The 3' Ig{kappa} enhancer contains RNA polymerase II promoters: implications for endogenous and transgenic {kappa} gene expression

Hong Ming Shen, Andrew Peters,, Daniel Kao and Ursula Storb3

1 Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA
Departments of Molecular Genetics and Cell Biology, and

Correspondence to: U. Storb


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have created a {kappa} transgene in which a polymerase (pol) III promoter replaces the pol II promoter. Two independent transgenic lines show somatic hypermutation of the transgene in B cells from hyperimmunized mice. Both lines transcribe transgenes from the pol III promoter in the liver. However, in spleen and spleen B cell-derived hybridomas, they also transcribe mRNA from pol II promoters located within the 3' {kappa} enhancer of the preceding transgene copy in a tandem transgene array. The findings demonstrate that in an array of multiple transgenes the expression (and somatic hypermutation) of an individual transgene copy must be considered in the context of the other copies. We also show that sequences around the 3' {kappa} enhancer in endogenous genes are transcribed. The possible role of these promoters in endogenous {kappa} gene expression is discussed. An unrelated finding in this study was a novel RNA splice in one hybridoma.

Keywords: Ig genes, non-consensus RNA splicing, RNA polymerase II, RNA polymerase III, somatic hypermutation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ig transgenes have been extensively used for the study of the control of V(D)J recombination, B cell development, class switching and somatic hypermutation (1). The findings with such mice are generally considered as representing the effects from a single transgene copy. However, most transgenic mice harbor more than one and often a large number of copies. Thus, there may be effects that must be attributed to the multicopy status of the transgenes. Some of these effects may be epigenetic, i.e. chromosome changes arising from the interplay of the integration site and the transgenes, with different strengths of the effects by the insertion site for proximate or more distant copies.

The study reported here shows that there is a direct effect of regulatory regions in one transgene on the expression of the adjoining transgene. We produced transgenic mice that harbor a {kappa} transgene whose polymerase (pol) II promoter has been replaced by a pol III promoter. The aim was to determine whether transcription from a pol III promoter is permissible for somatic hypermutation. Surprisingly, the transgenes were mainly expressed from previously unknown pol II promoters.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transgenic mice
The transgenes were derived from the V{kappa}167 light chain gene (2). The VC167tr construct differs from the V{kappa}167 sequence by having a stop codon introduced into the leader region and several engineered deletions. A 3.0 kb region upstream of the promoter, a 5 kb region between the C region and the 3' {kappa} enhancer, and a 200 bp region in the leader-variable region intron have all been removed from the original construct.

The VC167va transgene is identical to the VC167tr transgene except for the replacement of the V{kappa} promoter and additional upstream sequence with the RNA pol III promoter of the adenovirus VA1 gene (Fig. 1Go). The VC167va transgene was created by ligating a 170 bp Klenow blunted BamHI fragment containing 96 bp upstream of the RNA start and 74 bp corresponding to the 5' 74 nucleotides of the adenovirus 2 VA1 RNA (including the internal promoter) (3) (kindly provided by R. Roeder, Rockefeller University) into the VC167tr plasmid from which the entire upstream region, including the pol II promoter and the first 69 nucleotides of the transcribed sequences, including the 5' untranslated region and a portion of the leader, had been removed. The VA1 gene itself was shortened to 74 bases, thus excluding the transcription termination signal. The promoter of VA1 (as in other pol III genes) lies inside the transcribed portion of the gene between positions +9 and +72 (3), and is included in VC167va. There are two tracts of thymidine (T) residues within the leader-variable region intron of the original V{kappa}167 gene (2), which were removed with the 200-bp deletion in both the pol II (VC167tr) and pol III (VC167va) constructs. Clusters of four or more T residues have been shown to terminate transcription by RNA pol III (46). The next T tracts (four Ts, seven Ts and four Ts, all three within a total of 37 nucleotides) are located in the 5' end of the J–C region intron and were left intact in order to terminate transcription after the J region in pol III transcripts of the VC167va transgene. The VA1 promoter is a strong pol III promoter used quite extensively to study pol III transcription and should function normally in the context of a transgene.



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Fig. 1. Structure of the VC167tr (A) and the VC167va (B) transgenes. The transgenes were created from the MOPC167 {kappa} gene (2). The features that are different from the original MOPC167 {kappa} gene, notably the deletions and the insertion of the pol III promoter (VA1), are indicated. The bent arrow (at +1) indicates the transcription start. The relevant elements of the genes are labeled below the map. The map is not to scale.

 
Both constructs, VC167tr and VC167va, were prepared for microinjection by isolating the KpnI and EcoRI fragment to remove the plasmid vector. One line of mice carrying the VC167tr construct and six lines carrying the VC167va construct were generated. Transgene copy number was determined by Southern blot of mouse tail DNA (not shown).

