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Locus ‘decontraction’ and centromeric recruitmentcontribute to allelic exclusion of the immunoglobulinheavy-chain gene
Esther Roldan1, Martin Fuxa2, Winnie Chong1, Dolores Martinez3, Maria Novatchkova2, Meinrad Busslinger2
& Jane A Skok1
Allelic exclusion of immunoglobulin genes ensures the expression of a single antibody molecule in B cells through mostly
unknown mechanisms. Large-scale contraction of the immunoglobulin heavy-chain (Igh) locus facilitates rearrangements
between Igh variable (VH) and diversity gene segments in pro–B cells. Here we show that these long-range interactions are
mediated by ‘looping’ of individual Igh subdomains. The Igk locus also underwent contraction by looping in small pre–B and
immature B cells, demonstrating that immunoglobulin loci are in a contracted state in rearranging cells. Successful Igh
recombination induced the rapid reversal of locus contraction in response to pre–B cell receptor signaling, which physically
separated the distal VH genes from the proximal Igh domain, thus preventing further rearrangements. In the absence of locus
contraction, only the four most proximal VH genes escaped allelic exclusion in immature l-transgenic B lymphocytes. Pre–B
cell receptor signaling also led to rapid repositioning of one Igh allele to repressive centromeric domains in response
to downregulation of interleukin 7 signaling. These data link both locus ‘decontraction’ and centromeric recruitment to the
establishment of allelic exclusion at the Igh locus.
The diverse antigen receptor repertoire of lymphocytes is generated byV(D)J recombination, which assembles the variable regions of immu-noglobulin and T cell receptor genes from discontinuous variable (V),diversity (D) and joining (J) gene segments during B and T celldevelopment1,2. These gene segments are flanked by recombinationsignal sequences that function as recognition sites for the V(D)Jrecombinase consisting of recombination activating gene 1 (RAG1)and RAG2 proteins. After pairing of two compatible re-combination signal sequences, the RAG1-RAG2 complex introducesdouble-strand DNA breaks between the recombination signalsequences and flanking gene segments, followed by processing andreligation of the DNA ends by repair factors of the nonhomologousend-joining machinery1,2.
V(D)J recombination is tightly controlled in a lineage- and stage-specific way. Immunoglobulin and T cell receptor genes are rearrangedonly in B and T lymphocytes, respectively1,2. In the B lymphoidlineage, the immunoglobulin heavy-chain (Igh) locus undergoesrearrangements in pro–B cells before recombination of the genesencoding immunoglobulin light chains (IgL) in small pre–B andearly immature B cells1,2. Moreover, DH-JH rearrangements precedeVH-DJH recombination in the Igh locus, whereas, among the two IgLgenes, the Igk locus rearranges before the Igl locus3. The observed
temporal order of V(D)J recombination is determined mainly by theaccessibility of the different gene loci and segments to the V(D)Jrecombinase4,5, which is controlled at multiple levels, including sub-nuclear relocation6, DNA demethylation7, chromatin remodeling8,histone acetylation9,10 and germline transcription4 of the differentimmunoglobulin loci.
The approximately 200 VH genes of the Igh locus are spread over a2.4-megabase region and can be divided into 15 distal, central orproximal VH gene families according to their sequence similarity andposition relative to the proximal DH segments11. In non–B lymphoidcells and lymphoid progenitors, the two Igh alleles are present in anextended conformation at the potentially repressive periphery of thenucleus6, where they are anchored via the distal VHJ558 gene regionwith the proximal Igh domain facing toward the center of thenucleus12. This orientation of the Igh locus is likely to facilitateactivation of the proximal domain in lymphoid progenitors, thusresulting in DH-JH rearrangements10,13. Early pro–B cell developmentis characterized by relocation of the Igh alleles to central nuclearpositions6, histone acetylation of the distal VHJ558 genes in response tointerleukin 7 (IL-7) signaling10, antisense transcription along the entireVH gene cluster14 and long-range contraction of the Igh locus6,12,which ultimately results in VH-DJH recombination. The transcription
Published online 5 December 2004; doi:10.1038/ni1150
1Department of Immunology and Molecular Pathology, Division of Infection and Immunity, University College London, London W1T 4JF, UK. 2Research Institute ofMolecular Pathology, Vienna Biocenter, A-1030 Vienna, Austria. 3The Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK.Correspondence should be addressed to J.A.S. ([email protected]) or M.B. ([email protected])
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factor Pax5 has an essential function in regulating contraction of theIgh locus12. The central and distal VH genes are fully accessible in activechromatin and yet fail to rearrange in Pax5–/– pro–B cells15 becauseof the physical separation of these genes from the proximal DJH-rearranged domain in the absence of locus contraction12.
Successful rearrangement of the Igh locus leads to cell surfaceexpression of the m protein as part of the pre–B cell receptor (pre-BCR), which functions as an important checkpoint to signal proli-ferative expansion of populations of large pre–B cells, to inducesubsequent differentiation to small pre–B cells and to establish allelicexclusion at the second, DJH-rearranged Igh allele3,16,17. Feedbackinhibition of Igh recombination by the membrane-bound m protein(referred to as allelic exclusion) was initially noted in mice expressing am transgene, which efficiently prevents VH-DJH rearrangements atboth endogenous Igh alleles during B cell development18–21. RAGprotein expression is rapidly lost after pre-BCR signaling, which haltsall further V(D)J recombination and ‘prepares the ground’ for theestablishment of allelic exclusion in large pre–B cells22. Pre-BCRsignaling also leads to histone deacetylation and thus reduced acces-sibility of the VH genes in small pre–B cells, which has been consideredas a possible feedback mechanism underlying allelic exclusion23. These
chromatin alterations, however, could be an indirect consequence ofpre-BCR signaling, as they depend on the pre-BCR-induced down-regulation of IL-7 signaling23. It is therefore still unknown whatchanges occur on the DJH-rearranged Igh locus during the shortrecombinase-free ‘window’ in large pre–B cells so that this allele isunable to further rearrange after subsequent reexpression of the RAGproteins in small pre–B cells.
