Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Correspondence to: A. Martin
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Abstract |
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Keywords: antibody, B lymphocytes, Burkitt's lymphoma, somatic mutation
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Introduction |
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Transgenic and genetically deficient mice have been used to identify some of the trans-acting factors involved in V region mutation as it occurs in vivo and to eliminate many potential mechanisms of V region mutation (3). Genetically defective mice have recently revealed a role in the mutational process for mismatch repair (48) and for the activation-induced cytidine deaminase (AID) (9). Mice with genetically manipulated transgenes have also recently been used to show that double-stranded breaks in DNA occur early in the mutational process (1012). These and other studies have focused attention on error-prone DNA polymerases that might be recruited to the Ig genes during the mutational process (reviewed in 13,14).
The pursuit of the role of these polymerases and other potential trans-acting proteins illustrates the need for cultured antibody-forming cells that are undergoing V region mutation to allow the detailed analysis of the mechanisms involved in regulating and targeting V region mutation (15). Such cell lines have the obvious benefit of providing large relatively homogeneous populations of cells that can be easily transfected with many different engineered Ig genes and subjected to detailed biochemical analysis. Attempts to identify such cell lines have gone on for 30 years (16). Until recently, the few cell lines that mutated their V region mutations did it at rates that were much lower than those that occur in vivo and represented stages of B cell differentiation that do not normally undergo V region mutation in vivo (reviewed in 15,17). With identification of the Ramos (10), BL2 (18) and CL-01 (19) human Burkitt's lymphoma cell lines that have the markers of centroblasts and undergo high rates of mutation in culture, it has become possible to study V region mutation in cells that are more likely to reflect the normal process. As with all cell lines that carry out functions specific to a developmental stage, there are questions about the stability of this phenotype in vitro. Here we examine the clonal variation in rate and characteristics of somatic V region hypermutation in the Ramos cells and correlate this variation with the expression of some genes that could play a role in the mutational process.
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Methods |
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ELISA spot assay and fluctuation analysis
The assay was performed as previously reported (20). Briefly, 50 µl/well of goat anti-human IgM (Southern Biotechnology Associates, Birmingham, AL) diluted 1:500 in PBS was added to coat 96-well ELISA plates, then blocked with 2% BSA in PBS. Plates were washed in TBS with 0.1% Tween 20 (TBS-T), rinsed with IMDM, and 13x106 cells were added to 48 wells in IMDM feeding medium and incubated at 37°C for 1618 h. The plates were washed with TBS-T and 50 µl/well of biotin-conjugated goat anti-human IgM diluted 1:500 in 1% BSA TBS was added for 2 h at 37°C, washed and then treated with the Vectastain avidin phosphatase amplification system as per the manufacturer's specifications (Vector, Burlingame, CA). Spots were developed with 1 mg/ml substrate 5-bromo-4-chloro-3-indolyl phosphate (Amresco, Solon, OH) in 0.2 mg/ml MgCl2, 0.01% Triton X-405, 9.6% 2-amino-2-methyl-1-propanol (Sigma, St Louis, MO), pH 9.8. Reactions were stopped with distilled H2O. Rates were calculated by fluctuation analyses, as previously described (21,22). Statistics using the Wilcoxon signed-ranks test were performed on indicated data using SPSS for Windows (release 9.0).
Isolation of Ramos µ- cells and enrichment for µ+ cells
A 3-month culture of Ramos clone 1 was used to isolate µ- cells. Cells (5x106) were washed and suspended in 1 ml FACS buffer (0.1% NaN3, 1% BSA in HBSS without phenol red). Then 200 µl of goat anti-µFITC (Southern Biotechnology Associates) diluted 1:80 in PBS with 0.5% BSA/5 mM EDTA (MACS buffer) was added for 20 min at 4°C. The cells were washed with FACS buffer and suspended in 160 µl MACS buffer. After adding 40 µl anti-FITC Microbeads (Miltenyi Biotec, Auburn, CA) for 15 min at 4°C, the cells were washed with MACS buffer, resuspended in 1ml MACS buffer and loaded onto the autoMACS column (Miltenyi Biotec). The µ- cells were first collected, then sorted by a FACStar Plus machine (Becton Dickinson, Mountain View, CA) into a 96-well plate with 100 µl IMDM feeding medium in it. The same procedure was used to enrich for µ+ revertant cells from µ- cells, except that the positive portion was collected. The supernatants from each well were assayed for secreted µ heavy chain and light chain by ELISA. The V regions of µ- but
+ clones were sequenced.
