Hunter College and Graduate Center of The City University of New York, 695 Park Avenue, New York, NY 10021, USA
Correspondence to: L. A. Eckhardt
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Abstract |
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Keywords: antibodies, B lymphocytes, gene regulation, molecular biology
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Introduction |
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In the present study, we have identified the 3' IgH enhancers critical to IgH gene transcription in Ig-secreting cells, using a stably integrated IgH transgene as a target for enhancer deletions. As in the previous knockout studies, we were able to analyze enhancer function in the context of chromatin, but this IgH mini-locus system allowed us to circumvent difficulties we and others encountered in targeting the 3' IgH region for homologous recombination and, moreover, allowed us to directly examine 3' IgH enhancer function in the absence of the possibly compensatory effects of Eµ. Transgenes consisting of an Ig2b transcription unit linked to the 3' IgH enhancers were introduced into both an Ig-secreting plasmacytoma cell line and a surface Ig+ cell line. We used B lymphoid cell lines representing two different functional stages since earlier transient transfection studies (where reporters were not integrated into chromosomes) had revealed a pronounced difference in the behavior of the 3' IgH enhancers at these two stages (13). After stable expression of the integrated transgenes had been established, we deleted either hs1,2/hs3a or hs3b/hs4 from the transgenes of individual transformants and measured the effect on IgH expression levels.
As described below, we found that the hs3b/hs4 enhancer pair was essential for IgH gene expression from this IgH mini-locus when incorporated into the genome of an Ig-secreting cell line. In contrast, the function of this enhancer pair could be supplanted by the hs3a/hs1,2 enhancer pair in a surface Ig+ cell line. These studies constitute the first functional assessment of the hs3b and hs4 enhancers in a chromosomal context and reveal a critical, and apparently developmentally regulated role for these two enhancers in the control of IgH gene transcription.
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Methods |
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Cell lines 9921 and MPC11 were maintained in DMEM with 10% BCS. A20 was maintained in RPMI 1640 medium with 10% BCS.
Plasmid constructs
pBS185 is a cre recombinase gene expression vector (Gibco/BRL, Grand Island, NY; cat. no. 10347-011) in which cre is expressed under control of the major immediate early promoter from human cytomegalovirus (hCMV). Cre recombinase mediates loxP site-specific DNA recombination.
pEGFP-C1 encodes a red-shifted variant of green fluorescence protein (Clontech, Palo Alto, CA; cat. no. 6081). It was co-transfected with pBS185 in order to isolate cells that had taken up DNA as made evident by their fluorescence.
The following enhancer fragments were used in the construction of the IgH mini-loci diagramed in Fig. 2:
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The loxP sites inserted into the IgH mini-loci were derived from psk-2loxPneo (5.0 kb). This plasmid was constructed by inserting a loxP-flanked neomycin-resistance gene (neor) into pBS-SK+ (Stratagene, La Jolla, CA; cat. no. 21120). The loxP-flanked neor was obtained from ploxp2neo, a gift of Dr F. W. Alt (The Children's Hospital, Center for Blood Research, Harvard Medical School, Boston, MA).
A complete description of the mini-locus constructions is available upon request.
Stable transformations
Linearized plasmid DNAs were introduced into both 9921 and A20 cells by electroporation. Twenty micrograms of PvuI-linearized 2b-hs1-4loxPhs123a, PvuI-linearized
2b-hs1-4loxPhs3b4 or NotI-linearized, enhancerless psk-
2b were introduced into 9921 and A20 cells along with XhoI-linearized psk-2loxPneo (molar ratio of the
2b constructs:neor drug selection plasmid was 1:1). DNAs were combined with a 1 ml suspension of 107 9921 or A20 cells and the mixture was dispensed into a 0.4 cm (width) electroporation cuvette (BioRad, Hercules, CA). An electric pulse was delivered at 960 µF and 250 V by a BioRad Gene Pulser electroporator and Capacitance Extender (BioRad). G418 was added to the medium (1.5 mg/ml) 48 h after transfection and neor colonies were visible within ~2 weeks.