Here we have identified, using fluorescence in situ hybridization(FISH), two previously unknown mechanisms that are likely toestablish allelic exclusion during the recombinase-free transitionphase in large pre–B cells. ‘Decontraction’ of both Igh loci wasinitiated in large pre–B cells and was maintained at all subsequentdevelopmental stages. The reversal of Igh locus contraction is likely toprevent VH-DJH rearrangements in small pre–B cells, in analogy to theextended Igh conformation in Pax5–/– pro–B cells12. Pre-BCR signal-ing simultaneously induced rapid repositioning of one Igh allele torepressive centromeric domains. This monoallelic centromeric recruit-ment was transiently maintained in small pre–B and early immature Bcells, where it kept the nonfunctional Igh allele in an inactive stateduring IgL gene rearrangements. In contrast to the Igh alleles, the Igkgene underwent locus contraction specifically in small pre–B and
Pro-BLargepre-B
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0–0.3 µm
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1–1.5 µm
VHJ558 DH JH Cγ1Cµ
526A21 HE17
a
Distal Proximal
c
Figure 1 Decontraction of the Igh locus in large pre–B cells. (a) Igh locus (not drawn to scale), indicating the positions of bacterial artificial chromosome
(BAC) 526A21 (ref. 12) and plasmid HE17 (ref. 24), which were used to generate the VHJ558 (red) and Cg1 (green) probes, respectively. (b) Representative
confocal sections through the nuclei of B lymphocytes at various developmental stages (above images) in which three-dimensional DNA FISH analysis of
the Igh locus was done with VHJ558 (red) and Cg1 (green) probes. The two Igh alleles of each cell are presented on separate optical sections. Broken lines
outline the contours of the nuclei. Pre–B cells were identified as large or small cells under the microscope. (c) Separation of VHJ558 and Cg1 gene segments.
The distance (in mm) separating the VHJ558 and Cg1 segments was evaluated statistically for B lymphocytes of various developmental stages (horizontalaxis). Actual numbers and sample sizes are in Supplementary Table 1 online.
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immature B cells, thus demonstrating the general principle thatimmunoglobulin loci are in a contracted state only in rearranging Blymphocytes. Furthermore, ‘looping’ of individual subdomains wasresponsible for the contraction of the Igh and Igk loci. Finally, bothendogenous Igh alleles were in an extended state without havingundergone centromeric recruitment in pre–B and immature B cells ofm-transgenic mice. In the absence of locus contraction, only the fourmost proximal VH genes escaped allelic exclusion in m-transgenic Blymphocytes. These data strongly suggest that both locus decontrac-tion and centromeric recruitment establish allelic exclusion at the Ighlocus in response to pre-BCR signaling.
RESULTS
Decontraction of the Igh locus in early pre–B cells
To study the contraction state of the Igh locus throughout B celldevelopment, we used two-color three-dimensional DNA FISH ana-lysis to localize the distal VHJ558 and proximal Cg1 gene segments inthree-dimensionally preserved nuclei using confocal laser-scanningmicroscopy12,24. We isolated B lymphocytes of the following develop-mental stages by cell sorting: c-Kit+CD19+ bone marrow pro–B cells25,which we cultured on ST2 cells in the presence of IL-7; B220+CD25+
bone marrow pre–B cells25, which we visually identified as largecycling or small resting pre–B cells after FISH analysis; and splenic
IgM+ B cells, which we cultured in activating conditions24. Weanalyzed the sorted B lymphocyte populations using three-dimensional FISH with a labeled Cg1 probe in combination with aVHJ558 probe (Fig. 1a) that does not cross-hybridize with membersof other VH gene families12. The results of these three-dimensionalFISH experiments are presented as confocal images (Fig. 1b) and asa statistical evaluation of the distances between the proximal Cg1 anddistal VHJ558 gene segments of the Igh alleles (Fig. 1c). Consistentwith previous results6,12, most of the pro–B cells undergoing Ighrecombination had their Igh loci in a contracted conformation(Fig. 1b), as the proximal and distal domains were separated inonly 10% and 1% of the analyzed Igh alleles by a distance of 0.3–1 mmor 1–1.5 mm, respectively (Fig. 1c and Supplementary Table 1 online).In contrast, the large pre–B cells mainly contained the Igh locus inan extended state (Fig. 1b), as there was a distance of 0.3-1 mm or 1-1.5 mm separating the VHJ558 and Cg1 genes in 33% and 30% ofall Igh alleles, respectively (Fig. 1b,c). All subsequent developmentalstages contained the Igh locus in a similarly extended state (Fig. 1b,c).Therefore, Igh alleles undergo decontraction after the production of afunctional rearrangement at the onset of pre–B cell development.
The VHJ558 gene family at the distal end of the Igh locus comprisesabout 44% of all Igh VH genes11 and should give rise to the leastextension after decontraction in response to productive VH-DJH
b
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Cκ296M13
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a c
Figure 2 Contraction and decontraction of the Igk locus. (a) Igk locus (not drawn to scale), indicating the positions of BAC 296M13 (ref. 50) and plasmid
IgkC24, which were used to generate the Vk24 (red) and Ck (green) probes, respectively. (b) Representative confocal sections through B cells at various
stages of development (above images) in which three-dimensional DNA FISH analysis of the Igk locus was done with Vk24 (red) and Ck (green) probes. The
two Igk alleles of each cell are presented in separate optical sections. Flow cytometry sorting of the developmental stages is in Supplementary Fig. 1 online.
(c) Separation of Vk24 and Ck gene segments. The distance (in mm) separating the Vk24 and Ck segments was evaluated statistically for cells of various
developmental stages (horizontal axis). Actual numbers and sample sizes are in Supplementary Table 1 online. Imm.B, immature B cell.
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recombination, which juxtaposes the VHJ558gene family next to the Cg1 region within ashort distance of 100 kilobases (ref. 11) thatcannot be resolved by three-dimensionalDNA FISH analysis12. The signals of theVHJ558 and Cg1 genes were colocalized in37–45% of the Igh alleles analyzed in pre–Bcells. Large pre–B cells (‘preB-II’ cells)26 andsmall pre–B cells (fraction D)27 carryVHJ558-DJH rearrangements on 46% and41% of their Igh loci, respectively. The closecorrelation between the frequencies of colo-calized signals and VHJ558DJH-rearrangedIgh loci strongly suggests that the distanceseparating the VHJ558 and Cg1 genes reflectsthe recombination status of the decontractedIgh alleles in pre–B cells. Accordingly, rear-rangements involving genes from the middleof the VH gene cluster are likely to fall withinthe category of 0.3- to 1.0-mm gene separa-tion. Moreover, Igh alleles with proximalVHQ52, VH7183 and DJH rearrangementsshould give rise to the full extent of separation (1–1.5 mm), as no-ted before for the Igh alleles in Rag2–/–Pax5–/– pro–B cells, which areunable to undergo Igh locus contraction and V(D)J recombination12.
