© Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 200, Number 9, 1205-1211
Important Roles for E Protein Binding Sites within the Immunoglobulin
Chain Intronic Enhancer in Activating V
J
Rearrangement
Matthew A. Inlay,
Hua Tian,
Tongxiang Lin, and
Yang Xu
Division of Biological Sciences, University of California, San Diego, CA 92093
Address correspondence to Yang Xu, Div. of Biological Sciences, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0322. Phone: (858) 822-1084; Fax: (858) 534-0053; email: yangxu{at}ucsd.edu
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Abstract
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The immunoglobulin
light chain intronic enhancer (iE
) activates
rearrangement and is required to maintain the earlier or more efficient rearrangement of
versus lambda (
). To understand the mechanism of how iE
regulates
rearrangement, we employed homologous recombination to mutate individual functional motifs within iE
in the endogenous
locus, including the NF-
B binding site (
B), as well as
E1,
E2, and
E3 E boxes. Analysis of the impacts of these mutations revealed that
E2 and to a lesser extent
E1, but not
E3, were important for activating
rearrangement. Surprisingly, mutation of the
B site had no apparent effect on
rearrangement. Comparable to the deletion of the entire iE
, simultaneous mutation of
E1 and
E2 reduces the efficiency of
rearrangement much more dramatically than either
E1 or
E2 mutation alone. Because E2A family proteins are the only known factors that bind to these E boxes, these findings provide unambiguous evidence that E2A is a key regulator of
rearrangement.
Key Words: B cell development V(D)J recombination accessibility monospecificity transcription factor
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Introduction
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Each B lymphocyte generates a unique set of immunoglobulin heavy (IgH) and light (IgL) chain genes through the somatic rearrangement of V, D, and J gene segments. To ensure the monospecificity of each B cell, V(D)J recombination is regulated in such a lineage- and stage-specific manner that IgH and IgL rearrangement occur at distinct stages of development (1). Because V(D)J rearrangement of all Ig loci involves the same recombination machinery, cis elements within these antigen receptor loci must play decisive roles in regulating the accessibility of each locus to V(D)J recombination machinery (2). Within the
locus, several cis-acting elements were initially identified by their ability to activate transcription of
reporter constructs in B cell lines, including two enhancers, one within the J
-C
intron (iE
) and one 3' of C
(3'E
) (38). More recently, a putative third enhancer 8 kb downstream of 3'E
(Ed) was also discovered (9). The deletion of the intronic enhancer and matrix attachment region (MiE
) led to decreased
rearrangement and a lower
:
ratio. More importantly, MiE
is required for the earlier or more efficient rearrangement of
versus
loci. (10, 11). The matrix attachment region (MAR) within MiE
does not contribute to these activities since the deletion of the
MAR alone does not have an inhibitory effect on the overall level of
rearrangement (12). The deletion of the 3' enhancer (3'E
) results in a similar, though less dramatic decrease in the ratio of
:
B cells and
rearrangement (13). In addition, 3'E
appears to play an important role in activating
transcription in mature B cells (11, 13). Although the loss of either enhancer alone does not eliminate
rearrangement, deletion of both enhancers from endogenous
loci results in a complete block of
rearrangement (11). Therefore, MiE
and 3'E
together are the necessary elements for
rearrangement.
Several functional motifs within the intronic enhancer were identified through a battery of biochemical and cell line transfection studies. One such functional motif is the NF-
B binding site, denoted
B (14). The potential role of the
B site in
rearrangement was suggested by the finding that LPS could induce
germline transcription and rearrangement through an NF-
Bdependent pathway (1518). iE
also contains a class of protein-binding motifs referred to as E boxes, which are also identified in enhancers of other antigen receptor genes (19, 20). iE
contains three E boxes, labeled
E1,
E2, and
E3.
E2 was found to bind the E2A gene products E12 and E47, which are required for B cell development (2123). E2A gene products can also bind to
E1 but not to
E3 (Murre, C., personal communication). E2A can induce germline transcription and
rearrangement when cotransfected with the RAG genes in a nonlymphoid cell line (24). However, the role of E2A in the regulation of
rearrangement in the physiological context remains unclear.
To delineate the mechanism through which iE
activates
rearrangement, we employed homologous recombination and Cre/loxP-mediated deletion to produce independently targeted deletions of four functional motifs within the endogenous
intronic enhancer. Analysis of the effects of these mutations on
rearrangement revealed that the E2A-binding E boxes, but not the
B site, were quantitatively important for the activation of
rearrangement. In addition, the simultaneous mutation of both
E1 and
E2 sites impacts
rearrangement as severely as the deletion of the entire iE
, indicating that E2A-dependent pathways are the major mediator of iE
's activity in activating
rearrangement.