Soft agar cloning
Cell subcloning was performed using soft-agar containing 0.30.4% Seaplaque agarose (FMC Bioproduct). First, 4 ml of IMDM cloning medium (IMDM with 20% FCS) containing 0.4% SeaPlaque agarose was introduced into a 60-mm culture plate (Falcon-Becton Dickinson). After solidification at 4°C for 10 min, 1 ml of the same medium containing 1000 cells was laid over the top of the soft agar and put at 4°C for 10 min. Cells were grown at 37°C for 57 days. Clones were randomly picked and placed into a 96-well plate.
PCR Amplification of the Ig gene from genomic DNA
Genomic DNA was prepared using QIAamp DNA blood mini kit according to the manufacture's instruction (Qiagen, La Jolla, CA). Pfu polymerase (Stratagene) was used to amplify the various parts of the Ig gene from genomic DNA using 30 cycles of 95°C/15 s, 58°C/15 s, 72°C/1 min. The primers used were the following: VDJ region, 5' primer: 5'-AAAA-GCTAGCACAAGAACATGAAAC-ACC-3', 3' primer: 5'-CCATCGATGGCGGTACCTGAGGAGACGGTGACC-3'; 5'VDJ region, 5' primer: 5'-GAGTCTAGAGAATAAAACGCAATG-3', 3' primer: 5'-TAAATAGTACCTGAGA-GCTGCCC-3'; Cµ24 region: 5' primer: 5'-CGGACCAGGTGCAGGCTGAGGCC-3', 3' primer: 5'-CTCCCGCAGGTTCAGCTGCT-CCC-3'.
Cloning PCR product and sequencing
PCR products were cloned using the Zero BluntTOPO PCR cloning kit (Invitrogen, Carlsbad, CA). Minipreps were prepared from overnight bacterial cultures using the QIAPREP 8 miniprep kit (Qiagen) and sequenced on an ABI model 3700 using standard T3 and T7 primers. The accession numbers of the mutated sequences listed in Table 1 are AF385858AF385893.
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Results |
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To examine the characteristics of this apparent clonal heterogeneity more carefully, we needed an assay that would allow us to determine the mutation rate in many individual subclones after relatively short periods of growth. We have previously reported such an assay in which cells with a nonsense mutation in their V region were analyzed for reversion of that nonsense codon by using the ELISA spot assay to detect for individual IgM-secreting cells (20). This assay requires only a 20-day culture period (i.e. the time it takes for an individual cell to expand to ~1x106 cells required for the assay). At least 10 subclones are assayed for every clone and the rates of mutation are then calculated using fluctuation analysis (21,22). To obtain cells with a nonsense mutation in the V region of their endogenous heavy chain gene, the same Ramos clone described above was grown for 3 months and sorted for IgM- cells. The V regions from µ- clones were then sequenced. A total of 38 µ- + clones were obtained; 25 of these clones (66%) had nonsense mutations in the single copy of their endogenous V region (Table 2
). The identification of cells with nonsense mutations in their V regions shows that mutation was occurring in clone 1 and confirms the sequencing results (Table 1
, clone 1-2). By Southern blot analysis, four clones (11%) had deletions within the SµCµ region, while one clone (clone 5) had deleted the productive Ig heavy chain gene (data not shown). The remaining clones had no identifiable mutations within the coding region of the Ig gene and the basis for their loss of µ production has yet to be determined.
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To verify that the ELISA spots generated during the fluctuation analysis were due to cells that mutated the nonsense codon, eight individual subclones of clone 6 were cultured for 2 months, enriched for µ+ cells and the V regions sequenced. Of the 17 revertant sequences identified, 11 were independent because they were obtained from different subclones of clone 6. Despite the fact that the first two bases of the TAG stop codon in clone 6 are part of an AG TAG hotspot motif, all but one mutation occurred in the G at the third base which is not part of the hotspot. Seven G C and three G
T mutations were identified at the third base, while an A
G mutation arose at the second base. This is not surprising, considering that the overwhelming majority of mutations in Ramos target G/C bases (10) (Table 1
). Theoretically, this G at the third base can mutate to an A, T or C. However, since we enriched for µ+ cells, a G
A mutation would not be detected amongst revertants because it would produce a TAA nonsense codon.
