alt,F.W., Oltz,E.M., Young,F., Gorman,J., Taccioli,G. and Chen,J. (1992)Immunol. Today, 13, 306-314.

Anderson,S.J., Abraham,K.M., Nakayama,T., Singer,A. andPerlmutter,R.M. (1992) EMBO J., 11, 4877-4886.

Ariizumi,K., Wang,Z. and Tucker,P.W. (1993) Proc. NatlAcad. Sci. USA,in press.

Banerji,J., Olson,L. and Schaffner,W. (1983) Cell, 33, 729-740.Bird,A. (1992) Cell, 70, 5-8.Blackwell,T.K. and Alt,F.W. (1989) Annu. Rev. Genet., 23, 605-636.Cedar,H. (1988) Cell, 53, 3-4.Cockerill,P.N., Yuen,M.-H. and Garrard,W.T. (1987) J. Biol. Chem, 262,5394-5397.

4644

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Davis,M.M. and Bjorkman,P.J. (1988) Nature, 334, 395-401.Dickinson,L.A., Joh,T., Kohwi,Y. and Kohwi-Shigematsu,T. (1992) Cell,

70, 631-645.Early,P., Huang,H., Davis,M., Calame,K. and Hood,L.E. (1980) Cell,

19, 981-992.Engler,P., Haasch,D., Pinkert,C.A., Doglio,L., Glymour,M., Brinster,R.

and Storb,U. (1991) Cell, 65, 939-947.Ferrier,P., Krippl,B., Blackwell,T.K., Furley,A.J.W., Suh,H., Winoto,A.

Cook,W.D., Hood,L., Constantini,F. and Alt,F.W. (1990) EMBO J.,9, 117-125.

Gay,D., Saunders,T., Camper,S. and Weigert,M. (1993) J. Exp. Med.,177, 999-1008.

Gillies,S.D., Morrison,S., Oi,V. and Tonegawa,S. (1983) Cell, 33,717-728.

Hsieh,C.-L. and Lieber,M.R. (1992) EMBO J., 11, 315-326.Lennon,G.G. and Perry,R.P. (1985) Nature, 318, 475-478.Mansour,S.L., Thomas,K.R. and Capecchi,M.R. (1988) Nature, 336,348-352.

Mombaerts,P., Iacomini,J., Johnson,R.S., Herup,K., Tonegawa,S. andPapaloannou,V.E. (1992) Cell, 68, 869-877.

Nelsen,B. and Sen,R. (1992) nt. Rev. Cytol., 133, 121-149.Oettinger,M.A., Schatz,D.G., Gorka,C. and Baltimore,D. (1991) Science,

248, 1517-1523.Oltz,E.M., Alt,F.W., Lin,W.-C., Chen,J., Taccioli,G., Desiderio,S. and

Rathbun,G. (1993) Mol. Cell. Biol., in press.Reth,G.M., Jackson,S. and Alt,F.W. (1986) EMBO J., 5, 2131-2138.Rosenberg,N. and Baltimore,D. (1976) J. Exp. Med., 143, 1453-1463.Sakano,H., Kurosawa,Y., Weigert,M. and Tonegawa,S. (1981) Nature,

290, 562-565.Schatz,D.G., Oettinger,M.A. and Baltimore,D. (1989) Cell, 59,

1035-1048.Schlissel,M.S. and Baltimore,D. (1989) Cell, 58, 1001-1007.Schlissel,M.S., Corcoran,L.M. and Baltimore,D. (1991) J. Exp. Med., 173,711-720.

Serwe,M. and Sablitzky,F. (1993) EMBO J., 12, 2321-2327.Shinkai,Y., Rathbun,G., Lam,K.-P., Oltz,E.M., Stewart,V.,

Mendelsohn,M., Charron,J., Datta,M., Young,F., Stall,A.M. andAlt,F.W. (1992) Cell, 68, 855-867.

Smith,R.K., Zimmerman,K., Yancopoulos,G.D., Ma,A. and Alt,F.W.(1992) Mo. Cell. Biol., 12, 1578-1584.

Staudt,L.M. and Lenardo,M.J. (1991) Annu. Rev. Immunol., 9, 373 -398.Su,L.-K. and Kadesch,T. (1990) Mol. Cell. Biol., 10, 2619-2624.Takeda,S., Zou,Y.-R., Bluethmann,H., Kitamura,D., Muller,U. and

Rajewsky,K. (1993) EMBO J., 12, 2329-2336.Tiegs,S.L., Russell,D.M. and Nemazee,D. (1993) J. Exp. Med., 177,

1009-1020.Tonegawa,S. (1983) Nature, 302, 575-581.Tybulewicz,V.L.J., Crawford,C.E., Jackson,P.J., Bronson,R.T. and

Mulligan,R.C. (1991) Cell, 65, 1153-1163.Yancopoulos,G.D. and Alt,F.W. (1985) Cell, 40, 271-281.Yancopoulos,G.D., Blackwell,T.K., Suh,H., Hood,L. and Alt,F.W. (1986)

Cell, 44, 251-259.

Received on May 3, 1993; revised on August 4, 1993

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