Transient transfections and cell sorting
Thirty micrograms of pBS185 (cre-expressing plasmid) and 2.5 µg pEGFP-C1 (green fluorescence protein-expressing plasmid), molar ratio 8:1, were simultaneously introduced, by electroporation, into 9921 and A20 clones that carried a single copy of one of the 2b mini-loci. DNAs were combined with a 1 ml suspension of 5x106 9921 or A20 cells for electroporation (960 µF, 290 V). Forty-eight hours after transfection, fluorescent cells (expressing the EGFP gene) were identified and bulk sorted with a FACS Vantage (Becton Dickinson, San Jose, CA) and then subcloned by limiting dilution into 96-well culture plates. Up to 15 subclones were analyzed from each of 24 single-copy transformants transfected with cre. The overall frequency of CRE-mediated enhancer deletion was 10%, with a range from 0 to 12 deletion subclones isolated per 15 subclones analyzed. Data shown are for all of the single-copy transformants that yielded one or more CRE-mediated enhancer deletion subclones.
Southern blot analyses
Agarose gel electrophoresis, transfers to membranes and DNA hybridizations were performed essentially as described previously (20). In all Southern analyses, ~25 µg of restriction enzyme-digested DNA was loaded into individual lanes of an 0.7% agarose gel. Probes included pJ11HE and Hs3b (probes A and B respectively, Fig. 2). pJ11HE is a 1.5-kb HindIIIEcoRI fragment isolated from pJ11, a plasmid containing the JH gene region of BALB/cJ liver DNA (21). hs3b is a 1.2-kb XbaI fragment isolated from plasmid pHS3.4 (18).
Northern blot analyses
Total cellular RNA was isolated by Trizol reagent (Gibco/BRL; cat. no. 15596-026) according to the manufacturer's instructions. Approximately 25 µg of total RNA were analyzed per sample. Northern blots were performed as previously described (20).
Probes were a 0.3-kb SacI fragment containing the CH3 domain of C2b and which does not cross-hybridize to
2a transcripts (22), and ß-actin (Ambion, Austin, TX; cat. no. 7323) and GAPDH (Ambion; cat. no. 7330) probes for normalization.
ELISA
Microtiter plates were coated with purified rat anti-mouse 2b (PharMingen, San Diego, CA; cat. no. 02041D). Coated wells were then incubated with 50 µl cell lysate, which was prepared by lysis of 106 cells in 0.5% Nonidet P-40 lysis buffer (19). Mouse
2b heavy chains were then detected with alkaline phosphatase-conjugated rabbit anti-mouse
2b (Zymed, South San Francisco, CA; cat. no. 61-0322), using p-nitrophenol phosphate as the enzyme substrate.
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Results |
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Each linearized plasmid construct was co-transfected with a neor selectable marker gene (psk-2loxPneo, see Methods) into 9921, an IgG2a-secreting cell line, and into A20, a surface IgG+ cell line. Immediately following transfection, the cells were dispensed into 96-well plates. G418-resistant clones were recovered in 3040% of the wells in each plate so that each growing well represented, on average, a single transformation event (Poisson statistic). Isolated clones were tested by genomic Southern for the presence of the 2b reporter gene.
When DNAs were digested with HindIII and hybridized with probe B (see Fig. 2), a transgene-specific fragment of 7.9 kb was detected in all transformants (representative data for single-copy transformants shown in Figs 4 and 6
). This probe also detects fragments from the endogenous IgH loci of 9921 and A20 so that comparative densitometry measurements of the transgene fragment and the endogenous fragments provided information regarding copy number (data not shown). Additional information was obtained from Southern blot analyses involving HindIII-digested genomic DNA and probe A. Note that with this probe and enzyme, each transformant should have a novel HindIII fragment due to random integration into the genome (see Fig. 2
). Transformants carrying multiple copies of the transgene at several independent sites were identified by virtue of the fact that Southern analyses of their DNA revealed multiple transgene-derived fragments (data not shown). As with probe B, probe A also detects HindIII fragments derived from the endogenous IgH loci of 9921 and A20. Probe A is derived from JH region sequences and, in 9921, detects only the productively-rearranged IgH chromosome (23). In A20, probe A detects a fragment from each of the allelic IgH chromosomes. Using this information, along with densitometry measurements of the autoradiographs from Southern blots with both probes A and B, therefore, we were able to assign copy numbers for the transformants.
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hs3a/hs1,2 and hs3b/hs4 are functionally redundant in the 2b mini-loci of surface Ig+ cells
Having established that we could measure expression from a single copy of either 2b-hs1-4 mini-locus in multiple transformants, we chose several single-copy transformants of the A20 cell line (surface Ig+ cells) for further analysis of enhancer function. Since each transgene contained loxP sites flanking either the hs3a/hs1,2 or the hs3b/hs4 enhancer pair, we could induce deletion of these pairs without changing the chromosomal location of the transgene. In this way, we could test directly whether one or the other enhancer pair was critical to maintaining gene expression when in a chromosomal context.