Thus, our data demonstrate that the contraction of the Igh locus isreversible and occurs only during V(D)J recombination in pro–B cells.Decontraction in response to pre-BCR signaling is therefore likely toprevent further distal and central VH gene recombination in pre–Bcells, whereas it may still be compatible with proximal VH generearrangements on the extended Igh locus, analogous to the contrac-tion-deficient Pax5–/– pro–B cells12.
Igk locus contraction coincides with recombination
The Igk locus contains approximately 140 Vk genes spread over a3-megabase region28,29 and is thus as large as the Igh locus. Thequestion therefore arises as to whether transient contraction of the Igklocus also facilitates Vk-Jk recombination by promoting the interactionbetween distal Vk and proximal Jk gene segments. Although a few Vk-Jk rearrangements take place early in pro–B cells30, efficient recombi-nation occurs in small resting pre–B cells and continues in earlyimmature B cells31 undergoing receptor editing32. We thereforedivided the immature B cell population of the bone marrow by flow
cytometry into early (B220+IgMlo) and late (B220+IgMhi) immature Bcells (Supplementary Fig. 1 online) and used these cells together withbone marrow pro–B and pre–B cells and activated splenic B cells todetermine the contraction state of the Igk locus at various develop-mental stages. For this three-dimensional DNA FISH analysis, we usedprobes detecting the distal Vk24 gene family and proximal Ck genesegment at either end of the Igk locus (Fig. 2a). The results of theseFISH experiments indicated that the Vk24 and Ck genes wereseparated by a large distance of 1–1.5 mm in 85% of the Igk allelesof pro–B and large cycling pre–B cells (Fig. 2b,c and SupplementaryTable 1 online). In contrast, the Vk24 and Ck signals were colocalizedin most small pre–B cells (90%) and early immature B cells (78%).Late immature B cells were characterized by a notable increase inpartially separated (0.3–1.0 mm) or widely separated (1–1.5 mm) Igkalleles (24% and 32%, respectively). In splenic B cells, the Vk24 and Cksignals were once again separated by a large distance (1–1.5 mm) inmost Igk alleles (72%). The higher proportion of fully extended Igk(72%) versus Igh (31%) alleles noted in splenic B cells (Figs. 1c and2c) is explained by the different outcome of V(D)J recombination atthe two immunoglobulin loci. As all VH genes are oriented in the sametranscriptional direction as the DH and JH segments11, recombination
b
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113G24296M13
aVHJ558 VH11 DH Cµ Cγ1
Vκ24 Vκ21 Jκ Cκ
Cκ
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Cκ
JH
526A21 HE17282021
Vκ24 Vκ21 Cκ
VHJ558 VH11 Cγ1
Figure 3 Contraction by looping of the Igh and
Igk loci. (a) Igh locus, indicating the position
of the third VH11 probe (BAC 282021).
(b) Confocal section through the nucleus of a
Rag2�/� pro–B cell, which was analyzed by
three-dimensional DNA FISH with VHJ558 (blue),
VH11 (red) and Cg1 (green) probes. Middle,
enlargement of the Igh allele shown at left; right,looping configuration. (c) Igk locus, indicating the
position of the third Vk21 probe (BAC 113G24;
ref. 51). (d) Confocal image of an early immature
B cell showing the relative positions of the Vk24
(blue), Vk21 (red) and Ck (green) probe signals.
Middle, higher magnification of the Igk allele
shown at left; right, looping configuration. There
was similar looping of Igk and Igh loci in 20 and
24 cells, respectively. Additional alleles with Igk
and Igh looping are in Supplementary Fig. 2
online.
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invariably results in deletion of the intervening sequences at theIgh locus. In contrast, about two thirds of all Vk genes are presentin antisense orientation relative to the Jk segments29, which leadsto inversion and thus retention of the intervening sequence afterIgk recombination. Thus, our data unequivocally demonstrate thatIgk alleles are present in a contracted state only when they undergoVk-Jk recombination in small pre–B and immature B cells. Hence, ageneral principle emerges demonstrating that the contraction of Ighand Igk loci is reversible and occurs only in cells actively undergoingV(D)J recombination.
Locus contraction by looping
We next took advantage of three-color three-dimensional DNA FISHto further analyze the conformation of Igh and Igk loci in cells inwhich locus contraction occurs (Fig. 3). For these experiments, weused a third probe detecting the VH11 or Vk21 gene, which are locatedbetween the probes used in previous FISH experiments (Fig. 3a,c).DNA FISH analysis of Rag2–/– pro–B cells with VHJ558, VH11 and Cg1
probes identified several Igh alleles (n ¼ 24) for which the signals ofthe three probes contacted each other in an order that did not reflecttheir linear arrangement on the Igh locus (Fig. 3b and SupplementaryFig. 2 online). For example, the VHJ558 signal was located in the
middle between the VH15 and Cg1 signals in the Igh allele inFigure 3b, demonstrating that the VHJ558 gene region looped backonto the proximal Igh domain. Likewise, DNA FISH analysis of earlyimmature B cells identified Igk alleles (n ¼ 20) for which the relativearrangement of the Vk24, Vk21 and Ck signals was incompatible withtheir gene order in the Igk locus (Fig. 3c,d and Supplementary Fig. 2online). The Igk allele in Figure 3d showed a looping configurationthat brought the proximal Ck domain in contact with both the Vk24and Vk21 gene families. These data indicate that looping mediateslong-range interactions between distinct domains of the Igh or Igkloci, thus resulting in locus contraction.
