NF-{kappa}B elements associated with the Stat6 site in the germline {gamma}1 immunoglobulin promoter are not necessary for the transcriptional response to CD40 ligand

Michael T. Berton1, Leslie A. Linehan1, KeriLyn R. Wick1 and Wesley A. Dunnick2

1 Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA
2 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA

Corresponding author: M. T. Berton; Email: berton{at}uthscsa.edu


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Both germline transcription and switch recombination of heavy chain genes are likely to be regulated by cis elements binding transcription factors in the promoter regions of germline immunoglobulin genes. To identify cis-acting elements important in germline transcription of the murine {gamma}1 heavy chain gene, we have used a transgenic approach. Seventeen kb {gamma}1 immunoglobulin transgenes with mutations in three NF-{kappa}B sites in the {gamma}1 proximal promoter, a putative CD40 response element, are expressed well. Compared to wild-type transgenes, there is no deficiency in the expression of the transgenes with mutations of the three NF-{kappa}B sites after induction of splenic B cells with IL-4 alone, CD40L, or CD40L + IL-4. There may be a small reduction in the response of these mutant transgenes after induction with LPS + IL-4. We also prepared transgenes that were truncated at –150 (rather than –2100) and therefore included the wild-type Stat6 binding site at –123 and the three wild-type NF-{kappa}B sites. Nevertheless, {gamma}1 germline transcripts were not expressed from these transgenes. We conclude that the three proximal NF-{kappa}B sites are dispensable for expression of {gamma}1 germline transcripts under most conditions. However, cis-acting elements distal to –150 must be critical to this transcription.

Keywords: B lymphocyte, CD40 signaling, class switch recombination, germline transcription, NF-{kappa}B


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Class switch recombination is mediated by a DNA deletion between switch regions sequences upstream of each CH gene except C{delta} (1,2). Transcription of an upstream exon (the ‘I’ exon), the switch region, and the C region in their germline configuration precedes class switch recombination. Both germline transcription and switch recombination are regulated in a gene-specific manner by a combination of cytokines and B cell activators. It is generally accepted that the same DNA binding proteins that regulate the expression of germline transcription also regulate switch recombination either directly or via a mechanistic role for germline transcripts (1,2). Thus, an understanding of which factors regulate germline transcription, and the cis-acting elements to which they bind, is likely to illuminate the regulation, and perhaps mechanism, of class switch recombination.

Germline transcripts of the murine {gamma}1 CH gene are induced to a small extent by IL-4 alone or CD40L alone, and are induced substantially by the combination of LPS + IL-4 or CD40L + IL-4 (36). This induction has been studied extensively in vitro and by transient transfection/reporter gene assays. IL-4 regulates gene expression largely by the activation of Stat6, and the binding of Stat6 to a consensus Stat site at –123 bp (relative to the 5' most transcription start site) is known to play a critical role in IL-4-mediated activation of the germline {gamma}1 promoter in reporter gene assays (68).

The role of NF-{kappa}B family members in murine {gamma}1 germline transcription and switch recombination has been also intensely studied, but has yielded some apparently contradictory findings. Three NF-{kappa}B binding sites have been identified at –95, –71 and –53 bp relative to the 5' most start site for {gamma}1 germline transcripts (9,10). Ectopically expressed NF-{kappa}B activates transcription from reporter constructs that include the {gamma}1 promoter for germline transcripts (9). Mutation of these NF-{kappa}B sites eliminates responsiveness to CD40 ligation or to ectopic NF-{kappa}B by the same reporter constructs in some cell lines (9,11), but not in other cell lines (10). Mutation of these NF-{kappa}B sites can have an effect on the IL-4 responsiveness of {gamma}1 reporter constructs (10), consistent with interactions between NF-{kappa}B and Stat6 bound to closely linked sites (12,13).