Clonal instability of V region hypermutation
The finding that our original Ramos clone progressively lost the ability to mutate its V region when maintained in culture (Table 1) suggested that V region hypermutation is unstable in Ramos cells. This notion was supported by the observed drop in mutation rates between 1 and 2 month cultures of clone 6 (Table 1
). The shorter assay period used to determine mutation rates by fluctuation analysis allows the periodic quantification of the mutation rate as clones are propagated. Using this assay, it is therefore possible to determine whether mutation rates of the V region were stable or unstable in Ramos clones. Clones 6, 7, 30 and 71 were carried in culture and at various times subclones were removed and rates of mutation determined by fluctuation analyses. As a control for non-specific ELISA spotting, the µ- clone 5 that had deleted the Ig gene was examined and no IgM-secreting cells were detected (filled-in circles in Fig. 2
means that no ELISA spots were detected), indicating that there is little or no background in the ELISA spot assay. Clone 7 mutated at the same rate even after 3 months of culture (Fig. 2
, 7-0 versus 7-3). However, the rate of mutation of clone 6 decreased 10-fold after 1 month in culture and decreased a further 2-fold after 2 months in culture (Fig. 2
, 6-0 versus 6-1 and 6-2). The three subclones derived from the 2-month culture of clone 6 that did not produce ELISA spots (filled in spots in Fig. 2
) contained deletions that led to frame shifts within their V regions (data not shown) and were therefore not expected to revert at a detectable rate. The mutation rate of clone 71 dropped 35-fold after 3 months in culture, while that of clone 30 remained essentially unchanged after 3 months in culture (Fig. 2
). However, in clone 30 there appear to be two populations, one that retained the high mutation rate and the other that mutated at a lower rate, suggesting that clone 30 may be in the process of generating lower mutating clones as well. Different Ramos subclones can therefore exhibit relatively stable (at least within the assay period used) or unstable mutating phenotypes.
AID levels correlate with mutation rate
To investigate the basis for the clonal instability of hypermutation in Ramos clones, we examined the expression profiles of genes in mutating and non-mutating Ramos clones as a means to identify trans-acting factors that might account for this effect. We undertook this comparison using an RT-PCR analysis of candidate genes involved in hypermutation. These include the error-prone DNA polymerases (26),
(27),
(28), µ (29) and
(30), which are believed to be involved in translesional repair of damaged DNA, the putative cytidine deaminase AID (9) and the mismatch repair enzyme MSH2 (reviewed in 31). To determine whether expression differences of these genes exist between mutating and non-mutating clones, semi-quantitative RT-PCR was performed on clones 6-1 and 7-3, and the non-mutating clone 1-12. Simultaneous PCR reactions specific for GAPDH were first performed using 5-fold sequential dilutions of template cDNA to demonstrate linearity and normality between each sample (Fig. 3
). The expression levels of MSH2 and of the DNA polymerases
,
, and
were similar in all samples (Fig. 3A
). On the other hand, the levels of DNA polymerase µ were at least 25-fold lower in clone 7-3 than in clones 1-12 and 6-1, while the levels of DNA polymerase
were a little higher in clone 1-12 than in clones 7-3 and 6-1 (Fig. 3A
). Interestingly, AID cDNA levels were at least 5-fold lower in the non-mutating clone 1-12 than in clones 6-1 and 7-3 (Fig. 3A
). AID cDNA levels were also low in subclones derived from 1-12 (Fig. 3B
) and, as expected, were not detectable in the T-cell line HUT-78 (Fig. 3B
). In addition, the levels of AID cDNA decreased along with the progressively decreasing rates of mutation in clones 6 and 71 (Fig. 3C
). These data suggest that expression levels of AID account for the differences in the rate of mutation in Ramos clones.