Centromeric location of one Igh allele in pre–B cells
The transcriptionally silent Igh allele is located at centromericheterochromatin in activated splenic B cells, whereas the productivelyrearranged, expressed Igh allele is positioned away from centromericclusters24. To determine whether centromeric recruitment of oneIgh allele may contribute to allelic exclusion in pre–B cells, weexamined the nuclear position of Igh loci relative to centromeres atvarious developmental stages using three-color three-dimensionalDNA FISH (Fig. 4 and Supplementary Table 2 online). We used ag-satellite probe to visualize the centromeric foci, whereas VHJ558
a
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Figure 4 Monoallelic centromeric recruitment of the Igh locus during B cell
development. (a) Centromeric location of one Igh allele in bone marrow pre–B
cells. Large and small pre–B cells and activated splenic B cells were analyzed
by three-color three-dimensional DNA FISH with VHJ558 (red), Cg1 (green) and
g-satellite (g-sat; blue) probes. The relative positions of the three signals are
shown in confocal sections through the nuclei of these cells. (b) Association of
the distal VHJ558 gene domain with centromeric clusters. Enlargements show
the orientation and decontraction of the Igh locus at g-satellite clusters. Below
images, distance between the VHJ558 and Cg1 probe signals. (c) Monoallelic
recruitment of the Igh locus to centromeres. Data represent the percentage of
cells showing association of one Igh allele with centromeric heterochromatin
in various B cell developmental stages, sorted as indicated in Supplementary
Fig. 1 online. Actual numbers and sample sizes are in Supplementary Table 2
online. (d) Preferential location of widely separated Igh alleles at the
centromeres. The cells showing monoallelic centromeric recruitment weresubdivided into a population of cells containing an Igh allele with wide sepa-
ration (1–1.5 mm) of the VHJ558 and Cg1 genes. Data represent the percentage
of centromeric recruitment of the widely separated Igh allele in this cell
population for large and small pre–B cells and activated (Act.) splenic B cells.
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and Cg1 probes localized the distal and proximal Igh domainswithin the nucleus. There was infrequent centromeric recruitment(16%) in pro–B cells, as reported before24 (Fig. 4c). In contrast, wedetected association of one Igh allele with g-satellite clusters in66–75% of large and small pre–B cells and in 65% of early immatureB cells (Fig. 4a,c). During subsequent development, the frequencyof centromeric recruitment was decreased to 34% in late immatureB cells (Fig. 4c) and to 28% in resting splenic B cells24 (Fig. 5).Hence, centromeric recruitment of one Igh allele is initiatedtogether with allelic exclusion at the onset of pre–B cell developmentand is transiently maintained in B lymphocytes undergoing IgLgene rearrangements.
To gain insight into whether the DJH-rearranged or nonproduc-tively VHDJH-rearranged Igh allele is recruited to centromeres in pre–Bcells, we assessed individual Igh alleles for both centromeric recruit-ment and locus decontraction. For this, we evaluated all cells thatshowed centromeric recruitment and additionally contained one Ighallele with widely separated (1–1.5 mm) VHJ558 and Cg1 genes. Thewidely separated Igh allele in this cell population was recruited to thecentromere in 81–91% of small and large pre–B cells and in 88% ofactivated splenic B cells (Fig. 4d). The class of widely separated Igh lociis likely to consist of all Igh alleles with proximal VH7183-DJH,VHQ52-DJH and DH-JH rearrangements, whereas the recombinationof VH genes located in the central and distal Igh domains should giverise to only partial gene separation (0–1 mm) after decontraction inpre–B cells. The rearrangement status of Igh alleles was analyzed beforein large pre–B (preB-II) cells by single-cell PCR assay26. Reevaluationof those data demonstrated that DH-JH and nonfunctional VH-DJH
rearrangements accounted for 81% of all rearrangements within theproximal Igh domain compared with 26% of nonfunctional VH generearrangements in the central and distal Igh domains (SupplementaryTable 3 online). The close correlation between centromeric recruit-ment (81–91%) and nonproductive recombination (81%) in theproximal Igh domains strongly suggests that the DJH-rearranged ornonfunctionally VHDJH-rearranged Igh allele is recruited to thecentromere at the onset of pre–B cell development.
IL-7 signaling prevents centromeric Igh recruitment
As shown by three-dimensional FISH analysis, the recruited Ighlocus was oriented at the centromere in pre–B and activated B cellsin such a way that the distal VHJ558 gene family was positioned closerto the g-satellite cluster than the proximal VH7183 and Cg1 genes in96% (50 of 52) and 90% (102 of 113) of recruited Igh alleles,respectively (Fig. 4a,b and data not shown). Centromeric recruitmentof the VHJ558 gene family thus coincides with histone deacetylation ofthe distal Igh domain in pre–B cells23, suggesting a link between thesetwo processes. Deacetylation of the distal VH genes has been associatedwith downregulation of IL-7 receptor signaling at the pro–B cell–to–pre–B cell transition23. Moreover, the distal VHJ558 genes arehypoacetylated in resting splenic B cells, but can be acetylated duringB cell activation in response to IL-7 signaling23, which mimicsopening of the distal Igh domain in early pro–B cell development10.To investigate whether IL-7 signaling prevents centromeric recruit-ment of the Igh locus, we activated splenic B cells for 4 d with anantibody to CD40 (anti-CD40) in the presence or absence of IL-7,as described23. DNA FISH analysis with Cg1 and g-satellite probesshowed that B cell activation in the presence of IL-7 resultedin substantially reduced centromeric recruitment of the Igh locus(38%), which approximated the basal recruitment (28%) foundin resting B cells at day 0 (Fig. 5a,b and Supplementary Table 4online). The observed inverse correlation between IL-7 signalingand centromeric Igh recruitment in splenic B cells thus suggeststhat the downregulation of IL-7 receptor signaling in largepre–B cells23,33 may be required for the initiation of centromericrecruitment of the Igh locus at the onset of pre–B cell development.Treatment of activated B cells with IL-7, however, had no effect onrepositioning of the Igk allele24 to centromeric clusters (Fig. 5a,b),indicating that different factors regulate centromeric recruitmentat the Igh and Igk loci. In activated splenic B cells, the Igk locuswas recruited to the centromeres also via the distal Vk24 region(Supplementary Fig. 3 online), demonstrating that centromericrepositioning of immunoglobulin loci via their distal domain is amore general phenomenon.
a b
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Figure 5 IL-7 signaling prevents centromeric recruitment of the Igh
locus in splenic B cells. (a) Two-color three-dimensional DNA FISH
analysis of activated splenic B cells with (+IL-7) or without (Control)
IL-7, showing the proximity of the Cg1 (Igh) and Ck (Igk) signals (green)
with g-satellite clusters (red). Both Igh or Igk alleles are on the same
optical section. (b) Statistical analysis of cells with centromeric
association of one Igh or Igk allele in splenic B cells before and after
in vitro activation with anti-CD40 in the presence (+IL-7) or absence
of IL-7. Actual numbers and sample sizes are in Supplementary
Table 4 online.