A20 transformants carrying the 2b-hs1-4loxPhs123a transgene were transiently transfected with both a cre-expressing plasmid (pBS185) and a plasmid expressing green fluorescence protein (pEGFP-C1). Cells that had taken up the EGFP plasmid were identified and isolated by flow cytometry (see Methods, data not shown). We expected that most of these cells would have also incorporated the cre-expressing plasmid. The sorted cells were cloned by limiting dilution and growing clones were recovered 3 weeks later.
Four independent transformants yielded subclones with deletion of hs3a/hs1,2, as determined by Southern blot. Genomic DNA from the A20-2b-hs1-4loxPhs123a transformants was digested with HindIII and Southern blots hybridized with probe B (refer to maps, Fig. 2
). Two HindIII fragments detected with this probe are derived from the endogenous IgH loci of A20 (Fig. 4
). Just below the smaller of these two fragments is the 7.9-kb fragment derived from the transgene (migrates near the 8.0-kb marker, see legend to Fig. 4
). This fragment was detected in each transformant before enhancer deletion (Fig. 4
, `B' lanes = before deletion; right panel,
hs123a). After loxP-mediated deletion of hs3a/hs1,2, the 7.9 kb transgene fragment should be lost and a new HindIII fragment formed, its size dependent upon the site of transgene integration (see maps, Fig. 2
). As expected, therefore, in the transformant subclones that had undergone loxP-mediated enhancer deletion, the 7.9-kb HindIII fragment was missing and a new HindIII fragment was detected (Fig. 4
, `A' lanes = after deletion; right panel,
hs123a). In two of the deletion clones (P19 and P71), the new HindIII fragment co-migrated with one of the endogenous HindIII fragments. This was obvious from the difference in relative signal intensity of these two endogenous fragments in A20 and in the transformants prior to enhancer deletion when compared to the deletion subclones. P19 deletion subclone (`B' lane in Fig.4
) has a new HindIII fragment that co-migrates with the larger endogenous fragment; P71 subclone has a new HindIII fragment that co-migrates with the smaller endogenous fragment.
These Southern blots were also hybridized with probe A (see map, Fig. 2) to confirm that the enhancer deletion left the rest of the transgene intact. This probe hybridizes to a HindIII fragment that spans most of the
2b transcription unit and extends into adjacent DNA at the site of transgene integration. As shown in Fig.2
, this fragment is predicted to be at least 8.2 kb in size and to differ among transformants (because of differences in integration site). As shown in Fig. 4
, a fragment >8.2 kb was detected in each single-copy transformant before enhancer deletion (Fig. 4
, `B' lanes). Moreover, the same size fragment was detected in each enhancer-deletion subclone, demonstrating that enhancer deletion had not disturbed the region of the transgene containing the
2b transcription unit (Fig. 4
, `A' lanes).
Having achieved the desired enhancer deletions at four different chromosomal sites in four independent, single-copy transformant lines, we measured 2b mRNA levels to determine the impact of these deletions on
2b transgene expression.
2b mRNA from the initial transformants and their enhancer-deletion subclones was quantified by Northern blot. As shown in Fig. 5
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2b mRNA levels changed very little after hs3a/hs1,2 enhancer deletion (Fig. 5
, cf. B and A lanes; right panel,
hs123a). In two transformant pairs (P17 and P19), transgene expression increased slightly after enhancer deletion while in the other two transformant pairs (P69 and P71), transgene expression decreased slightly (a graph of quantified data obtained from blot in Fig. 5
is provided in Fig. 8A
).
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A dramatic effect is seen upon deletion of hs3b/hs4 in Ig-secreting cells
In total, 9921 single-copy transformants carrying either the 2b-hs1-4loxPhs123a mini-locus or the
2b-hs1-4loxPhs3b4 mini-locus were similarly co-transfected with the cre-expressing and EGFP-expressing plasmids. Cells expressing EGFP were sorted by flow-cytometry, cloned by limiting dilution, and individual enhancer-deletion clones recovered. As described for the A20 transformants, both types of 9921 transformant contained a 7.9-kb HindIII fragment that hybridized with probe B. This fragment migrated just below a HindIII fragment derived from 9921's endogenous IgH loci (Fig. 6
, `B' lanes; 7.9-kb fragment migrates above 8-kb marker, see legend). The difference in signal intensity for the endogenous and the 7.9-kb transgene fragment is explained by the presence of four copies of the 3' IgH enhancer region within endogenous IgH loci: one is associated with the expressed
2a locus and the other three are associated with translocated copies of the IgH locus that juxtapose this region with the oncogene c-myc (27).