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Materials and Methods
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Generation of m
EX Embryonic Stem Cells.
The 740-bp
intronic enhancer was cloned into pBluescript. The four sites were deleted and replaced with diagnostic EcoRI sites independently by site-directed mutagenesis. The primers used to replace each site are as follows: m
B, 5'-TGGCATCTCAACGAATTCAGAGCCATCTGG-3'; m
E1, 5'-GACTTTCCGAGAGAATTCAGTTGCTTAAGA-3'; m
E2, 5'-CAGTTCCTCCGAATTCGATTACAG-3'; and m
E3, 5'-TGGCTAAAAATTGA-ATTCAAACCATTAGAC-3'.
The m
E1/2 double mutations were generated by PCR-mediated mutagenesis. The
E1 and
E2 sequences are as follows, with the underlined C nucleotides mutated to A:
E1, CATCTGGC;
E2, CAGGTGGC. The MiE
deletion (m
D) was generated as described (10). All m
EX clones were inserted into the m
D targeting construct. 20 µg of each targeting construct was linearized with PvuI and transfected into embryonic stem (ES) cells. Transfectants were selected with G418 (300 µg/ml) and gancyclovir (1 µM). Homologous recombination was screened by Southern blot, and positive clones were then transiently transfected with 1020 µg of a plasmid containing the Cre gene to remove the PGK-neor gene as described (10). PGK-neordeleted ES clones were subcloned, confirmed by Southern blot, and analyzed by RAG-2deficient blastocyst complementation as described (10). m
E1/2 ES cells were microinjected into blastocysts before Cre transfection and then bred to Cre-expressing mice to generate m
E1/2 knock-in mice.
Quantitative Analysis of V
J
Rearrangements.
To detect V
J
rearrangements, genomic DNA from mature B cells (CD19+/IgM+) was purified and amplified by PCR using the degenerate V
primer (V
D) (15) and a primer within the kappa intronic enhancer (K1). PCR reactions were conducted under the following conditions: 95°C for 5 min, followed by 26 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 3.5 min, followed by an additional 10 min at 72°C. To control for the total amount of B cell DNA used in each PCR reaction, VDJH rearrangement was also analyzed using a primer for the J558 family (J558) and a primer that can bind to all four JH gene segments (JH(1+4)). The intensity of each PCR product was quantified with a Storm phosphorimager. Primers used are as follows: V
D, 5'-GGCTGCAGSTTCAGTGGCAGTGGRTCWGGRAC-3'; K1, 5'-CCAT-GACTTTTGCTGGCTGTAGATTTTACCTC-3'; J558, 5'-CTTCAGTGAAGCTGTCCTGCAAGGCTT-3'; and JH(1+4), 5'-CAGCTTACCTGAGGAGACGGTGA-3'. Hybridomas were generated and analyzed as described (10). The University of California, San Diego Animal Subject Committee approved all experiments that involved mice.
Quantitative Analysis of
Transcription by Real-Time PCR.
B cells from the spleens of WT and homozygous m
E1/2 mutant mice were purified by magnetic-activated cell sorting using
CD43 microbeads according to the manufacturer's protocol (Miltenyi Biotec). In m
E1/2 mutants,
+ B cells were removed by costaining with 0.5 µg/million cells biotin-conjugated
-mouse Ig
antibodies (clone R26-46; BD Biosciences) and then, after washing, staining with 0.5 µg/million cells
-biotin microbeads (Miltenyi Biotec). RNA from 1 million sorted cells was purified using the RNAeasy kit (QIAGEN) combined with on-column DNase digestion (QIAGEN) and converted into cDNA using the Superscript First Strand System (Invitrogen) according to the manufacturers' protocols. Primers for amplifying the constant regions of µ (Cµ1 and Cµ2) and
(C
1 and C
2) were designed using Primer Express software (Applied Biosystems) and used at concentrations of 200 and 400 nM, respectively. Real-time PCR reactions were performed using the SYBR Green PCR Master Mix (Applied Biosystems) in an ABI Prism 7000 Sequence Detection System according to the manufacturer's protocols. Relative transcription levels were calculated using ABI Prism 7000 SDS software using the standard curve method. Primer sequences are as follows: Cµ1, 5'-ACACCTGCCGTGTGGATCA-3'; Cµ2, 5'-GAGGAAGATGTCGGCAAAGG-3'; C
1, 5'-CAACTGTATCCATCTTCCCACCA-3'; and C
2, 5'-GGCACCTCCAGATGTTAACTGCT-3'.
