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The Journal of Immunology, 2008, 180, 7443 -7450
Copyright © 2008 by The American Association of Immunologists, Inc.
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Requirement for Enhancer Specificity in Immunoglobulin Heavy Chain Locus Regulation1

Igor I. Kuzin*, Ludmila Bagaeva{dagger}, Faith M. Young*,{ddagger},§ and Andrea Bottaro2,*,{ddagger},§

* Department of Medicine, {dagger} Department of Neurology, {ddagger} Department of Microbiology and Immunology, and § J. P. Wilmot Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The intronic Eµ enhancer has been implicated in IgH locus transcription, VDJ recombination, class switch recombination, and somatic hypermutation. How Eµ controls these diverse mechanisms is still largely unclear, but transcriptional enhancer activity is thought to play a central role. In this study we compare the phenotype of mice lacking the Eµ element ({Delta}Eµ) with that of mice in which Eµ was replaced with the ubiquitous SV40 transcriptional enhancer (SV40eR mutation) and show that SV40e cannot functionally complement Eµ loss in pro-B cells. Surprisingly, in fact, the SV40eR mutation yields a more profound defect than {Delta}Eµ, with an almost complete block in µ0 germline transcription in pro-B cells. This active transcriptional suppression caused by enhancer replacement appears to be specific to the early stages of B cell development, as mature SV40eR B cells express µ0 transcripts at higher levels than {Delta}Eµ mice and undergo complete DNA demethylation at the IgH locus. These results indicate an unexpectedly stringent, developmentally restricted requirement for enhancer specificity in regulating IgH function during the early phases of B cell differentiation, consistent with the view that coordination of multiple independent regulatory mechanisms and elements is essential for locus activation and VDJ recombination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The IgH locus intronic enhancer, Eµ, was one of the first transcriptional enhancers identified and remains one of the best characterized (reviewed in Ref. 1). It is organized around a central core element, cEµ, flanked by two matrix attachment regions (MARs)3 (reviewed in Ref. 1). In addition to its activity as a transcriptional regulator, Eµ has been implicated in VDJ recombination and class switch recombination (CSR) (2, 3, 4, 5, 6, 7). An involvement of Eµ in somatic hypermutation (SHM) has also been proposed, although clear cut evidence for this effect is still lacking (7, 8). A critical outstanding question regarding the multifaceted activities of Eµ is how a single regulatory element, albeit complex like Eµ, can be implicated in such a diverse array of mechanisms. In particular, we are interested in understanding whether Eµ multifunctionality reflects inherent functional subspecialization of individual enhancer elements or whether a common functional principle underlies the regulatory processes for all mechanisms.

The regulated process of VDJ recombination requires the sequential activation of variable (V), diversity (D), and joining (J) segments in Ag receptor gene loci at specific developmental stages (9, 10). The precise mechanisms that determine this stepwise regulation are being actively investigated, but in agreement with the "accessibility model" they seem to be linked to transcriptional activation and/or concomitant alterations in DNA methylation and histone remodeling at the targeted regions (9, 10, 11).

The role of cis-acting regulatory elements in VDJ recombination at the various Ig and TCR loci has been studied primarily using artificial recombination constructs in cell lines and transgenics as well as in gene-targeting models (reviewed in Refs. 10 , 12, 13, 14). In the case of Eµ, these experiments have conclusively shown that this element, although not absolutely required for IgH locus rearrangement, is necessary for efficient D-J and V-DJ recombination steps, the bulk of this activity being attributable to the core Eµ element rather than the flanking MARs (2, 3, 5, 7, 15). An additional IgH regulatory element that drives transcription of DQ52, the most Eµ-proximal DH segment, was shown to be dispensable for the initiation of D-JH recombination (15). Finally, Eµ has also been shown to play similarly non-necessary but important roles in CSR and, in the case of SHM, in artificial constructs and cell lines but not the targeted endogenous IgH locus (4, 6, 7, 8, 16, 17).