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Extended Igh alleles in l-transgenic B lymphocytes
Cell surface expression of a functional m protein is essential forinitiating of feedback inhibition of VH-DJH recombination and Ighallelic exclusion in pre–B cells3,16. Allelic exclusion of the Igh locus hasbeen studied extensively in m-transgenic mice, in which the endogen-ous Igh alleles rarely undergo VH-DJH rearrangements, in contrast toefficient DH-JH recombination18–21. To further investigate the relation-ship between centromeric recruitment, locus decontraction andVH-DJH recombination, we next analyzed the pre–B and B cells oftwo m-transgenic strains of mice, M54 (ref. 34) and MD4 (ref. 35).Flow cytometry analysis confirmed that the splenic B cells of M54 andMD4 mice expressed transgenic IgMa and no endogenous IgMb
(C57BL/6), as published36,37 (Fig. 6a and data not shown). Beforethree-dimensional FISH analysis, we tested the VHJ558 and Cg1 probeson metaphase spreads of activated splenic MD4 B cells to ensure thatthey detected only the endogenous Igh loci at the telomeres ofchromosome 12 instead of the m transgene (Fig. 6b and data notshown). Contrary to expectations, the three-dimensional DNA FISHexperiments failed to show any substantial association of the
endogenous Igh loci with g-satellite clusters in bone marrow pre–Band immature B cells and activated splenic B cells of either M54 orMD4 mice (Fig. 6c–e and Supplementary Table 2 online). Inaddition, three-color FISH analyses of pre–B and immature B cellsshowed that the Cg1 signals were separated from the VHJ558 signals bya distance of 1–1.5 mm in more than 84% of all endogenous Igh alleles(Fig. 6d,f and Supplementary Table 4 online). Hence, this situationresembles the noncontracted state of the germline Igh alleles in Rag2–/–
Pax5–/– pro–B cells12. We conclude therefore that the endogenous Ighalleles of m-transgenic B lymphocytes are present in an extendedconformation and undergo allelic exclusion in the absence of centro-meric recruitment.
Allelic inclusion of the most proximal VH genes
The data reported above suggest that decontraction rather thancentromeric recruitment is responsible for allelic exclusion of endo-genous Igh alleles in m-transgenic pre–B cells. The absence of locuscontraction furthermore suggests that proximal VH genes should beable to rearrange in m-transgenic pre–B cells in analogy to Pax5–/–
a Cγ1 PIb
f
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d MD4 large pre-B MD4 small pre-Bc MD4 splenic B
1–1.
5 µm
M54MD4
Figure 6 Absence of centromeric recruitment and locus contraction of endogenous Igh alleles in m-transgenic B cells. (a) Flow cytometry of IgM expression
on CD19+ B cells of C57BL/6 and M54 transgenic mice. The M54 transgene of the IgMa haplotype was derived from the BALB/c hybridoma 17.2.25
(ref. 34) and was crossed into the C57BL/6 mouse strain of the IgMb haplotype. Lymph node B cells from a 3-week-old M54 mouse express transgenic IgMa
but no endogenous IgMb, as published36,37. Numbers in dot plots indicate the percentages of cells in quadrants. The same degree of allelic exclusion was
noted for MD4 transgenic B cells35 (data not shown). (b) Metaphase spread of MD4 transgenic B cells. Specific hybridization of the Cg1 probe (green) is
detected at the telomeres of chromosome 12. Chromosomes were counterstained with propidium iodide (PI; red). (c) Confocal section through the nucleus
of an activated splenic MD4 B cell. The Cg1 regions (green) of both endogenous Igh alleles are not associated with g-satellite clusters (red), as visualized
by three-dimensional DNA FISH. (d) Confocal sections through the nuclei of large and small pre–B cells of the MD4 transgenic mouse. The Cg1 (green)
and VHJ558 (red) signals are widely separated and are not associated with g-satellite clusters (blue). (e) Statistical analysis of the monoallelic centromeric
association of endogenous Igh loci in B lymphocytes of wild-type (WT) and MD4 and M54 transgenic mice. ND, not determined. Actual numbers and sample
sizes are in Supplementary Table 2 online. (f) Statistical analysis of the distance separating the distal VHJ558 and proximal Cg1 regions of the endogenous
Igh loci in developing MD4 (red) and M54 (black) transgenic B lymphocytes (Supplementary Table 4 online). Light or dark shading indicates VHJ558-Cg1
gene separation of 0.3–1 mm or 1–1.5 mm, respectively.
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pro–B cells12. Indeed, mouse B cells expressing a human m transgenehave been shown to carry proximal VHQ52 gene rearrangements at arelatively high frequency20, although the results obtained with themouse M54 transgene are more ambiguous37. We therefore reinves-tigated the rearrangement status of endogenous Igh alleles in splenicM54 B cells, which we sorted for expression of the ma transgene(Supplementary Fig. 4 online). Rearrangements of the VHQ52 andVH7183 genes were readily identified in M54 B cells, albeit at a lowerfrequency than in wild-type B cells and Pax5–/– pro–B cells (Fig. 7).However, we were unable to detect VHGam3.8 and VHJ558 generearrangements in M54 B cells (Fig. 7), consistent with the factthat these genes depend on Igh locus contraction for efficientrecombination12. Hence, our results, together with earlier work20,unequivocally demonstrate that proximal VH genes fail to be allelicallyexcluded in B lymphocytes expressing either a mouse or a humanm transgene.