Hybridomas
Hybridoma preparation was according to the protocol in Current Protocols in Immunology (2.5.1–2.5.17). Briefly, transgenic mice were injected with 2x108 sheep red blood cells on day 1, given a second injection on day 20, boosted on day 23 and sacrificed on day 26. Mouse spleen cells were harvested and fused with a fusion partner (SP2/0) (from the Frank Fitch Monoclonal Antibody Facility, University of Chicago). Hybridomas were selected with HAT-HT (hypoxanthine/aminopterin/thymidine–hypoxanthine/thymidine; Sigma, St Louis, MO). Hybridoma secretions were screened by ELISA. Limiting dilution was performed twice to purify the IgG-secreting hybridomas. Four IgG-secreting hybridomas with intact transgenes were selected for further study.

RT-PCR
RNA was prepared using RNA STAT-60 (Tel-Test, Friendswood. TX). RT-PCR was done to test splicing and polyadenylation. The first-strand cDNA was generated by using an oligo-dT primer or antisense primer V{kappa}2083 (5'-ACA AGT TGT TGA CAG TAA TA-3') which anneals to the 3' end of the V region (Fig. 2AGo). The pair of primers used for PCR were sense primer VA1-1 (5'-aga att cTT CGC AAG GGT ATC ATG G-3') and antisense primer EPSAS1 (5'-ATA CAC ACC CAC ATC CTC AGC C-3'), which were designed for detecting both spliced and unspliced message (see Fig. 2AGo for primers).



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Fig. 2. Primers used in the analysis of the VA1 transgenes. The striped rectangle is the VA1 sequence, the black rectangles are the leader and VJ sequences. IVS, intron. T tract, multiple T tracts in the 5' end of the JC intron which terminate pol III transcripts. The primer sequences are shown in Methods.

 
Rapid amplification of cDNA ends (RACE) and RT-PCR to detect transcription initiation sites and promoters
RACE was carried out according to the manufacturer's instructions (Gibco/BRL, Gaithersburg, MD). In this experiment, V{kappa}2083 (see above) was used for reverse transcription to generate V{kappa}167 specific cDNA and three internal primers were used to run three consecutive PCR reactions (Fig. 2AGo). The primers were V{kappa}2058 (anti-sense, 5'-AC ATC CTC AGC CTT CAC TCG), V{kappa}1996 (anti-sense, 5'-A AAC CGG TCT GAG ACT CCT GAG) and RACEint1 (anti-sense, 5'-CA CAA TAT CCC CAC TGA CTC) (Fig. 2AGo).

Reverse transcriptions were carried out to determine promoters near the 3' {kappa} enhancer using anti-sense primers V{kappa}2141 (5'-TGT ACT TAC GTT TCA GCT) and V{kappa}2083 (see above) and sense primers 5' En (5'-AAA GCC TCA TAC ACC TGC TCC), 3' En526 (5'-CC ACA CCC TTT CAA GTT TCC), 3' En475 (5'-ACA TCT GTT GCT TTC GCT CCC), 3' En454 (5'-ACT GAA AAC AGA ACC TTA GGC) and 3' En432 (5'-GAT CAA GAA GAC CCT TTG AGG), and an anti-sense primer V{kappa}1996 (see above; Figs 2, 4A and BGoGo).





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Fig. 4. Analysis of the pol II promoters within and 3' of the 3' {kappa} enhancer. (A) DNA sequences surrounding the 3' {kappa} enhancer. The published (33) 3' {kappa} enhancer is in [ ] and the VA1 sequence is in { }. Bent arrows are transcript initiation sites and arrows below the sequence are primers used to detect the promoters. The primers are numbered according to the position of the 5' nucleotide in the sequence (the tip of the arrow is the 3' nucleotide of the primer). Capital letters are sequences around the predicted initiator promoter (near the 3' enhancer) and TATA promoter. Italics indicate the 3' {kappa} enhancer motifs (3436). SD, splice donor; SA, splice acceptor. (B) Identification of a hidden promoter in the 3' {kappa} enhancer by RT-PCR of RNA of hybridoma 10F. A pol II promoter is predicted in the 3' {kappa} enhancer. The positions of a perfect initiator-type promoter in the 3' {kappa} enhancer and the pol III promoter at the start of the transgene are shown in the map (bent arrows, left and right). The 5' PCR primers designed to test for transcripts surrounding the initiator and the 3' RT-PCR primers are shown at the bottom. Va21 mouse tail DNA was used as a control to confirm reliability of the primers and the DNA PCR products were run on an agarose gel next to the cDNAs derived from hybridoma 10 F. (C) Schematic diagram of the spliced transcripts originating at the promoter inside the 3' {kappa} enhancer. Three different transcripts were found. The two spliced forms shown have alternative splice acceptors in exon 3' {kappa}EII. The first intron is located between the 3' {kappa} enhancer exon 3' {kappa}EI and the exon 3' {kappa}EII (a or b); the second intron extends from 3' {kappa} EII to the V region of the next transgene. The three RNA bands shown in (B) are from the top: unspliced, the first intron spliced out and both introns spliced out.