The requirement for transcription in VDJ recombination, CSR, and SHM raises the possibility of a direct relationship between Eµ enhancer activity and these processes. Indeed, Eµ activity in artificial transgenic substrates for all three processes can be replaced by other transcriptional regulatory elements, including nonspecific enhancers (17, 18, 19, 20, 21, 22). Similarly, the replacement of enhancer elements for other Ag receptor loci (Ig L chains or TCR loci) with exogenous enhancer elements results in changes in timing and lineage specificity of VDJ rearrangement but a limited decrease in overall activity (23, 24).

Thus, the diverse functions of Eµ in IgH locus regulation could indirectly be attributed entirely to its activity as a transcriptional enhancer. To address this question, we generated mice bearing either a complete deletion of the Eµ core enhancer element or the strong, ubiquitous SV40 enhancer as a replacement and compared their B cell phenotypes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of targeted embryonic stem cell clones and mutant mice

{Delta}Eµ and SV40eR vectors were generated by replacing the 272-bp SspI-PpuMI fragment spanning the IgH Eµ core enhancer region with a LoxP-flanked pgk-neor gene (deletion) or with a 275-bp SV40 core enhancer sequence plus LoxP-flanked pgk-neor gene (replacement) (where pgk is phosphoglycerate kinase). The two flanking arms were a 6.2-kb EcoRI-SspI fragment at the 5' and a 1.6-kb PpuMI-HindIII fragment at the 3'. A thymidine kinase gene driven by pgk promoter was inserted at the 3'-end of the construct for double G418/gancyclovir selection of homologous recombinants. F1.11 IgHa/b heterozygous embryonic stem (ES) cells (a gift from B. Sleckman, Washington University, St. Louis, MO) were transfected by electroporation. Double G418/gancylovir-resistant clones were screened for homologous recombination by Southern blotting using a 0.8-kb XbaI-BamHI fragment 5' of the Cµ gene as a probe. Positive clones were injected into C57BL/6 blastocysts and the resulting chimeric mice were first bred for germline transmission with C57BL/6 mice. Heterozygous progeny carrying a mutated IgH allele were identified by PCR using the primers PGK-F and Smu-B (Table I) and bred with transgenic mice bearing a Cre recombinase gene driven by the adenovirus EIIA promoter (25) for LoxP-mediated deletion of the pgk-neor gene. Mice in which the neo gene had been deleted were identified by genomic DNA PCR using primers UDEµ-F and UDEµ-B (Table I). For analysis of IgH locus expression in pro-B cells, {Delta}Eµ and SV40eR mice were bred with RAG2-deficient mice (26). All experiments involving mice were conducted in accordance with local and federal guidelines for the humane treatment of experimental animals following protocols approved by the University of Rochester Institutional Animal Care and Use Committee (Rochester, NY).


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  		Table I. List of PCR primers and oligonucleotide probes

 
Flow cytometry

Total splenocytes were collected by mechanical disruption of spleens. Bone marrow (BM) cells were harvested by flushing femur and tibia with PBS containing 3% FCS. RBCs in cell suspensions were lysed by incubation in hypotonic solution. Cells were stained with fluorochrome-conjugated Abs specific for mouse IgM, IgD, B220 (eBioscience), CD2, and CD43 (BD Pharmingen). Samples were run on a FACScalibur flow cytometer and analyzed using CellQuest software (BD Biosciences).

LPS cultures

For in vitro LPS cultures, spleen B cells from 6- to 12-wk-old mutant and control mice were first positively selected with anti-CD19 magnetic beads using LS columns (Miltenyi Biotec) and then cultured for 3 days in complete RPMI 1640 medium with 10% FCS and 20 µg/ml LPS. Dead cells were removed by centrifugation on Lympholyte M gradient before DNA extraction from cultured cells.

Long-term BM cultures (LTBMC)

LTBMC were established from freshly harvested BM cells using IL7-supplemented RPMI 1640 medium with 10% FBS as described (27). Suspensions of cells from parallel cultures were repeatedly harvested from weeks 4 to 8 and used directly for RNA or DNA extraction or frozen at –80°C for long-term storage.