As the two overlapping VHQ52 and VH7183 gene families constitutethe proximal 320-kilobase region of the VH gene cluster11 (Fig. 8a),the question arises as to whether all of these genes or only a subset ofthem are able to undergo VH-DJH recombination in the absence of Igh
locus contraction. To investigate this, we compared the spectrum ofVHQ52-DJH and VH7183-DJH rearrangements in the presence of Ighlocus contraction in wild-type pro–B cells12 and in the absence of Ighlocus contraction in Pax5–/– pro–B cells12, M54 transgenic IgMa B cellsand ‘IkPax5/+’ pro–T cells12, which ectopically express a Pax5 ‘mini-gene’ under the control of the Ikaros (Zfpn1a1) locus38. We amplifiedVH-DJH4 rearrangements of pro–B and pro–T cells and VH-DJH3rearrangements of M54 (VHDJH4)34 transgenic B cells by PCR, thencloned and sequenced them and assigned the sequences to the variousmembers of the VHQ52 and VH7183 gene families (Fig. 8b,c).
VH7183
VHGam3.8
VHJ558
VHQ52
WT M54 Pax5–/–
ST2
Pro-B cells
12
3
Splenic B cells
12
3
1
2
3
12
3
Cµ
3x 3x 3x
Figure 7 Proximal VH-DJH rearrangements in M54 transgenic B cells. IgMa
CD19+ B cells expressing the ma transgene were isolated by flow cytometry
sorting from the spleens of M54 transgenic mice (Supplementary Fig. 4
online). PCR was used to determine the frequency of the different VH-DJH
rearrangements in these transgenic B cells versus Pax5–/– bone marrow
pro–B cells and wild-type B220+ splenocytes. Threefold (3�) serial DNA
dilutions were analyzed by PCR with VH family–specific forward primers and
a JH3 reverse primer12, which was unable to amplify the VHDJH4-rearrangedM54 transgene34. Bottom, input DNA was normalized by PCR amplification
of an Igh Cm fragment; far right lane, DNA of stromal ST2 cells (negative
control). Numbers along the left margin indicate rearrangements to
the JH1, JH2 and JH3 segments. The same result was obtained in two
independent experiments.
Distal
Rea
rran
gem
ent (
%)
Rea
rran
gem
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%)
a
b
c
VH71835′ 3′ Proximal
n1b1b2b3p
b4n2b5p
b6b7b8b9b10
b11
VHS
107.
b1
b2b3pb4n1b5pb6b7pb8b9
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5′ 3′
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IkPax5/+ pro-T cells
Pax5 –/– pro-B cells
WT pro-B cells
0
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60
0
20
40
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80
b5pb6b7b8b11
b22 b21 b20 b15 b12 b9 b6 b4 b2 b1p
b1b4 b2
Figure 8 The most proximal VH genes escape allelic exclusion in M54
transgenic B lymphocytes. (a) VHQ52 and VH7183 gene families of
the mouse C57BL/6 strain. The family members were identified by
annotation of the Igh sequences of the mouse genome database (http://
mendel.imp.univie.ac.at/SEQUENCES/VH/). The VH genes are numbered
according to published nomenclature11. b, C57BL/6; p, pseudogenes.
Our annotation identified previously unknown family members (n). Thick
horizontal black bars indicate a large sequence duplication; genes with high
sequence similarity are in the same color. The VH7183.b2 gene segment of
the C57BL/6 mouse corresponds to the VH81X gene segment of the BALB/c
strain. (b,c) Statistical analysis of VH-DJH rearrangements involving different
members of the VHQ52 (b) and VH7183 (c) gene families in wild-type
and Pax5–/– pro–B cells, IkPax5/+ pro–T cells and M54 transgenic IgMa
B cells. VH-DJH4 rearrangements of pro–B and pro–T cells and VH-DJH3
rearrangements of M54 B cells were amplified by PCR, cloned, sequenced
and assigned to the various family members. Data represent the percentageof rearrangements involving individual genes for each cell type. Total number
of distinct rearrangements analyzed: wild-type pro–B cells, 76 (VHQ52)
and 123 (VH7183); Pax5–/– pro–B cells, 61 (VHQ52) and 83 (VH7183);
IkPax5/+ pro–T cells, 73 (VHQ52) and 81 (VH7183); splenic M54 B cells,
71 (VHQ52) and 79 (VH7183).
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In the presence of locus contraction, VH genes across the entire320-kilobase region underwent VH-DJH recombination in wild-typepro–B cells, although with different efficiencies, as exemplified by thepublished high rearrangement frequency of the VH7183.b2 (VH81X)gene39,40. In contrast, only the two most proximal, functionalVH genes of the VHQ52 (b1 and b2) and VH7183 (b2 and b4) genefamilies were able to rearrange at a high frequency in M54B lymphocytes and IkPax5/+ pro–T cells, whereas Pax5–/– pro–B cellsshowed the same trend of preferentially rearranging the more proxi-mally located VH gene (Fig. 8b,c). These data indicate that only thefour most proximal VH genes of the Igh locus efficiently escape allelicexclusion in m-transgenic B lymphocytes.
DISCUSSION
Allelic exclusion is responsible for monoallelic expression of immu-noglobulin genes, which ensures the single-receptor specificity ofB cells, and yet the molecular mechanisms underlying this importantphenomenon are still mostly ‘enigmatic’3. Here we have describedcentromeric recruitment and locus decontraction as two previouslyunknown mechanisms that contribute to allelic exclusion of immu-noglobulin genes. Locus decontraction seems to be particularlyimportant, as all VH genes, with the notable exception of the fourmost proximal VH genes, were allelically excluded in the absence of Ighlocus contraction in m-transgenic B lymphocytes.