 
Additional primers used for Fig. 5Go were: 3' En1035 (5'- GCC TTG ACT TGA GTA GAA CC), 3' En367 (5'-CTA CCT TAT TGG GAG TGT CCC), 5' ß-actin (5'-GTGGGCCGCTCTAGGCACCAA), 3' ß-actin (5'-CTCTTTGATGTCACGCACGATTTC).



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Fig. 5. RT-PCR analysis showing transcripts originating near the 3' {kappa} enhancer in the endogenous {kappa} locus. RNAs from bone marrow (B), spleen (S) and thymus (T) of C57BL/6 mice. (A) Total RNA was used to directly run PCR to detect possible DNA contamination. Primers were 3' En367/3' En1035 for lanes 2–4 and 5' En/3' En1035 for lanes 5–7. (B and C) Oligo-dT primed cDNAs were used to run the PCRs. Primers in (B) as in (A); in (C) 5' ß-actin/3' ß-actin. (D) PCR with mouse genomic DNA as a template to test primer efficiency. Primers in lane 2 are 3' En367/3' En1035 and in lane 3 are 5' En/3' En1035.

 
Sequence analysis
Automated sequencing was carried out for all amplified gene fragments at the University of Chicago Cancer Research Center Sequencing Facility. Both strands of each clone were sequenced and analyzed with the Macintosh version 3.0 of Sequencher (GeneCodes, Ann Arbor, MI).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of the VC167tr (pol II) and VC167va (pol III) transgenes
Transgenic mice were generated containing either the VC167tr (pol II) construct or the VC167va (pol III) construct (Fig. 1Go). Both types of transgenes were mutated in PNAhi B cells of hyperimmunized mice (not shown). The VC167tr (pol II) transgene undergoes somatic hypermutation with a mutation frequency of 4x10–4 mutations/bp, >10 times higher than the Pfu polymerase background mutation frequency (2.8x10–5 mutations/bp for 30 cycles of PCR) and similar to the mutation frequency of the same region in another V{kappa}167 transgene (P5'C) (4.98x10–4 mutations/bp) (7). Thus, the alterations introduced into this transgene (see Methods) do not detectably affect somatic mutation.

The VC167va (pol III) transgene in two lines of transgenic mice, line va62 containing two and line va21 containing five copies of the transgene, also undergoes somatic hypermutation at frequencies of 9x10–4 and 1.7x10–4 mutations/bp respectively (not shown). Further analyses described below show that one must be critical in evaluating the role of promoters in somatic hypermutation of transgenes because of potential pol II promoters in many genomic regions.

Transcripts from a pol III promoter should be unspliced, but transcripts from a pol II promoter should be both unspliced and spliced. To detect spliced and unspliced RNAs PCR assays were done on total spleen RNA (not shown). With the pol II promoter transgene (tr 20) mostly spliced RNA was seen. With the pol III promoter transgenes, unexpectedly, spliced RNAs were also prominent in spleen, suggesting pol II products. This is further investigated below. Only unspliced, and therefore pol III-only, transcripts were seen in liver RNA from both va62 and va21 transgenic mice (not shown). Since in liver cells, in the absence of B cell transcription factors, pol II mRNA from the Ig transgene would not be produced, the liver transcripts confirm that the VA1 promoter was functioning as a pol III promoter and producing unspliced transcripts.

Transcripts of the va21 transgene in B cell hybridomas
To determine whether indeed pol II transcripts are produced from the pol III promoter transgene, hybridomas were made from spleen of the five copy va21 mouse line. Four IgG producing hybridomas were analyzed in detail. Full-length transcripts from a pol II promoter should be partially spliced, fully polyadenylated and capped.

RT-PCR was used to analyze splicing of hybridoma transcripts. A transgene-specific primer in the VJ region (V{kappa}2083, Fig. 2AGo) was used to generate the first-strand cDNA. The cDNAs were amplified using the primers EPSAS-1 and VA1-1 (Fig. 2Go). As expected, PCR generated two bands. The lower band represents the transcripts from which the Leader-V region intron has been spliced out and the higher band represents the unspliced transcripts (Fig. 3AGo). There is always less spliced than unspliced RNA, but the ratio of spliced transcripts to unspliced transcripts is variable among these hybridomas, perhaps reflecting different epigenetic effects in different cells.