Reverse transcription and quantitative PCR analysis

Total RNA was extracted from splenocytes and BM using TRIzol reagent (Invitrogen), contaminating DNA was removed with the "DNA-free" kit (Ambion), and reverse transcription was conducted with the SuperScript first strand cDNA synthesis kit (Invitrogen). Serial dilutions of cDNAs were amplified by PCR using the primer pairs listed in Table I. Quantitative real-time PCR (qPCR) with SYBR Green I fluorescent dye (Applied Biosystems) and primers specific for µ0 and mb-1 transcripts (Table I) was conducted using a Rotor-Gene real-time DNA amplification system (Corbett Research) and the following amplification parameters: 95°C for 15 min and then 42 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 30 s. Melting curve and data analysis of PCR products was performed using Rotor-Gene analysis software, and the comparative cycle threshold (CT) method was used for relative quantitation of the µ0 levels normalized to Mb-1 expression (28).

Methylation analysis

Genomic DNA from LTBMC or day 4 LPS-stimulated B lymphoblasts was extracted by SDS/proteinase K digestion and isopropanol precipitation. Purified DNA (30 µg) was first digested with HindIII endonuclease. After ethanol precipitation, one-third of the digested DNA was additionally treated with either MspI or its methylation-sensitive isoschizomer HpaII. Digested samples were resolved by gel electrophoresis using a 0.8% agarose gel and then blotted to nylon membranes and analyzed sequentially by Southern hybridization using either a 2.2-kb HindIII fragment spanning the region from 5' of DQ52 to the JH1 segment or a PCR-amplified 575-bp fragment mapping between DQ52 and JH1 (see primers in Table I), followed by a 0.7-kb EcoRI-HindIII fragment probe spanning the Iµ region immediately 3' of the Eµ enhancer site.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of {Delta}Eµ and SV40eR mice

Targeting constructs were generated using 129-strain genomic DNA and carrying either a complete deletion of the Eµ 272-bp SspI-PpuMI fragment ({Delta}Eµ) or its replacement with a 275-bp SV40 enhancer core fragment from a Clontech pEGFP-C2 vector (SV40eR) (Fig. 1A). The linearized constructs were transfected into the (129Sv x C57BL/6J)F1 ES cell line F1.11 (a gift from B. Sleckman, Washington University School of Medicine, St. Louis, MO). After selection and screening (Fig. 1B), cells from two ({Delta}Eµ) or three (SV40eR) targeted clones for each construct were injected into C57BL/6 blastocysts and the chimeras obtained were bred first with C57BL/6 mice to obtain germline transmission (Fig. 1C). Finally, germline mutant mice were bred with EIIA-Cre transgenic mice (25) to eliminate the G418 resistance gene, and the resulting progeny (Fig. 1D) were selected and bred for analysis. The results presented hereafter are from one germline {Delta}Eµ clone and two independent germline-transmitting, phenotypically indistinguishable SV40eR clones.


Figure 1
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  		FIGURE 1. Targeting and screening strategy. A, Maps of the IgH locus and targeting constructs. Relevant restriction enzyme sites (R, EcoRI; B, BamHI; S, SspI; P, PpuMI; H, HindIII) are indicated. Schematic 1: Structure of the endogenous IgH locus, with the position of DQ52 and JH segments, Eµ core (filled circle), and MARs (gray boxes), and Cµ exons 1 and 2. Schematic 2: Targeting construct for the {Delta}Eµ mutation, with LoxP sites (striped boxes) flanking a G418 resistance marker (neo) and a thymidine kinase negative selection marker (TK) outside of the 3' homology arm. The site of insertion of the SV40 enhancer (SV40e; open oval) is marked. Schematic 3: Structure of the {Delta}Eµ targeted locus (site of insertion of the SV40e in the SV40eR mutants is indicated below). PCR primers used for screening for germline transmission (PGK-F and Smu-B; Table I) are indicated by arrows. Schematic 4: Structure of the {Delta}Eµ targeted locus after Cre-mediated deletion of the neo gene (site of insertion of the SV40e in the SV40eR mutants is indicated below). PCR primers used for screening for deletion (UDEµ-F and UDEµ-B; Table I) are indicated by arrows. B, Southern blot analysis of targeted {Delta}Eµ (left) and SV40eR (right) ES cell clones. Genomic DNAs were digested with BamHI and hybridized with the 5'-Cµ probe (position shown in A, Schematic 1). IgH a and b alleles (IgHa and IgHb) in F1.11 cells generate fragments of ~8 and 9 kb, respectively, while successful integration of the targeting construct gene results in insertion of a new BamHI site and generates bands of 7.5 kb. IgHKO, IgH knockout; wt, wild type. C, PCR screening for germline transmission of the {Delta}Eµ-neo and SV40eR-neo mutations. Wild-type alleles do not amplify with the PGK-F and Smu-B primer pair, because the former maps to the neo marker and the latter is outside the construct homology region. D, PCR screening for Cre-mediated neo deletion. Using the UDEµ-F and UDEµ-B primer pair, wild-type (wt) es give rise to a 393-p band, {Delta}Eµ alleles to a 283-p band, and SV40eR to a 503-bp band.