Centromeric recruitment has been associated with transcriptionalsilencing of the nonfunctionally rearranged Igh allele in activatedB cells24. Here we have shown that centromeric repositioning of oneIgh allele was initiated by the onset of pre–B cell development and wastransiently maintained in small pre–B and immature B cells under-going IgL rearrangements. The close statistical correlation betweenfully extended Igh alleles at the centromere and alleles with DH-JH andnonfunctional VH-DJH rearrangements in the proximal Igh domainstrongly indicated that centromeric recruitment contributes to theinactivation of the nonproductively rearranged Igh allele in pre–B andimmature B cells. As the mechanism controlling the monoallelicrepositioning to centromeres discriminates between the two Igh alleles,the question arises as to which distinguishing feature of these allelesdetermines the choice of the nonproductively rearranged Igh locus forcentromeric recruitment. For example, 40% of all pre–B cells containone productive and one nonproductive VH-DJH rearrangement3.These two alleles functionally differ from each other only by a singleframeshift mutation resulting in transcripts with a premature termi-nation codon, which could be discriminated in the nucleus by the‘nonsense-mediated’ mRNA decay pathway41. As transcription,mRNA processing and monitoring of the nonsense codon take placein the proximal Igh region, it is conceivable that this information isalready transmitted in the contracted state to the distal VHJ558 generegion, which subsequently is recruited to centromeric clusters in pre–B cells. The Igk locus is also associated with centromeres via the distalVk24 region in activated splenic B cells, indicating that the centro-meric recruitment of immunoglobulin loci via their distal domain is amore general phenomenon. During the pro–B cell–to–pre–B celltransition, the distal VHJ558 genes are not only recruited to centro-meres but also undergo histone deacetylation, leading to reducedchromatin accessibility23 as a consequence of the downregulation ofIL-7 receptor signaling in pre–B cells23,33. The idea of a causal linkbetween histone deacetylation and centromeric recruitment of theIgh locus is supported by the fact that treatment of activated splenic Bcells with IL-7 interferes with centromeric recruitment of the Ighallele (as reported here) while simultaneously inducing histone ace-tylation of the distal VHJ558 genes23. These findings are in agreement
with the temporal coincidence of histone deacetylation and centro-meric recruitment during the establishment of heritable silencing atthe terminal deoxynucleotidyltransferase (Dntt) locus42. It is unclear,however, whether histone deacetylation is the cause or consequence ofsubnuclear repositioning to centromeres, which constitute an envi-ronment with abundant histone deacetylase complexes43. Finally,centromeric recruitment seems to be incomplete in pre–B cells, asonly 66–75% of all nonfunctional Igh alleles are positioned atcentromeres. However, the Igh alleles in the remaining approximately30% of cells were located at the nuclear periphery12 (data not shown),which may function, in addition to the centromeres, as a repressivesubnuclear compartment44.
In contrast to centromeric recruitment, locus decontraction equallyaffects the two Igh alleles at the onset of pre–B cell development, as itdisconnects the proximal region from the central-distal domains onboth Igh loci. This physical separation, however, has a functionalconsequence only for the incompletely DJH-rearranged Igh allele,where rearrangements of central and distal VH genes are probablyprevented, analogous to the situation described for Pax5–/– pro–Bcells12,15. Whereas the Igh locus is in a decontracted state in pre–B cellsand all subsequent developmental stages, the Igk locus specificallyundergoes contraction during the phase of Igk rearrangements insmall pre–B and immature B cells. Hence, distinct factors must beinvolved in controlling the contraction of Igh and Igk loci at differentstages of B lymphopoiesis. Our FISH analyses demonstrated the newgeneral principle that immunoglobulin loci actively undergo contrac-tion only in rearranging cells, whereas they are in the extended defaultstate at all other developmental stages. Our experiments furthermoreshowed that the looping of multiple subdomains is responsible forcontraction of both the Igh and Igk loci in rearranging B lymphocytes.At the molecular level, Pax5 has been identified as a key regulator,which induces Igh locus contraction in pro–B cells in cooperationwith an unknown factor ‘X’12. The inactivation of Pax5, however, isunlikely to be the cause of locus decontraction in response to pre-BCR signaling, as the function of this transcription factor is requiredthroughout B cell development45. Identification of factor ‘X’ will beneeded to test whether pre-BCR signaling induces the loss of thisregulator, thereby leading to decontraction of the Igh locus.
Immunoglobulin m transgenes have been used successfully to studyallelic exclusion of the endogenous Igh alleles during B cell develop-ment18–21. Our analysis of two different transgenes (M54 and MD4)has unequivocally demonstrated that the endogenous Igh alleles arepresent in an extended conformation but are not recruited tocentromeres in transgenic pre–B and immature B cells. The absenceof centromeric recruitment raises the question of how relevant the useof immunoglobulin m transgenes is for investigating the phenomenonof allelic exclusion. Indeed, early expression of a functional m transgeneis known to considerably shorten or even bypass pro–B cell develop-ment46, in which both Igh loci are normally made accessible at thechromatin level4,5,10 and undergo locus contraction6,12 before VH-DJH
recombination. As m-transgenic B cell precursors have only very lowexpression of VHJ558 germline transcripts (data not shown), the distalVH gene region may never be reorganized into accessible acetylatedchromatin, which, however, seems to be a prerequisite for subsequentcentromeric recruitment of the Igh locus in pre–B cells. Our experi-ments using B cells from m-transgenic mice thus suggest a function forcentromeric recruitment in reducing the accessibility of central-distaldomains of Igh loci that have previously been opened up during pro–B cell development. In contrast, the proximal Igh domain is activatedin both wild-type as well as m-transgenic pre–B and immature B cells,as germline transcription of the proximal VH7183 genes is as efficient
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in these cells as in pro–B cells (data not shown), and DH-JH
recombination of endogenous Igh loci occurs at a high rate intransgenic pre–B cells21. Unexpectedly, however, only the four mostproximal VH genes were efficiently rearranged in transgenic B cells,indicating that the absence of locus contraction interferes withVH-DJH recombination of all other VH genes in Igk-rearrangingpre–B and immature B cells. In wild-type mice, the rare allelicallyincluded B cells, which express two different IgM proteins on their cellsurface, show a disproportionately high frequency of proximalVHQ52-DJH and VH7183-DJH rearrangements47. Given our results,it is conceivable that the most proximal VH genes escaping allelicexclusion may give rise to some of these VH-DJH rearrangements notonly in pro–B cells but also in small pre–B and immature B cells.
In summary, our study has provided insight into how pre-BCRsignaling controls allelic exclusion at the Igh locus. Pre-BCR signalingresults in the rapid loss of RAG protein expression, thereby halting allfurther V(D)J recombination and ‘preparing the ground’ for theestablishment of allelic exclusion22. However, it is not known whichchanges the Igh locus would undergo during the short recombinase-free ‘window’ so that it could no longer be rearranged after subsequentre-expression of RAG proteins. As shown here, pre-BCR signaling inlarge cycling pre–B cells additionally induces rapid decontraction andcentromeric recruitment of the Igh locus, which are likely to preventVH-DJH rearrangement of the second Igh allele after re-expression ofthe V(D)J recombinase in small pre–B cells.