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Fig. 3. Transcripts in the va21 hybridomas. (A) Spliced and unspliced transcripts in hybridoma total RNAs. cDNAs were produced using the primer V{kappa}2083 for the reverse transcription reaction; primers VA-1 and EPSAS1 were used for the PCR reactions (see Fig. 2Go). Lane 1, marker DNAs (Hi-Lo DNA marker; Minnesota Molecular, Minneapolis, MN); lanes 2–5, PCR from total RNA as a control for DNA contamination; lanes 6–9, PCR from cDNAs showing unspliced and spliced products. The different hybridoma lines are listed at the top. (B) Spliced and unspliced transcripts in polyadenylated RNAs. cDNAs were produced using oligo-dT as primer. VA-1 and EPSAS-1 primers (see Fig. 2Go) were used for the PCR reactions. cDNA bands from unspliced and spliced RNAs are indicated. The first lane is the size marker (see legend to A).

 
Polyadenylation is expected to occur only in pol II-transcribed mRNAs (8). When polyadenylated RNA was analyzed we found that both spliced and unspliced transcripts were present (Fig. 3BGo). Compared with the analysis with VJ region-primed cDNA (Fig. 3AGo), oligo-dT-generated cDNA shows higher ratios of spliced transcripts over unspliced transcripts. Since pol III transcripts are not spliced (9), the relative ratios of spliced to unspliced RNAs in oligo-dT-primed cDNA (only pol II promoter derived RNAs) (Fig. 3BGo) versus VJ region-primed cDNA (both pol II and pol III promoter derived RNAs) (Fig. 3AGo) suggests that there are unspliced transcripts without poly A which could be transcribed from a pol III promoter.

Most of the stable polyadenylated RNAs in these hybridomas were capped (not shown), further supporting that they were transcribed by pol II.

Where do the pol II transcripts originate?
By 5' RACE we found a number of cDNA products (not shown). The 5' end of the largest one was in the 3' {kappa} enhancer. Using a reverse transcription primer from the 5' end of the JC intron (V{kappa}2141) followed by a 3' PCR primer in the V region (V{kappa}1996) (Fig. 4BGo) and various 5' primers in the 3' {kappa} enhancer, both spliced and unspliced transcripts were found in the RNA of hybridoma 10F (Fig. 4BGo). The transcripts depended on the reverse transcription reaction; no product was found when the RNA was directly amplified, indicating that there was no detectable DNA contamination (not shown). A positive control with mouse tail DNA always showed the expected full-length product that increased in length with increasing distance of the 5' PCR primer from the 3' primer (Fig. 4BGo). The predominant promoter is defined by the primers 5' En and 3' En526 (Fig. 4A and BGo) which amplify from cDNA a fragment of the predicted size, indicating that an RNA is made from this region. That it initiates here is shown by the absence of bands with PCR primers 432 and 454 that anneal 5' of this region. There is a perfect initiator-type promoter starting at the 5' end of 3' En526. The initiator consensus is Py–Py–A + 1–N–T/A–Py–Py (10). The initiator sequence in the 3' {kappa} enhancer is CCACACC. There must be another weak promoter between position 475 and 454, since the former primer, but not the latter, gives some cDNA products. There is a potential initiator promoter in this region with five of seven nucleotides agreeing with the initiator consensus sequence (469: TTAGGCA; consensus underlined).

Both unspliced and spliced transcripts are seen in RNA of hybridoma 10F (Fig. 4BGo) and hybridoma 12C (not shown). In the other two hybridomas, 5C and 11G, mainly unspliced RNAs were seen (not shown). The PCR products amplified from the unspliced or spliced forms were gel purified, sequenced and splice sites determined. There are splice donor and acceptor sites in the region 3' of the 3' {kappa} enhancer (Fig. 4AGo). The three spliced products are schematically shown in Fig. 4Go(C). The largest one is the unspliced transcript. The middle one has lost the small intron 1a or 1b, and the smallest one has lost both introns 1a and 2 or 1b and 2.

There is no evidence among these RNA species for an additional splice acceptor in the VA1 promoter in the 5' end of the transgene. This 5' transgene region is, however, clearly a part of an exon that can be spliced to the V region (Figs 2 and 3A and BGoGo). We searched therefore for the presence of another pol II promoter in this region and found a high consensus TATA promoter using the Promoter Prediction program (http://www.fruitfly.org), suggesting that there may be another promoter in the very 3' end of the transgene (Fig. 4AGo at the bent arrow at 1231).