 
B cell characterization in {Delta}Eµ and SV40eR mice

To initially characterize B cell development in the two strains, B cell populations from spleen and BM of homozygous {Delta}Eµ, SV40eR, and control C57BL/6 mice were stained with fluorochrome-labeled Abs to various B cell markers and analyzed by flow cytometry. As shown in Fig. 2 and Table II, both {Delta}Eµ and SV40eR mice displayed a significant decrease in absolute numbers and percentages of pre-B, immature B, and recirculating mature B cells in the BM and of IgM+B220+ B cells in the spleen, a deficiency that is slightly more severe in SV40eR than in {Delta}Eµ mice. This phenotype, consistent with previous observations in other Eµ-deficient mice (2, 3, 5, 7), points to a similar B cell developmental defect in {Delta}Eµ and SV40eR mice, suggesting that the SV40 enhancer cannot fully complement Eµ activity in B cell development.


Figure 2
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  		FIGURE 2. Flow cytometry of B cell populations in {Delta}Eµ and SV40eR mice. BM (top panels) and spleen (bottom panels) cells from {Delta}Eµ, SV40eR, and control mice were analyzed by flow cytometry with the indicated Abs. B220lowCD2+ pre-B cells were significantly decreased in both mutant strains and were mature B220+IgM+ cells in the spleen. Data are representative of 5–10 mice per strain.

 

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  		Table II. B cells in {Delta}Eµ and SV40eR BM and spleen

 
VDJ recombination analysis

The apparent block in B cell development in {Delta}Eµ and SV40eR mice is consistent with diminished VDJ recombination activity, as previously reported for other Eµ-deficient animals (2, 3, 5, 7). To assess the VDJ rearrangement status of {Delta}Eµ and SV40eR mutated alleles, we analyzed genomic DNA from magnetically sorted CD19-positive mature splenic B cells stimulated with LPS for 3 to 4 days. Although treatment with LPS may amplify various B cell subsets differentially, any such difference is unlikely to introduce bias with respect to the rearrangement status of the nonexpressed allele and serves to significantly increase the purity of the sample, thus reducing the contamination from nonrearranged alleles from non-B cell DNA, which is critical when dealing with low rates of unrearranged alleles in wild-type controls.

Southern blotting and densitometrical analysis was used to assess the retention of sequences upstream of the JH1 segment (deleted upon D-J rearrangement) and retention of the most distal DH segment, DFL16.1 (deleted upon V-DJ rearrangement), on the nonfunctional alleles of {Delta}Eµ, SV40eR, and control mice. The productively rearranged alleles having of course lost both sequences, the hybridization signal reflects specifically the retention of the sequences in question on the nonfunctional alleles. Hybridization with a probe specific for a nonrearranging gene, AID, was used for normalization.

In normal mature B cells only a small fraction of nonfunctional alleles (0–5%) are known to remain in germline configuration, whereas in various strains of Eµ-deficient mice this fraction is increased to 30–40% (2, 5, 7). Consistent with these previous observations, in our experiments 5'-JH1 sequences were essentially undetectable in normal B cells, while >30% of the hybridization signal was retained in {Delta}Eµ B cells (Fig. 3A). SV40eR B cells showed a similar picture to {Delta}Eµ mutants, indicating that a significant portion of nonfunctional alleles remained in germline configuration.