The 7.9-kb transgene fragment was lost after enhancer deletion in each of the enhancer-deletion clones and was replaced by a new HindIII fragment, its size dependent upon integration site (Fig. 6, `A' lanes). The enhancer deletion itself explains the difference in signal intensity for the 7.9-kb fragment (before deletion) versus the new HindIII fragment that replaces it (after deletion): in the 7.9-kb fragment, both hs3a and hs3b are present and homologous to this probe while in the new fragment, only one of these (hs3a or hs3b) is present.
Probe A was used on HindIII-digested DNA to confirm that the 2b transcription unit remained unchanged after enhancer deletion (lower panel, Fig. 6
). Consistent with that expectation, DNA from individual transformants yielded unique HindIII fragments that did not change after enhancer deletion (Fig. 6
, lower blot, cf. A and B lanes).
As shown in Fig. 7, the effect of hs3b/hs4 deletion on
2b transgene expression differed dramatically from that of hs3a/hs1,2 deletion. In all three independent, single-copy transformants carrying
2b-hs1-4loxPhs3b4, cre-mediated deletion of hs3b/hs4 resulted in a precipitous drop in
2b mRNA expression. In two of the clones, no
2b mRNA was detectable. In the third, it was reduced to ~8% initial levels (Fig. 8
). In contrast, deletion of hs3a/hs1,2 from
2b-hs1-4loxPhs123a transformants had no such effect. Enhancer-deletion subclones lacking hs3a/hs1,2 expressed the
2b transgene at levels that differed very little from those of the parental lines carrying all four enhancers (Figs 7 and 8B
). Hence, in this Ig-secreting cell line, the hs3b/hs4 enhancer pair has taken on a critical role in maintaining IgH transgene function.
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Discussion |
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Two mini-loci consisting of a 2b transcription unit and two different arrangements of the 3' IgH enhancers (hs14) were expressed in 115 out of 118 transformants analyzed from both a surface Ig+ cell line (A20) and an Ig-secreting cell line (9921). Without the 3' IgH enhancers, the same
2b transcription unit was inactive in all of 25 independent transformants isolated. Early studies of the individual 3' IgH enhancers showed that only hs4 had detectable activity in pre-B cells and surface Ig+ cells (17,18,3335). The remainder of the enhancers were believed to achieve activity only at the Ig-secreting stage of B cell development since all were active in plasmacytoma cell lines. Subsequently, transient transfection studies with the group of 3' IgH enhancers revealed pronounced synergistic activity in both surface Ig+ cell lines and primary B cells from mouse spleen (13,36). Consistent with the earlier studies, the 3' IgH enhancers (with the exception of hs4) had little or no detectable activity at this stage in development when assayed individually (13,36). In the present study, these findings are supported and extended by the demonstration that an IgH gene lacking the intronic enhancer Eµ and carrying only the 3' IgH enhancers is very efficiently expressed when integrated into the genome of a surface Ig+ cell line. The 3' IgH enhancer region, therefore, begins to play a role in IgH gene expression well before B cells differentiate into Ig-secreting plasmacytes.
The murine 3' IgH enhancers have been described as a LCR or, more recently, as having some of the attributes of an LCR (18,24). In the present study, site-independence was clearly demonstrated by active transgenes in all but three of 118 different chromosomal positions in two different B-lineage cell lines. There was no strict, copy number-dependence, however, and single-copy transgenes were expressed at varying levels that approached, but did not reach, that of an endogenous IgH locus. These findings are consistent with those of a transgenic mouse study (24). It is also worthy of note that in the latter study, the 3' IgH enhancers lay downstream of the reporter gene, rather than upstream as in our mini-loci, arguing against the notion that enhancer orientation relative to the promoter influenced LCR activity in our transgenes. Unlike previous studies, the reporter in the present study was an IgH gene, presumably the gene that the 3' IgH enhancers are meant to regulate. We conclude that the cloned 3' IgH enhancers, when fused into a single unit, are not capable of establishing a fully insulated and independently acting IgH locus. We have yet to identify, therefore, the minimal elements required for such locus autonomy. Possibly, the natural spacing among enhancers is required or, perhaps more likely, there are additional control elements that await discovery.