Transcripts originating near the 3' {kappa} enhancer in the endogenous {kappa} locus
It was important to determine whether the promoters found near the 3' {kappa} enhancer in the transgenes are active in the endogenous locus. RT-PCR reactions with RNA from bone marrow and spleen showed polyadenylated transcripts (reverse transcription priming was with oligo-dT) using a 5' PCR primer just downstream of the major initiator-type promoter in the 3' {kappa} enhancer (Fig. 5BGo, lanes 5 and 6). There was a small amount of RNA produced in the spleen, but not the bone marrow, with a more upstream primer (Fig. 5BGo, lanes 2 and 3). This is likely due to another weaker promoter 3' of C{kappa}. The transcripts are unlikely due to read-through from VJC{kappa} gene transcription, because they are polyadenylated. Any RNA polymerase continuing past the cleavage/polyA addition site just 3' of C{kappa} would have lost the RNA processing components (11,12). Thymus also showed some RNA originating from the major initiator-type promoter (Fig. 5BGo, lane 7). This is presumably due to contamination with B cells, since C{kappa} transcripts were seen in the thymus sample (not shown). The RT-PCR products were indeed from RNA, since no DNA contamination was observed by directly amplifying the RNA samples without a reverse transcriptase reaction (Fig. 5AGo). The transcripts were confirmed as derived from this region by sequencing several DNA clones derived from a spleen cDNA PCR band (Fig. 5BGo, lane 6) (not shown).

An unusual RNA splice product in one hybridoma
Curiously, hybridoma 5C shows an unusual splice in the spliced cDNAs sequenced. The splice results in an insertion of 20 nucleotides compared with normally spliced RNA (Fig. 6Go). This does not seem to be due to the alteration of the classical leader-V splice donor and acceptor, as these sequences are normal in 14 sequences of unspliced RNAs from this hybridoma (not shown). The larger spliced fragment is also obvious when the cDNA is displayed by electrophoresis (Fig. 3A and BGo). It is clearly a pol II-transcribed RNA, since the oligo-dT primed cDNA contains the unusual splice product (Fig. 3BGo). It appears to be an RNA derived by RNA splicing since a Southern blot of hybridoma DNA does not show a transgene copy with a deletion of 184 nucleotides (not shown). Amplification of the hybridoma DNA across the splice sites indicates further that there is no deletion within a transgene copy which would give rise to this aberrant transcript without splicing (not shown). A number of non-consensus splice donor and acceptor dinucleotides have been found with a variety of genes (1317), but the combination of CU and AC as in the hybridoma 5C has not been reported. It is unlikely to be a splice due to U12 snRNA-promoted splicing, since the –3 and +12 nucleotides surrounding the donor splice site and the 13 nucleotides surrounding the possible branch point do not match the sequences in U12-type introns (18). These nucleotides do match the splice donor and branch sites of less frequently used U2 splice sites, except for the C at position +1 of the donor splice site. It is possible that in the hybridoma 5C an unusual splicing complex can form which recognizes the CU splice donor and AC splice acceptor. It has also been found that during a PCR reaction non-homologous recombination products between different exons or within an exon can be formed (14). However, in those reactions the majority of the products were canonical splice products, whereas in the hybridoma 5C all spliced molecules appear to have the same aberrant splice (Fig. 3A and BGo). This suggests that the spliced molecules were formed in the cell. Unless due to an unsuspected artifact, this may indicate an interesting splicing mode in this hybridoma.



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Fig. 6. Unusual RNA splice in hybridoma 5C. The arrows on top and bottom of the sequence show the conventional and hybridoma 5C splice sites respectively. The over- (under-) lined sequences are the conventional and hybridoma 5' and 3' intron end dinucleotides respectively. The sequence between the top arrows (including 173 nucleotides that are not shown) is absent from the normal spliced RNA; the sequence between the bottom arrows is absent from the hybridoma 5C spliced RNA. There are some repeated sequences 5' of the 5' and 3' splice sites. The splice sites shown were chosen because they and their flanking sequences are the best match for known splice sites.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The VC167va transgene arrays are transcribed by RNA pol III and II
Analysis of B cells from the va62 and va21 pol III transgenic mice shows that the pol III transgenes are expressed as pol III-, as well as pol II-transcribed RNAs. The adenovirus pol III promoter is functional in these transgenes as shown by the expression of the transgenes as unspliced RNAs in liver, which does not express pol II-driven Ig genes. However, since in splenic B cells and B cell hybridomas capped, spliced and polyadenylated RNAs were prominent, pol II transcripts are also produced.

Transcripts from RNA pol III-driven promoters have several characteristics distinguishing them from pol II transcripts. The major difference is the lack of processing of pol III RNA since processing appears directly linked to pol II activity (8). pol III RNA lacks the 5' mRNA cap, the splicing out of introns and the polyadenylated tail (8,19). Even when typical pol II-transcribed genes were placed under the control of a pol III promoter, the introns were not spliced out (9,20). Apparently, RNA pol II itself is required for efficient pre-mRNA splicing, polyadenylation and capping. In the absence of the C-terminal domain of the largest subunit of pol II these events cannot be detected (8,11,12,21). Thus, the presence of capped, spliced and polyadenylated transcripts suggests that there are pol II promoters that are active in the VA1-{kappa} transgenes.