The role of specific NF-{kappa}B members in transactivation of the {gamma}1 gene has been studied. Some forms of the NF-{kappa}B heterodimers or homodimers are better than other forms at activating the {gamma}1 germline promoter in reporter constructs (14). Likewise, deficiencies in various components of NF-{kappa}B have different effects on germline transcription of and switch recombination to the {gamma}1 gene (1519). In normal B cells, CD40 ligation induces different and longer lasting NF-{kappa}B forms than does LPS activation (14). These results are consistent with the expression of endogenous {gamma}1 germline transcripts after CD40 ligation, but not after LPS treatment.

To test the role of the NF-{kappa}B binding sites in the proximal germline {gamma}1 promoter, we chose to express mutant forms of a {gamma}1 transgene in mice. One advantage of this approach is that the mutations could be studied in the context of a large {gamma}1 gene, extending from –2100 bp through the I exon, S{gamma}1, C{gamma}1 and 2 kb 3' of C{gamma}1. Another advantage is that transcription of the mutated genes could be studied in normal, rather than transformed, B cells that are at the correct developmental stage for maximal induction of heavy chain genes. A third advantage is that the genes are chromosomally integrated in copies ranging from 1 to 30, rather than the thousands of copies of extrachromosomal genes typically found in a transient transfection assay. For these reasons, we hypothesized that the transcriptional activity we measured would be relevant to the endogenous genes in normal B cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Production of transgenic mice
All nucleotides are numbered relative to the 5'-most start site for germline transcripts (20), which is residue 1490 of GenBank accession no. M12389. Two new lines of transgenic mice with the wild-type 17 kb {gamma}1 transgene [in p{gamma}1/HE17, (21)] were prepared with a GATC insertion in the BglII site at +197 in I{gamma}1. The transgene in these two lines (589 and 601) as well as the 17 kb {gamma}1 transgene without the insertion in the BglII site (lines 45 and 46) are collectively designated ‘wild type’ in this publication.

A KpnI/BglII fragment from pKB350lucM1-2-3 (10) that included the three mutated NF-{kappa}B sites from the proximal germline {gamma}1 promoter was joined to the HindIII (–2100)/KpnI (–150) fragment (lacking the BglII site at –1490), using a partial digest to avoid cutting at an upstream KpnI site (–1200). The mutations in the three NF-{kappa}B binding sites (shown in Fig. 1 at –95, –71 and –53 bp relative to the 5'-most start site for {gamma}1 germline transcripts) were constructed previously by oligonucleotide-directed mutagenesis as described (10). Each NF-{kappa}B site was mutated at three consecutive nucleotide positions known to be critical for NF-{kappa}B heterodimer binding (22). The sequences of the three mutated sites are ttcCTCCCCC, AGAAAaaaCC and tttGAACCCT, respectively (10). EMSA demonstrated that these mutations completely abrogated binding of NF-{kappa}B heterodimers to these sites (data not shown). The resulting HindIII/BglII fragment was cloned into the HindIII and BglII sites of p{gamma}1/HE17. The BglII site at +197 was filled in, inserting GATC, and the transgene was designated ‘NF-{kappa}B TM’.



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Fig. 1. Structure and copy number of transgenes. (A) The various transgene constructs used in this study, founder numbers of transgenic lines, and copy numbers of transgenic lines, are shown compared to the structure of the {gamma}1 gene and germline transcripts (top). Exons are depicted by solid boxes and the S{gamma}1 region by a hatched box. The S{gamma}1 region in our transgenic constructs has a 3 kb deletion relative to the S{gamma}1 region in BALB/c DNA. The Stat6 binding site at –123 is depicted as an oval and three NF-{kappa}B binding sites at –95, –71 and –53 are depicted as open boxes. ‘X’ in the boxes denotes mutation of the NF-{kappa}B sites. Key restriction sites are shown: H, HindIII; K, KpnI; B, BglII; E, EcoRI. The BglII site in I{gamma}1 in the bottom three constructs is filled in, resulting in a GATC insertion, loss of the BglII site (no ‘B’) and creation of a TaqI site. (B and C) Transgene copy number. Southern hybridization experiments were used to determine transgene copy number. Founder numbers of transgenic lines are shown above the lanes. An S{gamma}1 probe (the BamH1 Y fragment) was used to detect polymorphisms between the transgene (7 kb SstI or 2.2 kb BamHI fragment) and the endogenous genes (5 kb SstI or 1.7 kb BamHI fragment) (23). A constant region probe was used to distinguish the transgene (which has a 4 kb BamHI fragment in each head to tail copy) and the endogenous gene (in a 13 kb BamHI fragment). Copy numbers were calculated as described in Methods.