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Results and Discussion
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Generation of m
EX ES Cells.
The 740-bp HindIIIAflII region spanning the
intronic enhancer and associated matrix attachment region (MiE
) was cloned into pBluescript. The NF-
B,
E1,
E2, or
E3 sites were each individually replaced with a diagnostic EcoRI restriction site by sitedirected mutagenesis (Fig. 1 F). These iE
mutants, collectively referred to as m
EX and individually as m
B, m
E1, m
E2, and m
E3, were independently inserted into the targeting construct used previously to delete MiE
, so that homologous recombination between the targeting vector and the endogenous locus would replace the endogenous MiE
with m
EX (10) (Fig. 1 B). Homologous recombination events were screened by Southern blotting with EcoRI digestion and hybridization to probe A (Fig. 1, A and C; not depicted). The PGK-neor gene was excised from the targeted allele by transient expression of the Cre gene in positive ES clones (Fig. 1 D). ES clones with the PGK-neor deleted were subcloned and confirmed by Southern blot with EcoRI digestion and hybridization to probe A (Fig. 1 E, lanes 25).
Analysis of V
J
Recombination in m
EX B Cells.
The effects of m
EX mutations on
rearrangement in B cells were assayed by recombination activating gene-2deficient (RAG-2/) blastocyst complementation as described (10). Since heterozygous mutant ES cells were used, this enabled us to compare the rearrangement frequency between the WT and mutant
alleles. B cells were sorted from m
EXRAG-2/ mice and analyzed for V
J
recombination using a quantitative PCR assay as previously described (11, 12). This assay could detect rearrangement to all four functional J
gene segments and distinguish PCR products derived from the WT allele from those from the mutant allele due to an additional 150 bp within the mutant allele (Fig. 2 A). Therefore, the efficiency of V
J
1-5 rearrangement of the mutant
allele could be compared with that of the WT allele in the same PCR reaction (Fig. 2 C). Our analysis indicated that neither the NF-
B (m
B) nor
E3 (m
E3) site mutations had any apparent effect on
rearrangement (Fig. 2 B). However, mutation of the
E1 (m
E1) or
E2 (m
E2) site reduced the rearrangement efficiency of the mutant
allele. The mutation of the
E2 site had a more inhibitory effect on
rearrangement (Fig. 2 B, lane 4). In this context, the ratio of the rearrangement frequency of V
to J
1, J
2, J
4, and J
5 gene segments of the mutant allele versus that of the WT allele was 52.1, 31.9, 26.2, and 27.9%, respectively (Fig. 2 C). In m
E1 B cells, the rearrangement efficiency of the mutant allele was approximately twofold reduced for each of the four possible V
J
rearrangements compared with that of the WT allele (Fig. 2 B, lane 3, and C).
Analysis of V
J
Recombination in m
EX
+ Hybridomas.
To further confirm the impact of m
EX mutations on
rearrangement efficiency and analyze the rearrangement frequency of mutant and WT alleles in individual B cells, we generated hybridomas from the spleen cells of all four m
EXRAG-2/ mice as described (10). Genomic DNA derived from
+ hybridomas was analyzed by Southern blotting to detect V
J
rearrangements at the WT and mutant alleles. Consistent with the data from the PCR analysis, we observed the most dramatic decrease in the rearrangement frequency of the mutant allele in m
E2 hybridomas (Fig. 3 B). In this context, of the 67
+ hybridomas analyzed 48 had rearrangements on only the WT allele, 10 had rearrangements on only the mutant allele, and 9 had rearrangements on both alleles (Fig. 3 C). The ratio of the rearrangement frequency of the WT allele versus that of the mutant allele was
3:1. A less dramatic decrease was observed in m
E1 hybridomas (Fig. 3 C). Consistent with the data derived from the PCR analysis, little difference was detected in the rearrangement frequency of the WT and mutant alleles in hybridomas derived from the m
B and m
E3 B cells (Fig. 3, A and C).
To determine the contribution of the
E1 and
E2 sites to the full activity of iE
, hybridomas were also generated from B cells in which the entire intronic enhancer was deleted from one allele (m
D). Only 3 of 43
+ m
D hybridomas analyzed harbored a V
J
rearrangement on the mutant allele, whereas the WT allele was rearranged in all 43 hybridomas (Fig. 3 C). Therefore, the reduction of the rearrangement efficiency caused by the deletion of the entire iE
was much more dramatic than that caused by
E1 or
E2 mutations alone. In addition, these data indicate that the rearrangement of the mutant allele occurs only after the rearrangement of the WT allele in m
D B cells.