Figure 3
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  		FIGURE 3. Analysis of IgH rearrangement in {Delta}Eµ and SV40eR mice. Genomic DNA from day 4 LPS-stimulated B cell cultures from mutant and wild-type (WT) mice and from parental F1.11 ES cells were digested with HindIII, blotted, and hybridized with probes for AID and a region upstream of JH1 (A) or digested with EcoRI, blotted, and hybridized with probes for AID and a region upstream of DFL16.1 (B). Each experimental lane contained DNA from two pooled parallel cultures after magnetically sorting for CD19+ cells and Ficoll gradient purification of dead cells. The graphs show averages and SD values from three independent experiments (six experimental mice). Hybridization signals from AID and IgH sequences were quantified using ImageQuant software after exposure to fluorescent screens, and the signal ratios of IgH/AID in each lane was calculated and normalized to ES cell signal ratio (100% retention because of lack of rearrangement). Note the almost complete D-JH rearrangement of nonexpressed alleles in wild-type cells (~0% residual signal for 5'-JH1) vs ~30% 5'-JH1 retention in {Delta}Eµ and SV40eR cells (A). Similarly, {Delta}Eµ and SV40eR nonexpressed alleles show almost complete lack of VH-DJ rearrangement (~50% retention of 5'-DFL16.1 signal), whereas wild-type alleles show substantial VH-DJ rearrangement (20% 5'-DFL16.1 retention, 60% rearrangement).

 
With respect to V- to DJ rearrangement, in normal B cells we found that ~40% of nonfunctional alleles only display rearranged DJ segments with no VH segment involvement, resulting in 5'-DFL16.1 hybridization signal retention of 20%. In {Delta}Eµ and SV40eR B cell DNAs, on the contrary, ~50% of the 5'-DFL16.1 hybridization signal was retained, indicating that the nonfunctional alleles almost entirely lacked V-DJ rearrangements (Fig. 3B).

These results indicate that SV40eR and {Delta}Eµ alleles display a significant decrease in VDJ recombination activity at both the D-J and the V-DJ recombination step. However, the leakiness of the rearrangement on both backgrounds is sufficient to allow detectable if diminished B cell development.

Transcriptional activity of {Delta}Eµ and SV40eR alleles

Expression of surface Ig and differentiation of B lymphocytes in {Delta}Eµ and SV40eR homozygotes clearly indicates that both types of mutations are compatible with gene transcription through the IgH locus. However, this may be the result of intrinsic selection for rare transcriptionally active alleles, because the lack of Ig expression is known to cause apoptosis of mature B cells (29). To control for this possibility, we analyzed CD19+ sorted mature B cells from {Delta}Eµ and SV40eR mice for expression of the nonfunctional alleles, which are under no known selective constraint, by using RT-PCR and qPCR with primers specific for µ0 germline transcripts. Significant levels of transcripts were observed in both mutant strains, with SV40eR mutants showing 1.7- to 3-fold higher expression compared with {Delta}Eµ, consistent with the possibility of transcriptional enhancement from the SV40e element (Fig. 4, B and D). A similar, slightly higher mRNA level was observed for mature rearranged transcripts of the VH J558 and 7183 families (Fig. 4B and not shown).


Figure 4
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  		FIGURE 4. IgH germline transcript expression in {Delta}Eµ and SV40eR mice. A, Schematics of the location of transcripts analyzed by RT-PCR in these experiments (not in scale). Top, Germline (gl) region; bottom, rearranged J558 region. Note that the µ0 and VHJ558-Cµ transcript-amplified regions span spliced exons. B, RT-PCR analysis of IgH transcripts in CD19-purified mature splenic B cells. Serial 10-fold dilutions of cDNA samples were analyzed with the following primer pairs: µ0, Mu0 and GLµR; Cµ, CµAH-F and CµAH-B; VHJ558-Cµ, VHJ558S and GLµR; Mb-1, Mb1-F and Mb1-B (see also Table I). Two of three independent experiments (Exp.) are shown. C, RT-PCR analysis of IgH transcripts in BM pro-B cells of {Delta}Eµ/RAG2–/–, SV40eR/RAG2–/–, and RAG2–/– controls. Primer pairs were as follows: µ0, Mu0 and GLµR; germline (gl) VHJ558, VHJ558 and J558Sp2; {lambda}5, {lambda}5-F and {lambda}5-B (Table I). VHJ558 germline transcript RT-PCR products were blotted and hybridized with the internal J558-specific oligonucleotide probe VHJ558Sc. D, qPCR analysis of µ0 expression in SV40eR and {Delta}Eµ bone marrow (left) and spleen (right) samples obtained as described in A and B. Note how SV40eR alleles express 7- to 10-fold lower µ0 transcripts compared with {Delta}Eµ in the BM but 1.7–3-fold higher levels in mature B cells.