 
The transgene truncated at –150 was constructed by cloning the KpnI (–150)/BglII (+197) fragment into pGEM. This fragment was extracted by HindIII and BglII digestion and cloned into the HindIII and BglII sites of p{gamma}1/HE17, effectively deleting the sequences from the HindIII site to the KpnI site at –150. The structure of the promoter region of this construct was verified by sequencing, and the BglII site at +197 was filled in, inserting GATC. This transgene was designated ‘–150’. For the production of the three types of transgenic mice, the 15 or 17 kb HindIII/EcoRI insert was purified and injected into C57BL/6xSJL (F2) fertilized eggs. To establish transgenic lines, transgene positive mice were bred to non-transgenic littermates or to C57BL/6 mice.

Copy number of transgenes was determined by Southern hybridization, using BamHI digestion and probes for S{gamma}1 [p{gamma}1/B.Y (23)] or C{gamma}1 (the 3.5 kb BamHI/EcoRI fragment with C{gamma}1). Hybridization to the endogenous genes was used to calibrate hybridization to the transgenes. A correction was made for blots with the S{gamma}1 probe, as it hybridizes 2.3-fold better to ‘a’ allele (transgenic) fragments than ‘b’ allele (endogenous) fragments.

Cell culture
Single cell suspensions of splenic B cells were purified as described (6). B cells (106/ml) were cultured in RPMI 1640 supplemented with 10% FBS (Hyclone) for 24–72 h with Sf9 cells (2 x 105/ml) expressing mouse CD40 ligand (CD40L) (6,24), IL-4 (500 U/ml) and LPS (Escherichia coli; Sigma) (20 µg/ml) as indicated. All cultures were maintained in a 6% CO2 atmosphere at 37°C.

Assay for germline transcripts
RNA was prepared using the one-step method (25). Germline {gamma}1 transcripts were detected by RNase protection using a probe (WD252) that distinguishes the transcripts from the transgene and endogenous genes due to the four bp insertion in the transgene (21). Alternatively, {gamma}1 germline transcripts were amplified (1 min, 95°C; 1 min, 54°C; 1 min, 72°C; 30 cycles) in the presence of [32P]dATP from cDNA (5', I{gamma}1: GACGGCTGCTTTCACAGCTT and 3', C{gamma}1: TAGTTTGGGCAGCAGATC). The purified 440 bp product was digested with TaqI, which cuts products from the endogenous gene once, and cuts products from the transgene twice due to the GATC insertion in I{gamma}1. Single nucleotide primer extension (SNuPE) was also used to detect germline transcripts (21). All radioactive gels were imaged with a Typhoon PhosphorImager and the results were quantified with ImageQuant software (Amersham BioSciences, Piscataway, NJ). The levels of transgenic germline transcripts detected in each assay were expressed as a percentage of the total germline transcripts (transgenic plus endogenous) and were reported as ‘percent transgene expression’.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The structure of the germline {gamma}1 transcripts and the wild-type and mutant transgene constructs generated in this study are shown in Fig. 1. The first major transcription start site lies 197 bp upstream of the 3' BglII site in the germline locus with additional start sites occurring over a 150 bp region downstream (20,26). The transgene constructs are shown aligned with the germline locus. Figure 1(A) also lists the founder numbers for each of the lines generated. Transgene copy number was estimated from Southern blots of tail DNA; examples are presented in Fig. 1(B).