METHODSMice. IkPax5/+, Pax5+/– and Rag2–/– mice38,48,49 and m-transgenic M54 and
MD4 mice34,35 were maintained on the C57BL/6 background and were
genotyped as described34,35,38,48,49.
Flow cytometry sorting and analysis. Antibodies to the following, coupled to
fluorescein isothiocyanate (FITC), phycoerythrin or allophycocyanin, were used
for flow cytometry: B220 (RA3-6B2), CD4 (L3T4), CD8a (53-6.7), CD19
(1D3), CD25/IL-2Ra (PC61), CD117/c-Kit (2B8), IgMa (DS1) and IgMb (AF6-
78; PharMingen), and goat polyclonal anti-mouse IgM (Southern Biotech).
Wild-type bone marrow was stained with the appropriate antibody
combination, and pro–B cells were isolated on a MoFlo cell sorter (Dako-
Cytomation) as c-Kit+CD19+ cells; pre–B cells, as B220+CD25+ cells; early
immature B cells, as B220+ IgMlo B cells; and late immature B cells, as
B220+IgMhi cells. The purity of the sorted cells was verified by flow cytometry
reanalysis (Supplementary Fig. 1 online).
Activation of splenic B cells. Splenic B cells of BALB/c mice were isolated by
elimination of non–B cells by magnetic cell sorting with anti-CD43 MACS
beads (Miltenyi Biotec) and then were activated with anti-CD40 (FGK-45) as
described24. The activated B cells were grown with or without 1% conditioned
supernatant of IL-7–producing J558L cells48 and were collected after 4 d for
three-dimensional DNA FISH analysis.
Probes for FISH. The locus-specific DNA probes were prepared from the
published bacterial artificial chromosomes CT7-526A21 (VHJ558)6, RP24-
282021 (VH11), CT7-296M13 (Vk24)50 and CT7-113G24 (Vk21)51 and plas-
mids HE17 (Cg1) and IgkC (Ck)24 and were labeled by nick translation with
dUTP-indodicarbocyanine, digoxygenin-dUTP or biotin-dUTP (Roche/Enzo
Biochem). The g-satellite probe was prepared from a plasmid containing eight
copies of the g-satellite repeat sequence24 and was labeled directly with dUTP-
rhodamine or dUTP-indodicarbocyanine (Amersham Pharmacia).
Three-dimensional DNA FISH and confocal analysis. Cells sorted by flow
cytometry were washed three times in PBS and then were fixed onto poly-L-
lysine-coated slides for two- and three-color three-dimensional DNA FISH
analysis as described in detail12,24,52. Digoxygenin-labeled DNA probes were
detected with sheep rhodamine-coupled anti-digoxygenin (Roche/Enzo
Biochem), followed by further signal amplification with Texas red–coupled
anti-sheep (Vector). Biotinylated DNA probes were detected with FITC-avidin
followed by further signal amplification with biotinylated FITC-coupled anti-
avidin and FITC-avidin (Vector). Cells were analyzed by confocal microscopy
on a Leica SP2 AOBS (acoustica optical beam splitter) system. Optical sections
separated by 0.3 mm were collected, and only cells with signals from both alleles
(typically 90%) were analyzed.
V(D)J recombination assay. B220+c-Kit+ pro–B cells were sorted from the
bone marrow of 2-week-old Pax5–/– mice; B220+ splenocytes, from wild-type
mice; and IgMa CD19+ splenocytes, from M54 transgenic mice at the age of
4.5 months (Supplementary Fig. 4 online). DNA was isolated and analyzed for
the presence of VH-DJH rearrangements (Fig. 7) by PCR assay with published
primers as described12.
PCR cloning and sequencing of VH-DJH rearrangements. Pax5+/+ and Pax5–/–
bone marrow pro–B cells as well as IkPax5/+ CD4+CD8+ thymocytes were
isolated by flow cytometry sorting as described12, followed by DNA preparation
and PCR amplification of VHQ52-DJH4 and VH7183-DJH4 rearrangements
with the following primers27: 5¢-CTCACAGAGCCTGTCCATCAC-3¢ (forward
VHQ52 VHB), 5¢-GTGGAGTCTGGGGGAGGCTTA-3¢ (forward VH7183 VHE)
and 5¢-TCTCAGCCGGCTCCCTCAGGG-3¢ (reverse JH4A). IgMa CD19+ sple-
nocytes were isolated from 4.5-month-old M54 mice as shown in Supplemen-
tary Fig. 4 online. As the m transgene M54 carries a JH4 rearrrangement34, we
amplified the rearrangements of endogenous Igh alleles from M54 transgenic
B cells using the forward VH7183 VHE or VHQ52 VHB primer in combination
with the JH3 primer12 5¢-GTCTAGATTCTCACAAGAGTCCGATA-
GACCCTGG-3¢. The VH-DJH4 or VH-DJH3 PCR fragment was gel-purified
and was cloned into the pGEM-T easy vector (Promega), followed by DNA
sequencing of individual clones. The 250– to 300–base pair 3¢ sequence of each
VH gene was assigned by sequence comparison to its corresponding VHQ52 and
VH7183 family member. For this purpose, we annotated the entire VHQ52 and
VH7183 gene region of the mouse C57BL/6 strain (http://mendel.imp.univie.
ac.at/SEQUENCES/VH/) based on the Igh sequences of the mouse genome
database (www.ensembl.org; release NCBIm32). Only VH sequences with
distinct VDJ joints were used for statistical analysis, with identical sequences
being counted only once.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTSWe thank F. Batista for providing MD4 mice, and A. Corcoran andN. Mitchison for critically reviewing the manuscript. Supported by a WellcomeTrust University Award, Boehringer Ingelheim and the Austrian GEN-AUinitiative (financed by the Bundesministerium fur Bildung, Wissenschaft undKultur).
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
Received 1 October; accepted 16 November 2004
Published online at http://www.nature.com/natureimmunology/
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