Another feature of the 3' IgH enhancers that has attracted attention is the palindrome that extends from hs3a to hs3b with hs1,2 at the axis of symmetry (25,26). While none of the assays of 3' IgH enhancer function published to date have re-created this palindrome in its entirety, our finding that two very different configurations of the 3' IgH enhancers yield equivalent and substantial activity argues against a strong dependence upon this palindromic arrangement for enhancer function. Notably, the palindrome found in the murine 3' IgH enhancer region is not conserved in the comparable region of human IgH loci (8).
The primary goal of the present study was to assess IgH 3' enhancer function in the context of chromatin, comparing IgH gene activity at a single chromosomal site before and after deletion of 3' IgH enhancer pairs. The experimental system allowed for the analysis of gene expression among multiple, independent clones, all of which were expressing the transgenes at appreciable levels before enhancer deletion. Enhancer deletion, therefore, provided a means for identifying the control elements required to sustain locus activity. Our results establish a critical and probably stage-restricted role for the hs3b/hs4 pair in IgH gene expression. In the Ig-secreting cell line, 9921, loss of this pair led to a dramatic decrease in (or loss of) IgH reporter gene expression. In contrast, in the surface Ig+ cell line A20, hs3a/hs1,2 and hs3b/hs4 were redundant with respect to their ability to enhance IgH gene transcription. Each pair sustained high levels of transgene expression in the absence of the other. The dramatic effect of hs3b/hs4 deletion in all three, independent transformants of 9921, each carrying a single copy of the 2b-hs1-4loxPhs3b4 mini-locus, and the lack of effect in all four, single-copy A20 transformants carrying the same mini-locus argues strongly for a cell-specific and against a chromosomal site-dependent mechanism. Prior work by us and others has shown 9921 and A20 to mirror the behavior of other cell lines at their respective stages, making these appropriate representatives of Ig-secreting and surface Ig+ cells respectively (13,17,35). We think it most likely, therefore, that the dramatically different effect of the hs3b/hs4 deletion in 9921 as compared to A20 cells reflects a stage-specific shift in the function of the 3' IgH enhancers. Experiments are underway to extend these findings to other cell lines and to determine whether the effect of the hs3b/hs4 paired deletion can be mimicked by a single-enhancer deletion (of hs3b or of hs4), further delineating the unique functions of these 3' IgH control elements.
The fact that the hs3b/hs4 deletion can be tolerated in the A20 transformants but not in 9921 transformants also suggests that hs3a/hs1,2 lose some aspect of their activity by the time cells reach the Ig-secreting stage. This was not apparent from transient transfection experiments in which hs3A and hs1,2 were active as an enhancer pair in Ig-secreting cell lines as well as in surface Ig+ cell lines and splenic B cells (J. Ong et al., unpublished data and 13,36). In these and other studies, hs3a and hs1,2 had low, but significant activity, even when assayed individually in Ig-secreting cell lines (13,17,33,37). These differences underscore the critical difference between assaying enhancer function within extra-chromosomal plasmids, as in transient transfections, and assaying enhancer function within a chromosome. Proteins that can bind enhancers on plasmids are not necessarily capable of penetrating a locus embedded in chromatin. We hypothesize that while there are transcription factors available in Ig-secreting cells that are capable of binding to and therefore mediating hs3a/hs1,2 enhancer function, this enhancer pair is unable, alone, to recruit the necessary chromatin-remodeling co-activators to allow transcription factor access to the transgene. Presumably, such remodeling factors that operate through interactions with hs3a and/or hs1,2 are available in surface Ig+ cells. This hypothesis will be explored in future experiments.
The mini-locus results presented here also shed light on our previous experiments in which hs1,2 was replaced with pgk-neor in 9921 cells (9). Given that deletion of hs3a/hs1,2 had no effect on mini-locus activity in 9921 cells while the hs3b/hs4 deletion had a pronounced effect, we conclude that the earlier hs1,2-deletion/neor-replacement experiments in 9921 constituted a technical knockout of all of the 3' IgH enhancers. Promoter competition led to re-direction of hs3b/hs4 activity away from the IgH promoter and toward that of pgk-neor, depriving the endogenous Ig2a gene of its essential, positive control elements (hs3b/hs4). It remains to be determined whether diversion of this enhancer pair was responsible, as well, for the aberrant class-switching phenotype seen in mice in which hs1,2 (or hs3a) was replaced with pgk-neor (11).
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Acknowledgments |
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Abbreviations |
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LCR locus control region |
CSR class switch recombination |
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Notes |
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Received 15 February 2001, accepted 27 April 2001.
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References |
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