Expression of {gamma}1 transgenes with mutations in the three germline {gamma}1 promoter NF-{kappa}B binding sites
As stated previously, three NF-{kappa}B binding sites have been identified at –95, –71 and –53 bp relative to the 5'-most start site for {gamma}1 germline transcripts (9,10). Ectopically expressed NF-{kappa}B activates transcription from reporter constructs that include the germline {gamma}1 promoter (9) and mutation of these NF-{kappa}B sites eliminates responsiveness to CD40 ligation or to ectopic NF-{kappa}B by the same reporter constructs in some cell lines (9,11), but not in others (10). We examined the role of these three sites in vivo by determining the expression of a germline {gamma}1 transgene (‘NF-{kappa}B TM’) in which all three NF-{kappa}B sites have been mutated to prevent the binding of NF-{kappa}B heterodimers (9,10). We determined expression of germline {gamma}1 transcripts in splenic B cells from wild-type and NF-{kappa}B TM transgenes in multiple mouse lines by three different assays described in the Methods.

We employed a semi-quantitative assay, RNase protection, which measured the amount of transgenic and endogenous germline transcripts directly (Fig. 2A). Previous studies have shown that the 17 kb wild-type {gamma}1 transgenes are regulated similarly to endogenous {gamma}1 genes by LPS, CD40L and IL-4 (21,27). Transcripts from the NF-{kappa}B TM transgenes are readily detected by protection of a transgenic (252 bp) fragment, compared to no protection of this size using RNA from non-transgenic B cells (Fig. 2B, compare lanes 5–10 with 1–3). In parallel to the endogenous genes, almost no transgenic germline transcripts are expressed by B cells cultured in LPS alone (lanes 13 and 17). Small quantities of transgenic transcripts are induced by treatment with CD40L, and greater quantities are induced by CD40L ± IL-4 treatment (lanes 6 and 7, 9 and 10, 15 and 16, 19 and 20). Therefore, to a first approximation, the NF-{kappa}B TM transgenes, in spite of the mutations in the three proximal NF-{kappa}B sites, are induced by the same treatments that induce the endogenous {gamma}1 gene. Virtually identical results were obtained from RNase protection assays of lines 594 and 622 (not shown). We did observe, however, that smaller quantities of transgenic germline transcripts are induced by LPS ± IL-4 than by CD40L ± IL-4 in NF-{kappa}B TM B cells (lanes 5 and 7, 8 and 10, 14 and 16, 18 and 20). This is different from the endogenous genes, where approximately equal amounts of germline transcripts result from stimulation with LPS ± IL-4 and with CD40L ± IL-4 (lanes 1 and 3). Thus, relative to the endogenous genes, the NF-{kappa}B TM transgene is expressed less by induction with LPS ± IL-4 (for example, 30% transgene expression for line 592 cells) than by induction with CD40L ± IL-4 (57% transgene expression for line 592).



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Fig. 2. Detection of transgenic {gamma}1 germline transcripts by RNase protection. (A) A 252 nt fragment of the WD252 probe (21) is protected from RNase digestion when annealed to transcripts from the transgene. However, when annealing occurs to the endogenous transcripts, RNase cuts at the 4 nt bubble in the probe created by the insertion at the BglII site in the I{gamma}1 exon. Thus, a 166 nt fragment of the probe is protected. (B) RNase protection was performed on RNA derived from the indicated transgenic B cells cultured as noted at the top of each lane. A probe for mouse actin was included in the assays for normalization. Percent transgene expression is shown below each lane.

 
We also noted that the addition of many extra {gamma}1 genes as transgenes does not increase the total amount of germline transcripts by a significant amount. Relative to actin protection, the amount of transgenic plus endogenous germline transcripts induced by LPS and IL-4 or CD40L and IL-4 in line 592, with 30 to 50 extra {gamma}1 genes, ranges from only 1.2 to 3.8 times that in non-transgenic B cells (Fig. 2B, lanes 5 and 7 compared to lanes 1 and 3). This is consistent with our hypothesis that the total amount of germline transcripts, at least in cells treated with LPS + IL-4 or CD40L + IL-4, is limited by the availability of some transcription factor(s) (21).