Putative pol II promoters were confirmed to give rise to transcripts originating in the preceding transgene copy. It is unlikely that the transcripts which contain 3' {kappa} enhancer sequences are derived from read-through from a more 5' promoter, since no transcripts were detected with primers located 5' of the 3' {kappa} enhancer.

Concerning the need for pol II in somatic mutation, the study is suggestive. In a similar study in which a pol II promoter was replaced by a pol I promoter by homologous recombination in the heavy chain locus, somatic mutation was also found (22). Spliced and polyadenylated transcripts were also seen with the pol I promoter, but capping was not investigated. It was not shown that pol I transcripts were actually produced. If they were, they would not be expected to be polyadenylated (23). It is likely that germline D region pol II promoters (24) were responsible for the transcripts. Thus, it is not clear which promoter drove the somatic hypermutation seen in these experiments. It is possible that the presence of the pol I promoter helped to concentrate TBP [this protein is shared for pol I, II and III promoter activation (25)] near the adjacent pol II promoters. Taken together with our findings these considerations suggest, but do not prove, that only pol II can deliver the postulated mutator factor for somatic hypermutation (26). In any case, the test of a requirement for a pol II promoter can apparently not be done in vivo and will have to await a cell-free somatic hypermutation system.

Significance of the 3' {kappa} enhancer as a pol II promoter
In the absence of the normal Ig promoter, the two {kappa} enhancers present in the transgene appear to mediate transcription through the non-V gene pol II promoters located at the 3' end of the VA1-{kappa} transgenes. The enhancers clearly play a role in the production of pol II transcripts from these transgenes and therefore of spliced mRNAs, since spliced transcripts are only detected in the spleen where the enhancers are active, but not in liver where they are inactive.

What is the significance of the previously undiscovered promoters in the 3' region of the {kappa} gene? Since transcripts from this region were also found in non-transgenic mice they may play a role in endogenous gene expression. Mouse sequences 3' of our data (Fig. 4AGo) have not been published. There are no known human ESTs published from this region, but it is not known whether B cell expression was tested. We considered that the function of the 3' {kappa} transcripts may be to open the chromatin around the RS element which is located 3' of C{kappa} and is implicated in eliminating C{kappa} in {lambda}-producing B cells (27). However, the promoters around the 3' {kappa} enhancer are ~16 kb away from the RS element (28). There is the interesting possibility that there may be an unknown gene downstream of C{kappa}. There are two AUGs downstream of the two initiator promoters within the 3' {kappa} enhancer. They are both in a non-consensus Kozak environment and both are in-frame with a stop codon. However, the stop could be eliminated by an RNA splice just 5' of it (Fig. 4AGo). Finally, the 3' {kappa} promoters may be able to squelch enhancer activity, thus competing with the V{kappa} promoter. This may play a role in some stages of B cell development, depending on the types of trans-activating factors present. In summary, the role of these 3' {kappa} promoters in endogenous gene expression is currently not known.

On the other hand, these promoters also function in {kappa} transgenes and this has consequences in cases where multiple transgene copies are arranged in tandem. First, if an RNA splice occurs from an exon in the 3' {kappa} enhancer to the V region in the next transgene copy (see Fig. 4CGo), the leader sequence would be eliminated and, unless a leader is provided by a 3' {kappa} region exon, the resulting mRNA would encode a {kappa} protein that cannot be translated on membrane-bound ribosomes for assembly with a heavy chain. Furthermore, if transcription started from the TATA promoter at the very 3' end of most {kappa} transgenes (just 5' of an EcoRI site that is often used to linearize the transgenic DNA) there is a first AUG very near the start site (Fig. 4AGo). It would then depend on the sequences at the 5' end of the transgene whether an in-frame {kappa} protein can be produced from the adjacent transgene copy.

Translatability of the transgene is of course irrelevant if the {kappa} transgene is used as a passenger transgene for somatic mutation studies. In that case, protein products of the transgene are not essential. It is possible that the additional promoters just upstream of the mutable VJ region provide additional entry sites for a postulated mutator factor that becomes associated with the transcription complex (29). This raises the question whether deletion of the 3' {kappa} enhancer (and its 3' flank) which results in a significant decrease in somatic hypermutation of a multicopy transgene does so only because of the overall decrease of enhancer activity (30) or perhaps also because of the elimination of the additional promoters.