cDNA microarray analysis revealed few differences in gene expression between mutating and non-mutating Ramos clones
While the RT-PCR analyses suggests AID expression is responsible for the difference in mutation rates between different clones, other genes whose expression was not analyzed might also contribute partially or fully to this effect. To examine this, and to determine how different the mutating and the non-mutating clones were in their general pattern of gene expression, we performed a more extensive survey of gene expression differences using cDNA microarrays. For this analysis, two sets of comparisons were made between mutating and non-mutating clones: 6-1 (mutating) versus 1-12 (non-mutating) and 7-3 (mutating) versus 1-12A (sublone of 1-12; see Fig. 3B). Approximately 9000 unique human genes are imprinted on the cDNA microarrays used. With the exception of DNA polymerase
and GAPDH, none of the genes assayed in the RT-PCR experiment shown above are present on our cDNA microarray. Of the ~9000 genes, 42 and 27 differences existed for 6-1 versus 1-12 and 7-3 versus 1-12A respectively (data not shown). Differences that were shared between both sets are presented in Table 3
. Only eight genes showed consistent differences in expression between both sets of mutating and non-mutating clones. Four genes were over-expressed while four genes were under-expressed in the mutating clones (Table 3
). RT-PCR analysis confirmed a low level of SKAP55 expression in the mutating clones 6-1 and 7-3, thus supporting the differences observed by the cDNA microarray analysis (data not shown). The significance of these eight differences to the mutational process is presently not known.
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Discussion |
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The clonal variation and instability of Ramos clones was documented using two different techniques, each of which has limitations, but together they proved to be informative. The benefit of sequencing unselected V regions is that it samples codons throughout the whole V region and reveals biochemical characteristics of the mutational process. However, if this approach were used for the analysis of many clones, a very large number of sequences would have to be determined to achieve statistical significance. Furthermore, the long culture period required for V regions to accumulate enough mutations to allow for a quantification of mutation rate by sequencing could be associated with the outgrowth of non-mutating clones leading to an underestimation of mutation rate. Thus, mutation rates calculated in this manner are at best averages for the culture period, leaving open the possibility that some clones may have had much higher mutation rates sometime during this period. Reversion and fluctuation analyses allow a more rapid evaluation of many subclones and provide results that can be tested for statistical significance. However, the important observation is that both forms of analysis reveal rates of mutation that are similar and together provide strong support for clonal variation and for the different characteristics of each subclone.
By obtaining IgM- Ramos clones for the purpose of fluctuation analyses, we found that all such clones were highly mutating (Table 1 and Fig. 1
), which was surprising since the culture that these cells were derived from was mutating at low levels (Table 1
, clone 1-2). This suggested to us that this procedure allows one to purify mutating clones from a largely non-mutating population. We speculate that cells which have introduced a nonsense codon into the V region had to be undergoing V region mutation at a relatively high rate and thus by sorting for IgM- cells, we essentially separated mutating from mostly non-mutating cells. It is thus possible that mutating clones of uninduced CL-01 and BL-2, which normally require surface IgM cross-linking and T cell help to mutate (18,19), can be obtained by the same methods used in this study.
The mutational process in Ramos resembles that which occurs in vivo in that mutations are more frequently introduced into RGYW and the complementary WRCY sequences (10). This is an important characteristic of the mutational process since it probably reflects a general property of the mutational machinery which might provide clues of the enzymes involved. To ascertain the relative influence that the core RGYW/WRCY sequence confers to mutation rate, it is important to compare this motif to a non-hotspot at the same codon and, therefore, in the context of the same flanking sequences. This is because sequences that neighbor hotspot motifs may have an influence on mutation rates (34,35), which is supported by the fact that not all RGYW/WRCY sequences in the V region behave as mutational hotspots (3638). Fluctuation analysis is perfectly suited for such an analysis because it would not require sequencing hundreds of VDJ sequences that would make a comparison between a hotspot and a non-hotspot at the same codon valid. In this study, fluctuation analyses using two sets of clones (i.e. clones 6 and 62, and clones 10 and 8; Fig. 1) showed that some RGYW/WRCY hotspot motifs are indeed preferentially targeted for mutation by >4-fold over non-hotspot motifs at the same codon.