We wanted to study transcripts in B cells treated with IL-4 only, and explore further transcripts of the NF-{kappa}B TM transgene in B cells treated with LPS ± IL-4. To do this, we turned to two PCR-based assays that are well-suited for comparing the relatively small quantities of endogenous and transgenic germline transcripts in B cells treated with IL-4 only, and, at the same time, can compare with some accuracy the relative quantity of endogenous and transgenic transcripts in B cells treated with LPS ± IL-4. The SNuPE assay takes advantage of a polymorphism between the endogenous genes (C57BL/6) and transgenes (BALB/c) in the I exon (see schematic in Fig. 3A). If the primer used hybridizes to a PCR product of endogenous germline transcripts, a radiolabeled ‘T’ is added to it by Taq polymerase. If the primer hybridizes to a PCR product of transgenic transcripts, a radiolabeled ‘A’ is incorporated. We use this assay to determine the amount of transgenic germline transcripts relative to the amount of endogenous germline transcripts. In all SNuPE assays, quantities of RT–PCR product (determined in a preliminary experiment) were tested that would yield similar T incorporation for each sample. Therefore, we used larger amounts of cDNA for analysis of IL-4 only samples than for LPS ± IL-4 or CD40L ± IL-4 samples. By this experimental design, we normalized the transgene expression to the expression of the endogenous gene, and reported the results as percent transgene expression.



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Fig. 3. Detection of transgenic {gamma}1 germline transcripts by SNuPE and RT–PCR/Taq. (A) The schematic illustrates the central features of the SNuPE assay. (B) A SNuPE assay was performed using RNA from transgenic B cells cultured with the indicated agents. RNA from non-transgenic BALB/c and C57BL/6 mice was also analyzed to demonstrate the specificity of the SNuPE assay for the specific polymorphism used to distinguish transgenic and endogenous germline {gamma}1 transcripts. The percent transgene expression of {gamma}1 germline transcripts, calculated as described in Methods, is shown below each lane. (C) The relative levels of endogenous and transgene-derived germline transcripts in total RNA from transgenic B cells immediately after preparation (without culturing, ‘ex vivo’) or cultured with the indicated agents were determined by digestion of RT–PCR products with TaqI as described in Methods. The schematic illustrates how TaqI digestion differentiates transcripts from the transgene and the endogenous {gamma}1 gene. TaqI-digested RT–PCR products derived from the endogenous {gamma}1 gene and from the transgene are indicated. The percent transgene expression of {gamma}1 germline transcripts, calculated as described in Methods, is shown below each lane (nd, not detectable).

 
If the transgene expression parallels that of the well-characterized regulation of the endogenous {gamma}1 genes (36) (Fig. 2), then the percent transgene expression should be the same regardless of B cell treatment. This is approximately the case for two wild-type {gamma}1 transgenes (lines 45 and 46, Fig. 3B). If the {gamma}1 transgenes also paralleled the endogenous genes quantitatively, then the percent transgene expression should reflect the transgene copy number. For example, in line 45, 60% (three of five) of {gamma}1 genes in the B cells are transgenic and 55–61% of the transcripts are from the transgene. This indicates that in the four treatments tested in Fig. 3(B), the transgenes in line 45 are expressed just like the endogenous {gamma}1 genes. Of the small quantity of {gamma}1 germline transcripts known to be induced by IL-4 alone (5), almost 60% come from the transgene. Much larger amounts of germline transcripts are induced by treatment with CD40L ± IL-4 (Fig. 2B) (6), but the transgene still contributes almost 60% of the total transcripts.

Like line 45, the percent transgene expression was consistent for other wild-type transgenes across all treatments (with some small reductions discussed below). However, line 45 was unique in that each copy of the transgene expressed transcripts as well as an endogenous gene. For all other lines, expression per gene was 10–20% the amount of the endogenous gene (e. g. line 46 in Fig. 3B). The expression of transcripts from the transgene does increase, however, with transgenic copy number. Since the NF-{kappa}B TM transgenic mice that we obtained tended to have more transgene copies (7–50 copies), the percent transgenic expression was also greater (lines 619 and 305, Fig. 3B). One exception to this 43–89% expression by the NF-{kappa}B TM transgene was B cells treated with LPS ± IL-4, in which the transgenic expression was consistently lower, 30 and 27% in Fig. 3(B).