 
To better elucidate the role of IgH transcriptional activity in VDJ recombination of {Delta}Eµ and SV40eR alleles, we bred both mutations into the RAG2-deficient background in which B cell differentiation is arrested at the pro-B cell stage (26) and analyzed the expression of µ0 transcripts at this critical stage. Strikingly, little or no IgH locus expression was detected in SV40eR mutants while {Delta}Eµ pro-B cells, as already reported for similar mutants, express reduced but clearly detectable transcripts (Fig. 4, C and D). qPCR quantifies the reduction of µ0 transcripts in SV40eR BM compared with {Delta}Eµ as 7- to 10-fold (Fig. 4D). Expression of germline VH transcripts from 7183 VH family segments was comparable in controls and targeted B cells, while the large VH J558 gene family showed some interindividual variability, possibly due to genetic background, and was expressed at partially lower levels in both mutant strains compared with controls (Fig. 4C).

DNA methylation changes associated with the {Delta}Eµ and SV40eR mutations

To further investigate the functional status of the IgH locus in the {Delta}Eµ and SV40eR mutants, we decided to analyze the levels of DNA methylation. To obtain sufficiently pure material from early B cell precursors, we established IL7-supplemented, LTBMC from RAG2–/–, {Delta}Eµ, and SV40eR mutants. After 4–8 wk in culture, virtually all (>98%) suspension cells in these cultures consist of CD19+CD43+ pro-B cells (not shown). Genomic DNA methylation status at specific IgH sites (Fig. 5A) was established using MspI/HpaII restriction analysis. Hybridization with probes specific for either the DQ52-JH1 region or the Iµ exon (immediately 3' of Eµ or the introduced mutations) showed that both SV40eR and {Delta}Eµ loci were almost completely methylated at both JH and Iµ-proximal sites (Fig. 5B). When DNA methylation at the same sites was assayed in mature B cell cultures, however, SV40eR mutants showed complete demethylation of both JH and Iµ-proximal sites contrary to {Delta}Eµ mutants, in which ~10% of sites in both regions remain methylated (Fig. 5C).


Figure 5
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  		FIGURE 5. IgH locus DNA methylation in {Delta}Eµ and SV40eR B cells. IgH locus DNA methylation was analyzed by digestion of genomic DNAs from the indicated sources with HindIII alone or HindIII and either MspI or its methylation-sensitive isoschizomer HpaII. Resistance to digestion with HpaII compared with MspI indicates methylation of the site. A, Map of the relevant sites in the wild-type (wt) and mutant IgH loci (H, HindIII; M/H, MspI/HpaII; not in scale) and probes position. B, Southern blot analysis of DNA from cells harvested from IL7-supplemented LTBMCs of the indicated mice (>95% B220+CD43+ pro-B cells). Blots were sequentially hybridized with probes spanning either the entire DQ52-JH1 region (top) or the Iµ exon (bottom). Unlike IgH wild-type B cells, both SV40eR and {Delta}Eµ alleles show only limited demethylation at the JH locus. C, Southern blot analysis of DNA from magnetically sorted CD19+ LPS-stimulated splenic B cells from the indicated mice (>95% B220+ B cells). Digestion patterns are as indicated. Blots were sequentially hybridized with a probe spanning either the DQ52-JH1 interval (to avoid hybridization with DQ52-JH rearranged alleles) (top) or the Iµ exon (bottom). Both SV40eR and wild-type (WT) alleles are completely demethylated at both JH and Iµ-proximal sites, while {Delta}Eµ alleles show incomplete, although significant, demethylation. Note that the wild-type sample shows little DQ52-JH signal because of almost complete deletion of this region by VDJ rearrangement. One of two independent experiments is shown.