A comparison of the genes expressed in the high and low mutating clones could shed new light on the mechanism of V region mutation as well as explain the basis for the clonal variation in stable and unstable subclones. We have taken two approaches to identify genes that are differentially expressed in the high and low mutating clones. cDNA microarrays containing ~9000 genes were used to compare gene expression between the mutating clones 7-3 and 6-1 to the non-mutating clones 1-12 and 1-12A. Each of these comparisons revealed 3040 differences, indicating that clones that had been grown separately for 1 year were quite similar to each other in these gene expression patterns. When these two data sets were combined, only eight reproducible differences of gene expression were observed (Table 3). SKAP55, an adaptor protein involved in antigen receptor signaling in lymphocytes (39), was expressed at lowest levels in the mutating clones by this analysis. In contrast, an expressed sequence tags (EST) was over-expressed at highest levels in mutating clones. Whether the changes in expression of each of these genes are a response to the mutational process rather than the cause of mutation is not known. However, what is most striking from this analysis is how similar these clones are to each other with respect to gene expression differences.
As an alternative approach, the expression of genes thought to play a role in somatic hypermutation was examined by RT-PCR. This analysis revealed that the expression levels of AID correlated with mutation rates. AID cDNA levels were >5-fold lower in the non-mutating clone 1-12 than in the mutating clones 6-1 and 7-3 (Fig. 3A). In addition, when clones 6 and 71 were propagated in culture, the mutation rates and the levels of AID were observed to decrease in parallel (Fig. 3C
). The levels of AID are similar between clones 6-0 and 71-0 (Fig. 3C
), while 6-0 has a 3-fold higher rate of mutation than 71-0. While this could suggest that AID levels do not necessarily correlate with mutation rates, mutation rates for 6-0 and 71-0 were calculated by reversion analysis of different nonsense codons. Rates measured in this way are expected to be affected by the sequence context of the nonsense codon (see Fig. 1
and above), producing apparent differences in rate between these two clones. Thus, it is important to compare how the levels of AID correlate with mutations rates within the same clone. This was not necessary in Fig. 3(A
), since mutation rates for all three different clones were measured by sequencing V regions.
AID shows sequence identity to the RNA-editing deaminase family and has been observed to possess cytidine deaminase activity in vitro (40). Knockout studies show that loss of AID expression produces mice that mutate their V regions at least 10-fold lower than controls, implicating this enzyme in the hypermutation process (9). The data presented here, although only associative, supports the role of AID as an important player in this process in human cells. Whether the reduced expression of AID is the cause of the observed decreased in mutation rate is not clear. However, if this is true, it would be surprising that a 5-fold reduction in AID results in a >100-fold decrease in V region mutation. This might argue against AID having a direct effect on mutation, and would suggest that it is involved in an upstream process such as the editing of mRNA for proteins required for mutation. Thus, small changes in protein level could produce amplified effects. On the other hand, AID could be directly involved in mutation, such as by deaminating cytosine residues in the V region. In this case, small differences in AID expression might also produce large effects if it were to function as a dimer (or multimer) or was part of a large complex of proteins possibly involving error-prone polymerases and mismatch-repair proteins.
Error-prone DNA polymerases have long been implicated in hypermutation (41). With the recent cloning and characterization of these mammalian enzymes (27,29,30,42,43), much of the work is presently focused on assessing whether they are indeed involved (13,4446). We found that the cDNA levels of the error-prone DNA polymerase µ and varied in different clones (Fig. 3
), while that of the other error-prone DNA polymerases
,
and
were relatively uniformly expressed. The lack of correlation of polymerase µ expression with mutation suggests that it is not required for somatic hypermutation in Ramos. On the other hand, DNA polymerase
was found to be expressed at a slightly lower level in mutating Ramos clones, which supports a finding that the expression of this polymerase decreases when CL-01 cells are induced to mutate (46). It is tempting to speculate that the lower expression of DNA polymerase
in mutating Ramos clones is the reason for the targeting bias of G/C residues in mutation (Table 1
) in light of the fact that this polymerase has been implicated as being the A/T mutator (44).
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Acknowledgments |
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Abbreviations |
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AID activation-induced cytidine deaminase |
EST expressed sequence tags |
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Notes |
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Received 7 May 2001, accepted 13 June 2001.
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References |
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