These results were verified in a third assay in which {gamma}1 germline transcripts were amplified by RT–PCR. PCR products derived from transgenic and endogenous transcripts were distinguished by digestion with TaqI (Fig. 3C). The RT–PCR/TaqI digestion assay is very similar to the SNuPE assay, the only difference being in how the two types of transcripts are distinguished in the final step. We have used 30 cycles for the amplification step, which is a compromise. The small amounts of {gamma}1 germline transcripts in B cells treated with LPS, and even those in B cells treated with IL-4 only, are amplified to modest amounts of product for analysis. On the other hand, the relatively abundant {gamma}1 germline transcripts in B cells treated with LPS ± IL-4 or CD40L ± IL-4 are amplified to saturation. Because amplification is outside the semi-quantitative range, the absolute amount of the transgenic product detected in B cells under one culture condition cannot be compared to the absolute amount of product detected under another culture condition. However, because the transgenic and endogenous transcripts, which differ by only a few bp, are amplified in proportion to their initial concentration, even if the PCR goes to saturation (21), it is informative to compare the percent transgene expression among various treatments.

Consistent with the established regulation of the endogenous {gamma}1 gene, it was difficult to amplify {gamma}1 germline transcripts from non-transgenic, line 589, or line 619 B cells that were lysed immediately after purification (‘ex vivo’) or that were cultured in LPS only (Fig. 3C, lanes 1, 3, 7, 9, 13 and 15). The small quantity of {gamma}1 germline transcripts in B cells treated with IL-4 was easier to amplify (lanes 2, 8 and 14). Since the percent transgene expression is almost identical in ex vivo B cells, in B cells cultured in LPS, and in B cells cultured in IL-4, the results support the data from the RNase protection assay (Fig. 2B) and demonstrate that the transgenes are poorly transcribed in unstimulated B cells and are induced, in parallel to the endogenous genes, by IL-4. Also consistent with the results of the RNase protection assays and the SNuPE assays, line 619 with 15 to 25 copies of the NF-{kappa}B TM (e.g. line 619 in Fig. 3C) had greater percent transgene expression in B cells treated with LPS ± IL-4, CD40L alone, or CD40L ± IL-4 than did line 589 with 7–12 copies of the wild-type transgene. However, the expression in NF-{kappa}B TM B cells treated with LPS ± IL-4 (41%) was consistently less than that in B cells treated with IL-4, CD40L, or CD40L ± IL-4 (69–88%).

To summarize all the expression data with the NF-{kappa}B TM transgenes, we pooled data from all three assays and several experiments (Fig. 4). With all treatments, the transgenes with the mutations in each of the three proximal NF-{kappa}B binding sites are expressed well. Consistent with previous observations (21), there is a correlation between transgenic copy number and percent transgene expression for lines 46, 601, 589, 305, 619, 622 and 592 induced by IL-4 alone—the more copies of the transgene, the greater the transgene expression (Fig. 4A). Superimposed on this copy number dependence is some contribution by the insertion site of the transgenes, as demonstrated by line 45. B cells from line 45 routinely express more transcripts per copy than do other lines.



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Fig. 4. Summary of NF-{kappa}B TM transgene expression. (A) Mean transgene expression, with standard deviation bars, is shown for four lines of wild-type transgenic mice, for non-transgenic mice, and for four lines of NF-{kappa}B TM mice. For each treatment and transgenic line, these means represent a pool of 4–18 SNuPE, RT–PCR/TaqI digestion, and RNase protection assays. Within statistical variation, each of the three assays yielded the same percent transgene expression. (B) Normalized transgene expression. For each transgenic line, the mean percent expression from LPS + IL-4, CD40L, or CD40L + IL-4 cultures were each divided by the mean percent transgene expression from IL-4 only cultures.