 
Thus, the observed decrease in VDJ recombination in SV40eR mutants is associated with the active inhibition of transcription and deficient demethylation of IgH loci in pro-B cells caused by the SV40e. However, as development progresses the suppression mechanism is released and SV40e becomes active as an enhancer, generating µ0 transcripts and driving proximal DNA demethylation to a larger extent than the {Delta}Eµ mutants.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A large body of evidence supports the critical role of enhancer elements of Ig and TCR loci in mediating not only transcriptional regulation but also the stage and lineage specificity of VDJ recombination, as well as additional mechanisms such as somatic hypermutation and class switch recombination (reviewed in Refs. 10 and 12, 13, 14). Despite this extensive literature, however, the precise mechanisms by which these elements exert their varied functions are still only partially understood. In particular, enhancer dependence and specificity as assayed in recombination substrates and transgenic systems have often proven to be more stringent than the corresponding activities of endogenous sequences. As a consequence, a general picture has emerged which suggests that the regulation of endogenous Ig and TCR loci is the result of complex interactions between multiple elements and signals rather than of individual regulatory sequences.

In this study we set out to address the critical issue of whether generalized transcriptional enhancer activity is sufficient to mediate at least some of the activities conducted by the intronic Eµ enhancer or whether a specificity requirement exists for this element. The evidence provided clearly points to the second conclusion, albeit in an unexpected fashion.

Although SV40e is known to provide a strong, general enhancer activity in almost every cell type tested, including lymphoid cells (30, 31 ; reviewed in Ref. 32), our observations indicate that it is not capable of complementing a deletion of the Eµ enhancer with respect to IgH locus function. Contrary to what would have been our prediction, however, this is not associated simply with a lack of activity of SV40e or its inability to mediate some of the more specialized functions of Eµ, such as recruitment of the VDJ rearrangement machinery, but to suppression of IgH germline transcription early during B cell development. In fact, the data presented here show that SV40e replacement of Eµ exerts an active inhibitory effect on germline transcription that goes beyond the simple lack of Eµ sequences. This situation is reminiscent of the negative effect of the insertion of pgk promoter-driven selection cassettes in various positions within the IgH locus, including upstream of Eµ (2, 3, 33, 34, 35, 36). However, while those findings were attributed to promoter competition effects, this is unlikely the case for SV40e, which is not known to harbor any intrinsic promoter activity. Thus, the most likely explanation is that not promoter competition alone but the disruption of a higher order IgH control system, possibly mediated by the interplay of multiple regulatory elements, is responsible for pro-B cell-specific IgH transcriptional suppression by the inserted SV40e.

There are two components to the SV40e-mediated negative effects. First, SV40e appears unable to mediate the extensive Eµ-dependent DNA demethylation required for full activation of IgH. This finding mirrors data by Forrester and colleagues showing that SV40e can replace Eµ as a transcriptional enhancer on demethylated artificial constructs in vitro but, unlike Eµ, it cannot cooperate with MAR sequences to drive long-range demethylation of artificially methylated sequences (37). Indeed, methylation of IgH was essentially unchanged in SV40eR vs {Delta}Eµ LTBMC pro-B cells as opposed to the largely demethylated normal loci.

The observation of low but detectable VDJ rearrangement in the absence of substantial µ0 transcription in endogenous pro-B cells is surprising, although not completely unprecedented. Untranscribed gene segments undergo active V-DJ rearrangement in both artificial substrates and endogenous loci, and insertion of a neor gene upstream of Eµ suppresses IgH transcription but allows detectable locus rearrangement (36, 38, 39). Furthermore, Eµ has been shown to be able to mediate locus accessibility in the absence of active transcription (40). Thus, the cis-acting structural and topological modifications that determine VDJ recombination accessibility can be uncoupled from the process of transcription. A second, not mutually exclusive possibility is that IgH expression in the SV40eR and neor insertion mutants is variegated, as was recently demonstrated for IgH transgenes bearing weakened enhancer elements (41). A very small fraction of SV40eR cells transcribing IgH at normal levels would still be able to progress to the pre-B cell stage and undergo expansion while the majority would be transcriptionally silent and recombinationally inactive. The fact that B cell development is somewhat more severely inhibited in SV40eR than in {Delta}Eµ mice, although the recombination rates on the nonfunctional alleles are similar between the two mutants, would seem to support the latter scenario with the difference between the mutants residing in the fraction of recombinationally active cells rather than in a stochastic reduction of recombination efficiency across all cells.

