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
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Abstract |
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Keywords: Ig genes, non-consensus RNA splicing, RNA polymerase II, RNA polymerase III, somatic hypermutation
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Introduction |
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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 We produced transgenic mice that harbor a transgene whose polymerase (pol) II promoter has been replaced by a pol III promoter.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.
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Methods |
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The VC167va transgene is identical to the VC167tr transgene except for the replacement of the V promoter and additional upstream sequence with the RNA pol III promoter of the adenovirus VA1 gene (Fig. 1
). 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
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 JC 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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Hybridomas
Hybridoma preparation was according to the protocol in Current Protocols in Immunology (2.5.12.5.17). 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/thymidinehypoxanthine/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 V2083 (5'-ACA AGT TGT TGA CAG TAA TA-3') which anneals to the 3' end of the V region (Fig. 2A
). 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. 2A
for primers).
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Reverse transcriptions were carried out to determine promoters near the 3' enhancer using anti-sense primers V
2141 (5'-TGT ACT TAC GTT TCA GCT) and V
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
1996 (see above; Figs 2, 4A and B
).
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Results |
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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 9x104 and 1.7x104 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 (V2083, Fig. 2A
) was used to generate the first-strand cDNA. The cDNAs were amplified using the primers EPSAS-1 and VA1-1 (Fig. 2
). 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. 3A
). 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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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' enhancer. Using a reverse transcription primer from the 5' end of the JC intron (V
2141) followed by a 3' PCR primer in the V region (V
1996) (Fig. 4B
) and various 5' primers in the 3'
enhancer, both spliced and unspliced transcripts were found in the RNA of hybridoma 10F (Fig. 4B
). 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. 4B
). The predominant promoter is defined by the primers 5' En and 3' En526 (Fig. 4A and B
) 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 PyPyA + 1NT/APyPy (10). The initiator sequence in the 3'
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. 4B) 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'
enhancer (Fig. 4A
). The three spliced products are schematically shown in Fig. 4
(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 B). 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. 4A
at the bent arrow at 1231).
Transcripts originating near the 3' enhancer in the endogenous
locus
It was important to determine whether the promoters found near the 3' 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'
enhancer (Fig. 5B
, 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. 5B
, lanes 2 and 3). This is likely due to another weaker promoter 3' of C
. The transcripts are unlikely due to read-through from VJC
gene transcription, because they are polyadenylated. Any RNA polymerase continuing past the cleavage/polyA addition site just 3' of C
would have lost the RNA processing components (11,12). Thymus also showed some RNA originating from the major initiator-type promoter (Fig. 5B
, lane 7). This is presumably due to contamination with B cells, since C
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. 5A
). The transcripts were confirmed as derived from this region by sequencing several DNA clones derived from a spleen cDNA PCR band (Fig. 5B
, 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. 6). 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 B
). It is clearly a pol II-transcribed RNA, since the oligo-dT primed cDNA contains the unusual splice product (Fig. 3B
). 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 B
). 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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Discussion |
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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- 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' enhancer sequences are derived from read-through from a more 5' promoter, since no transcripts were detected with primers located 5' of the 3'
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' enhancer as a pol II promoter
In the absence of the normal Ig promoter, the two 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-
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 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. 4A
) 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'
transcripts may be to open the chromatin around the RS element which is located 3' of C
and is implicated in eliminating C
in
-producing B cells (27). However, the promoters around the 3'
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
. There are two AUGs downstream of the two initiator promoters within the 3'
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. 4A
). Finally, the 3'
promoters may be able to squelch enhancer activity, thus competing with the V
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'
promoters in endogenous gene expression is currently not known.
On the other hand, these promoters also function in 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'
enhancer to the V region in the next transgene copy (see Fig. 4C
), the leader sequence would be eliminated and, unless a leader is provided by a 3'
region exon, the resulting mRNA would encode a
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
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. 4A
). It would then depend on the sequences at the 5' end of the transgene whether an in-frame
protein can be produced from the adjacent transgene copy.
Translatability of the transgene is of course irrelevant if the 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'
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 transgene that contains the same 3' end as our transgenes and only ~100 nucleotides upstream of the known V
transcription start site, a 2- to 3-fold increase of somatic hypermutation over an endogenous
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
promoter was eliminated with the 3 kb upstream deletion. However, they found no `unusual sequence common to other V
genes'. It appears quite possible, that a promoter around the 3'
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.
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Acknowledgments |
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Abbreviations |
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pol polymerase |
RACE rapid amplification of cDNA ends |
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Notes |
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Received 10 October 2000, accepted 8 February 2001.
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References |
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