It has not been tested in a systematic way if multiple promoters in tandem increase somatic hypermutation levels, but, if potential loading sites of a mutator factor are limiting, one may expect such an outcome. In fact, there is quite suggestive indirect evidence that this may occur. Comparing a multicopy {kappa} transgene that contains the same 3' end as our transgenes and only ~100 nucleotides upstream of the known V{kappa} transcription start site, a 2- to 3-fold increase of somatic hypermutation over an endogenous {kappa} gene or a transgene with additional 3 kb at the 5' end was seen (31). The authors suggested that perhaps a negative element upstream of the V{kappa} promoter was eliminated with the 3 kb upstream deletion. However, they found no `unusual sequence common to other V{kappa} genes'. It appears quite possible, that a promoter around the 3' {kappa} enhancer in a preceding transgene copy in these multicopy transgenic mice enhanced the chance for initiating a mutagenic transcription complex. This would only work if there is no occlusion of the downstream promoter by a transcription complex coming from an upstream promoter. This is likely not to be a problem, as promoter occlusion has not been observed in Ig genes (7,32).

In conclusion, effects from flanking transgene copies have to be taken into account when the expression and somatic mutation of tandem Ig transgene arrays are investigated.


    Acknowledgments
 
We are grateful to G. Bozek for breeding the mice, N. Michael for Southern blot analysis, L. Degenstein for production of the transgenic mice, J. Auger for help with fluorescent cell sorting, R. Roeder for the gift of the VA1 DNA clone, J. Pelletier for the gift of a GST-eIF4E plasmid and C. McShan for the gift of the fusing cell line SP2/0. We thank J. Staley for suggestions concerning the unusual RNA splice, and P. Engler, N. Michael and N. Kim for critical reading of the manuscript. This work was supported by NIH grants GM38649 and AI47380. The DNA sequencing, cell sorting and transgenic facilities are partly supported by the University of Chicago Cancer Research Center NIH Cancer Center support grant, 3P30-CA14599-26S1.