Regardless of the mechanism allowing IgH VDJ recombination in SV40eR pro-B cells, the inhibitory effect exerted by SV40e is specific to early B cell progenitors, as mature SV40eR B cells not only express high levels of functional Ig transcripts but also high levels of µ0 transcripts emanating from nonrearranged alleles, which are under no known selective constraint. Indeed, µ0 levels in SV40eR mature B cells are higher than those in {Delta}Eµ mature B cells, suggesting not only that the transcriptional suppression observed at the pro-B cell stage is relieved at this stage but that SV40e has assumed an active enhancer role in these cells. Consistent with this possibility, SV40eR mutant mature B cells show complete demethylation of IgH loci, unlike {Delta}Eµ mutants.

DNA methylation has been shown to play an important role in VDJ rearrangement, regulating allelic exclusion of the Ig{kappa} locus as well as accessibility to recombination of transgenic substrates (42, 43, 44). Although direct evidence for any role of DNA methylation at the IgH locus is missing, Abelson virus-transformed precursor B cell lines from mice bearing a replacement of Eµ with a neor gene showed that the lack of demethylation on the targeted alleles is associated with the block in VDJ recombination (3). We show in this study that although IgH is almost completely methylated in pro-B cells lacking Eµ, de-methylation accrues as B cell development progresses even in the absence of the enhancer and is significant, although still incomplete, in mature {Delta}Eµ B cells. Also importantly, residual methylation is found equally on rearranged and nonrearranged {Delta}Eµ alleles, because the fraction of methylated, unrearranged alleles (that is, ~15% methylated loci of the 15–20% left in germline configuration, i.e., up to 3% of total) cannot account for the total fraction of loci methylated at Iµ-proximal sites (>10%). This suggests that demethylation is not an absolute prerequisite for either VDJ recombination or IgH expression. Unlike {Delta}Eµ loci, demethylation is complete on SV40eR allele mature B cells, indicating a direct role of SV40e in this process, albeit at a later differentiation stage than pro-B cells.

Although persistent IgH DNA methylation can be at least partly responsible for the inhibition in VDJ recombination observed in {Delta}Eµ and SV40eR mutants, it does not explain the additional inhibition of transcription caused in pro-B cells by SV40e. The evidence for differential and stage-dependent (early inhibitory vs late activating) SV40e activity strongly argues for a model of IgH regulation involving the compatibility of multiple control elements in coordinated locus expression. For instance, IgH locus expression has been correlated with the temporal localization of the expressed alleles in specific nuclear compartments (45). In addition, specific holocomplex formation between distant regulatory elements has been implicated in regulating the function of the TCRβ locus (46). Differential effects of transcription factor binding on locus accessibility in different contexts and at different developmental stages have been shown for PU.1 activity at the Eµ and 3'-E{kappa} enhancers (47, 48). Any perturbation of these or analogous higher order processes by inappropriate transcription factor recruitment and/or local chromatin modifiers may result in further inhibition beyond that caused by the simple lack of the Eµ element. The SV40eR mutants therefore offer a unique opportunity to probe these elusive mechanisms by establishing the basis for their inhibition.


   Acknowledgments
 
We are grateful to Barry Sleckman for kindly providing ES cells, Jianzhu Chen for IgH genomic DNA clones, and Craig Jordan and John Manis for critically reviewing the manuscript. We also thank David Heinrich for technical support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01 AI45012 (to A.B.). Back

2 Address correspondence and reprint requests to Dr. Andrea Bottaro, University of Rochester Medical Center 695, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address: andrea_bottaro{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: MAR, matrix attachment region; BM, bone marrow; CSR, class switch recombination; ES, embryonic stem; LTBMC, long-term BM culture; pgk, phosphoglycerate kinase; qPCR, quantitative real-time PCR; SHM, somatic hypermutation. Back

Received for publication August 31, 2007. Accepted for publication March 3, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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