Critical Roles of
E1 and
E2 in Mediating iE
's Function in Activating
Rearrangement.
Although the reduction of
rearrangement caused by the
E1 or
E2 mutations alone was significantly less than that caused by the deletion of the entire enhancer, it is possible that these two sites have redundant functions since both sites can be bound by E2A family proteins. To test this hypothesis, we mutated both
E1 and
E2 sites simultaneously through homologous recombination and assayed for effects on
rearrangement. The mutation introduced at both sites was a single nucleotide (C to A) mutation that destroyed the canonical basic helix-loop-helix binding site (CANNTG) (Fig. 4 A). To determine the effects of the
E1/2 mutation on
rearrangement, we used the same PCR assay described in Fig. 2 to analyze
rearrangement of WT and mutant alleles in
+ m
E1/2 B cells purified from the spleens of RAG/ chimeric mice or heterozygous mutant mice. The reduction in
rearrangement caused by the m
E1/2 mutation was much more severe than that caused by either
E1 or
E2 mutations alone (Fig. 4, B and C). Analysis of
+ hybridomas derived from m
E1/2 B cells revealed a more than 10-fold reduction in the rearrangement efficiency of the mutant allele, comparable to that caused by the deletion of the entire enhancer (Fig. 4 D). In addition, similar to findings in m
D hybridomas, the m
E1/2 allele only rearranged after the WT allele had already rearranged.
To rule out the possibility that the greatly reduced rearrangement efficiency of m
E1/2 alleles observed in splenic B cells is due to the impaired expression of rearranged
at m
E1/2 alleles during the transition from preB cells to immature B cells, we examined the rearrangement frequency of WT and mutant alleles in preB cells (B220+/CD43/IgM) sorted from the BM of m
E1/2 mice (Fig. 4 E). Similar to that observed in splenic B cells, the rearrangement frequency of the m
E1/2 allele was significantly reduced in preB cells compared with the WT allele. To further determine whether the m
E1/2 mutation affects
expression in B cells, we analyzed
expression in
1 B cells purified from the spleens of WT mice and homozygous m
E1/2 mutant mice. Similar levels of
mRNA were detected in WT and m
E1/2
1 B cells, indicating that the m
E1/2 mutation had no apparent effect on
transcription (Fig. 4 F). Therefore,
E1 and
E2 play synergistic roles in iE
's function in activating
rearrangement.
Sequential rearrangement of the IgH and IgL chain genes is critical to ensure B cell monospecificity. The
intronic enhancer plays an important role in this process (10, 11). By analyzing the functional motifs within iE
in vivo, we demonstrated that the
B site mutation had no apparent impact on
rearrangement. The
B site is bound by NF-
B family members and was found to play an important role in activating
rearrangement of recombination substrates in cell line transfection and transgenic studies (16, 25). However, the findings that the impacts of
E1 and
E2 double mutation on
rearrangement are very similar to the deletion of the entire iE
rule out any important roles of other protein-binding motifs within iE
, such as the NF-
B binding site, in activating
rearrangement. Based on the findings that iE
and 3'E
are the essential elements activating
rearrangement and that there is no NF-
B binding site within 3'E
, it remains unclear how NF-
B family transcription factors might activate
rearrangement. Based on the findings that NF-
B is activated during B cell activation induced by cross-linking of the antigen receptor (for review see reference 26), the potential roles of the
B site in receptor editing and somatic hypermutation of
should be examined.
Our findings provide unambiguous evidence that the
E1 and
E2 sites play critical roles in activating
rearrangement. E2A family transcription factors are the primary transcription factors binding to the
E1/2 sites, and when coexpressed with the RAG proteins, are able to activate
rearrangement in a nonB cell line (24). Therefore, our findings provide a mechanism for how E2A family transcription factors might function in activating
rearrangement. Based on the findings that E2A can interact with histone acetyltransferases such as p300/CBP and the SAGA complex (2729),
E1/2 might activate
rearrangement by increasing histone acetylation of the
locus.
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Acknowledgments
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The authors thank David Baltimore, Cornelis Murre, and Ann Feeney for helpful discussion and critical reading of the manuscript.
This work was supported by a National Institutes of Health grant (AI44838) to Y. Xu.
The authors have no conflicting financial interests.
Submitted: 7 June 2004
Accepted: 23 August 2004
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