 
To control for both transgene copy number and any insertion site effects, we chose to normalize transgene expression using the IL-4 only-induced expression (Fig. 4B). The percent transgene expression values (Fig. 4A) do not indicate an effect of the three NF-{kappa}B site mutations on IL-4-induced expression of the transgene. Hence, we assumed that the quantity of IL-4-only transgene expression reflected both the copy number and any insertion site effects, without an influence of the NF-{kappa}B mutations. By dividing expression values for LPS + IL-4, CD40L and CD40L + IL-4 by the value for IL-4 only, we would be able to normalize the eight different lines of transgenic mice for copy number and insertion site effects, and thus isolate the effect of the NF-{kappa}B mutations. When normalized to IL-4 only expression, expression of WT and TM transgenes in CD40L-induced or CD40L + IL-4-induced B cells is reduced somewhat, consistent with the hypothesis that {gamma}1 transgene expression is limited by the availability of necessary transcription factors (see above, and Discussion). Importantly, the normalized transgene expression, on average, is virtually identical for the wild-type and NF-{kappa}B TM transgenes. This implies that there is no effect of the NF-{kappa}B site mutations on induction of the {gamma}1 gene by CD40 ligation. On the other hand, the normalized expression by NF-{kappa}B TM B cells cultured in LPS + IL-4 is reduced somewhat compared to wild-type transgenes (average for the four wild-type lines, 0.75 ± 0.35, compared to the average for the four NF-{kappa}B TM lines, 0.35 ± 0.10, P < 0.066). Although {gamma}1 transgenes with mutations in the three proximal NF-{kappa}B sites are expressed well after culture with LPS + IL-4, the inability of NF-{kappa}B to bind these sites does reduce that expression by a small increment that does not quite reach statistical significance.

Expression of {gamma}1 transgenes truncated at –150
We tested the possibility that the proximal promoter region sequences including the Stat6 binding site and the NF-{kappa}B binding sites were sufficient for the expression of transgenic {gamma}1 germline transcripts. In transient transfection assays, constructs truncated at –150 are induced by both IL-4 and CD40L (6,7,9). We prepared lines of transgenic mice that included the entire I{gamma}1, S{gamma}1 and C{gamma}1 regions used in our other constructs, but were truncated at the KpnI site at –150, and hence deleted ~2000 bp of promoter region sequence. B cells from these transgenic mice did not express germline transcripts from the transgene, regardless of the activators and cytokines used in vitro. For example, B cells from lines 834 and 994 (with 10–14 copies of the –150 transgene) did not express transgenic germline transcripts after culture in CD40L alone, CD40L + IL-4, or LPS + IL-4 (Fig. 5A; see also Fig. 2B, lane 4). Controlling for the viability of the B cells, induction by CD40L or IL-4, RNA preparation, etc., B cells from the same transgenic lines did express germline transcripts from the endogenous {gamma}1 gene (see the ‘T’ panel). A summary of expression data from four lines with the –150 {gamma}1 transgene is presented in Fig. 5(B). Even though mice with the –150 transgene had copy numbers of 7–14, their expression of {gamma}1 germline transcripts was between 1–5%, and was not significantly different than the expression from non-transgenic mice. The results of these experiments strongly indicate a role for sequences upstream of –150 in the induced transcription of the germline {gamma}1 gene.



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Fig. 5. Lack of germline transcripts from –150 transgenes. (A) A SNuPE assay was performed using RNA from transgenic B cells cultured with the indicated agents. (B) Mean percent transgene expression (with standard deviation bars) for 1–6 SNuPE and RT–PCR/TaqI assays for four lines of –150 transgenes are presented. Means and standard deviations for the four wild-type mice are reproduced from Fig. 4(A).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Proximal elements and {gamma}1 germline transcription
We draw three conclusions from this study. The first is that mutation of the three proximal NF-{kappa}B binding sites has little effect on {gamma}1 transgene expression. Comparing expression of the wild-type and NF-{kappa}B TM transgenic mice, loss of NF-{kappa}B binding at these three sites had no measurable effect on {gamma}1 germline transcription induced by IL-4 alone (Fig. 4A), by CD40L alone, or by CD40L + IL-4 (Fig. 4B). There was a small effect on induction by LPS + IL-4 (Fig. 4B). The lack of a dramatic effect on transcription is surprising, given the location of the NF-{kappa}B sites and the effects observed in transient transfection/reporter gene assays (911). There are three differences between our study of transgenes and those of reporter constructs that may contribute to these discrepant results. (i) The transgenes described in this study are expressed in normal B cells that may express a qualitatively or quantitatively different array of transcription factors compared to specific B cell lines used for reporter gene assays. (ii) The transgenes are integrated into chromatin, as opposed to extrachromosomal reporter constructs. This is likely to change the requirements for factor binding and activation. (iii) The mutations of the NF-{kappa}B sites we test here, in the context of the entire {gamma}1 gene may be compensated by the binding of NF-{kappa}B, or other transcription factors, to sites elsewhere in the gene. Previously described reporter constructs (9,10) which extend from –954 (or –150) to +202 may lack some of these compensating sites.