    Abbreviations
 
pol polymerase
RACE rapid amplification of cDNA ends

    Notes
 
Transmitting editor: K. Knight

Received 10 October 2000, accepted 8 February 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Storb, U. 1995. Ig gene expression and regulation in Ig transgenic mice. In Honjo, T. and Alt, F., eds, Immunoglobulin Genes, p. 345–363. Academic Press, New York.
  2. Selsing, E. and Storb, U. 1981. Somatic mutation of immunoglobulin light-chain variable-region genes. Cell 25:47.[ISI][Medline]
  3. Fowlkes, D. M. and Shenk, T. 1980. Transcriptional control regions of the adenovirus VAI RNA gene. Cell 22:405.[ISI][Medline]
  4. Bogenhagen, D. and Brown, D. D. 1981. Nucleotide sequences in Xenopus 5S DNA required for transcription termination. Cell 24:261.[ISI][Medline]
  5. Mazabraud, A., Sherly, D., Mueller, F., Rungger, D. and Clarkson, S. 1987. Structure and transcription termination of a lysine tRNA gene from Xenopus laevis. J. Mol. Biol. 19:835.
  6. Maraia, R., Chang, D., Wolffe, A., Vorce, R. and Hsu, K. 1992. The RNA polymerase III terminator used by a B1-Alu element can modulate 3' processing of the intermediate RNA product. Mol. Cell. Biol. 12:1500.[Abstract]
  7. Peters, A. and Storb, U. 1996. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity 4:57.[ISI][Medline]
  8. Minvielle-Sebastia, L. and Keller, W. 1999. mRNA polyadenylation and its coupling to other RNA procesing reactions and to transcription. Curr. Opin. Cell Biol. 11:352.[ISI][Medline]
  9. Sisodia, S., Sollner-Webb, B. and Cleveland, D. 1987. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol. Cell. Biol. 7:3602.[ISI][Medline]
  10. Kaufman, J. and Smale, S. 1994. Direct recognition of initiator elements by a component of the transcription factor IID complex. Genes Dev. 8:821.[Abstract]
  11. McCracken, S., Fong, N., Yankulov, K., Ballantyne, S., Pan, G., Greenblatt, J., Patterson, S., Wickens, M. and Bentley, D. 1997. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385:357.[ISI][Medline]
  12. McCracken, S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G., Siderovski, D., Hessel, A., Foster, S., Program, A. E., Shuman, S. and Bentley, D. 1997. 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11:3306.[Abstract/Free Full Text]
  13. Li, W. and Shaw, J. E. 1993. A variant Tc4 transposable element in the nematode C. elegans could encode a novel protein. Nucleic Acids Res. 21:59.[Abstract]
  14. Zaphiropoulos, P. G. 1998. Non-homologous recombination mediated by Thermus aquaticus DNA polymerase I. Evidence supporting a copy choice mechanism. Nucleic Acids Res. 26:2843.[Abstract/Free Full Text]
  15. Hall, S. and Padgett, R. 1994. Conserved sequences in a class of rare eukaryotic nuclear instrons with non-consensus splice sites. J. Mol. Biol. 239:357.[ISI][Medline]
  16. Jackson, I. J. 1991. A reappraisal of non-consensus mRNA splice sites. Nucleic Acids Res. 19:3795.[ISI][Medline]
  17. Durkin, M., Gautam, M., Loechel, F., Sanes, J., Merlie, J., Albrechtsen, R. and Wever, U. 1996. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript. J. Biol. Chem. 271:13407.[Abstract/Free Full Text]
  18. Burge, C., Padgett, R. and Sharp, P. 1998. Evolutionary fates and origins of U12-type introns. Mol. Cell 2:773.[ISI][Medline]
  19. Gunnery, S. and Mathews, M. 1995. Functional mRNA can be generated by RNA polymerase III. Mol. Cell. Biol. 15:3597.[Abstract]
  20. White, R. and Kunkel, G. 1993. Pre-messenger RNA splicing of transcripts synthesized from human small nuclear RNA gene promoters. Biochem. Biophys. Res. Commun. 195:1394.[ISI][Medline]
  21. Misteli, T. and Spector, D. 1999. RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. Mol. Cell 3:697.[ISI][Medline]
  22. Fukita, Y., Jacobs, H. and Rajewsky, K. 1998. Somatic hypermutation in the heavy chain locus correlates with transcription. Immunity 9:105.[ISI][Medline]
  23. Smale, S. and Tijan, R. 1985. Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters. Mol. Cell. Biol. 5:352.[ISI][Medline]
  24. Alessandrini, A. and Desiderio, S. V. 1991. Coordination of immunoglobulin DJH transcription and D-to-JH rearrangement by promoter-enhancer approximation. Mol. Cell. Biol. 11:2096.[ISI][Medline]
  25. Trivedi, A., Vilalta, A., Gopalan, S. and Johnson, D. L. 1996. TATA-binding protein is limiting for both TATA-containing and TATA-lacking RNA polymerase III promoters in Drosophila cells. Mol. Cell. Biol. 16:6909.[Abstract]
  26. Storb, U., Peters, A., Klotz, E., Kim, N., Shen, H. M. S., Hackett, J., Rogerson, B. and Martin, T. E. 1998. Cis-acting sequences that affect somatic hypermutation of Ig genes. Immunol. Rev. 162:153.[ISI][Medline]
  27. Durdik, J., Moore, M. W. and Selsing, E. 1884. Novel kappa light-chain gene rearrangements in mouse lambda light chain-producing B lymphocytes. Nature 307:749.
  28. Muller, B., Staooert, H. and Reth, M. 1990. A physical map and analysis of the murine C kappa-RS region show the presence of a conserved element. Eur. J. Immunol. 20:1409.[ISI][Medline]
  29. Storb, U., Klotz, E., Hackett, J., Kage, K., Bozek, G. and Martin, T. E. 1998. A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript. J. Exp. Med. 188:689.[Abstract/Free Full Text]
  30. Betz, A., Milstein, C., Gonzalez-Fernandes, R., Pannell, R., Larson, T. and Neuberger, M. 1994. Elements regulating somatic hypermutation of an immunoglobulin K gene: critical role for the intron enhancer/matrix attachment region. Cell 77:239.[ISI][Medline]
  31. Wu, P. and Claflin, L. 1998. Promoter-associated displacement of hypermutations. Int. Immunol. 10:1131.[Abstract]
  32. Atchison, M. L. and Perry, R. P. 1986. Tandem kappa immunoglobulin promoters are equally active in the presence of the kappa enhancer: implications for models of enhancer function. Cell 18:253.
  33. Meyer, K. and Neuberger, M. 1989. The immunoglobulin kappa locus contains a second, stronger B-cell-specific enhancer which is located downstream of the constant region. EMBO J. 8:1959.[Abstract]
  34. Pongubala, J. and Atchison, M. 1991. Functional characterization of the developmentally controlled immunoglobulin {kappa} 3' enhancer: regulation by Id, a repressor of helix-loop-helix transcription factors. Mol. Cell. Biol. 11:1040.[ISI][Medline]
  35. Pongubala, J., Nagulapalli, M., Klemsz, S., McKercher, S., Maki, R. and Atchison, M. 1992. PU.1 recruits a second nuclear factor to a site important for immunoglobulin {kappa} 3' enhancer activation. Mol. Cell. Biol. 12:368.[Abstract]
  36. Liu, X., Prabhu, A. and Van Ness, B. 1999. Developmental regulation of the kappa locus involves both positive and negative sequence elements in the 3' enhancer that affect synergy with the intron enhancer. J. Biol. Chem. 274:3285.[Abstract/Free Full Text]