Snapper and colleagues have shown that activation of the {gamma}1 gene by LPS + IL-4 requires binding of specific NF-{kappa}B members to NF-{kappa}B sites (17,18). The data in Figs 2–5GoGoGo would suggest that some of those NF-{kappa}B binding sites are outside the proximal promoter, since mutation of those in the proximal promoter resulted in only a partial reduction in germline transcripts induced by LPS + IL-4.

A second conclusion is that sequences distal to –150 are indispensable for expression of {gamma}1 germline transcripts. Some of the compensating NF-{kappa}B binding sites might lie distal to –150. The compensating sites may also bind other transcription factors, for example ATF2, which is induced in B cells by CD40 ligation (11).

An upper limit for {gamma}1 germline transcription
Thirdly, with this study, we also extended the conclusion that the amount of total {gamma}1 germline transcripts has an upper limit, at least in cells treated with LPS + IL-4 or CD40L + IL-4 (21). We studied two new wild-type transgenes (589 and 601) and four NF-{kappa}B TM transgenes with high copy numbers and good expression. Despite the increase in total {gamma}1 genes per cell to 7–12 (line 589) or 30–50 (line 592), the amount of total {gamma}1 germline transcripts did not increase by 5-fold or 20-fold. In fact, the amount of {gamma}1 germline transcripts did not increase at all, or at most by ~4-fold. Thus, it appears that the {gamma}1 genes in a B cell compete for limiting amounts of one or more transcription factors. In B cells treated with IL-4 alone, the transgenes compete reasonably well for the limiting factors, probably because the amount of transcription from the endogenous genes is small. For copy numbers in excess of 15, the majority of the germline transcripts come from the transgene, and can reach as high as 90% of the total transcripts in IL-4-induced B cells (Fig. 4A). On the other hand, in B cells treated with LPS + IL-4 or with CD40L + IL-4, transcription of the endogenous genes is increased several-fold, and the transgenes do not compete as well. The percent transgene expression drops below 40% for lower copy number mice, and to ~50% for the high copy number mice. The single exception is line 45, which may be in a particularly favorable insertion site. It is expressed at levels consistent with its copy number, and competes favorably with the endogenous {gamma}1 genes in B cells treated with LPS ± IL-4 or CD40L ± IL-4 (Figs 3 and 4). Thus, sequences that lie outside the 17 kb {gamma}1 transgene but that are found in the endogenous locus apparently help the endogenous gene to compete for limiting transcription factors during high level transcription. Consistent with this interpretation, we have found that the {gamma}1 gene, within a 230 kb transgene of the entire constant region locus, expresses germline transcripts almost equal to that of the endogenous locus on a per gene basis (Dunnick et al., submitted).


    Acknowledgements
 
We thank Jian Shi, David Tyson and Tracy Sturm for technical assistance, and Maggie Van Keuren, Wanda Filipiak and Mark Berard for assistance in preparing transgenic mice. This work was supported by National Institutes of Health grants AI36310 and A150850 to M.T.B. and by grants from the National Cancer Institute, CA39068 to W.A.D. and CA46952 in support of the University of Michigan Transgenic Animal Model Core.


    Notes
 
Transmitting editor: P. Kincade

Received 5 May 2004, accepted 22 September 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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