The TATA binding protein, c-Myc and survivin genes are not somatically hypermutated, while Ig and BCL6 genes are hypermutated in human memory B cells
Hong Ming Shen,
Nancy Michael,
Nayun Kim1 and
Ursula Storb
Departments of Molecular Genetics and Cell Biology, and
1 Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA
Correspondence to:
U. Storb
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Abstract
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Immunoglobulin (Ig) genes are hypermutated in mature B cells after interaction with antigen and T cells in a germinal center reaction. We and others have recently shown that the human BCL6 gene is also hypermutated in human peripheral blood memory B cells and tonsils. A preliminary analysis of other non-Ig genes (c-Myc, S14 and AFP) suggested that they were not mutated in memory B cells. We have now performed an in-depth analysis of three non-Ig genes that are expressed in germinal center B cells in two human donors in whom BCL6 is highly mutated. It was found that the TATA binding protein (TBP), c-Myc and survivin genes are not hypermutated. This lack of targeting by the Ig hypermutation mechanism must be due to the lack of regulatory DNA elements, since the primary sequences of the three tested genes have at least as high intrinsic mutability indices as the BCL6 gene.
Keywords: BCL6, Ig genes, mutation, non-Ig genes, somatic hypermutation
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Introduction
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The somatic hypermutation mechanism has the potential to create an enormous variability in the antibody repertoire by introducing point mutations in the coding sequence of the variable (V) domains of Ig genes. The process is likely due to the interaction of a specific mutator factor with the RNA polymerase at the promoter of Ig genes and deposition of the mutator factor onto the Ig gene during its transcription (reviewed in 1). Ig enhancers appear to be required (reviewed in 2), but it has not been determined if other enhancers can permit somatic hypermutation when replacing Ig enhancers within an Ig gene. An Ig gene-specific promoter, on the other hand, does not seem to be required, at least in the presence of Ig enhancers. When the Ig promoter is replaced by a ß-globin or B29 promoter, the mutability of Ig transgenes is retained (3,4). Furthermore, when translocated into the Ig heavy chain locus, the c-Myc gene becomes hypermutable; in this situation, c-Myc appears to be transcribed from its own promoter (5).
These findings prompted us to investigate if other genes that are expressed in B cells undergoing somatic hypermutation may be mutable by this process (6). Memory B cells were isolated from the peripheral blood of four normal individuals. It was found that the BCL6 gene was highly mutable in three of the four donors tested, whereas the c-Myc gene, the ribosomal protein gene S14 and the
-fetoprotein gene appeared not to be mutated in the one donor where they were tested. c-Myc and S14 are expressed in germinal centers, and if mutated at that stage, should have been found mutated in memory B cells.
-Fetoprotein served as a control for a gene that is not expressed in germinal centers.
In the present study we have made an in depth study of the possible hypermutation of three non-Ig, non-BCL6 genes that are expressed in germinal center B cells. The study was carried out with the DNA from the memory B cells and, as control, virgin B cells of the two donors who had the highest levels of BCL6 mutations and in whom no other non-Ig genes had been previously investigated for somatic mutation (6).
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Methods
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Isolation of DNA from memory and virgin B cells
Peripheral blood IgMIgD and IgM+IgD+ B cells were isolated from human donors, and DNA prepared as described (6). In the present study the analysis of the TBP, c-Myc and survivin genes was done on the same DNA samples of donors C and D as used in the previous study in order to allow a direct comparison with the mutation values found for BCL6. The Ig gene analysis of donor A was done on RNA from total peripheral blood lymphoid cells (PBL).
Single-strand conformational polymorphism (SSCP) assay, DNA cloning and DNA sequencing
Mutations in the TATA binding protein (TBP), c-myc and survivin genes were first assessed by SSCP.
PCR primers were used to generate cloning fragments as following: for TBP, 5'-GGA AGA GAC TAG GAA TTG GCG-3'/5'-TTG GAA GTG CAG TAT GAA CAG-3'; for c-Myc, 5'-CCG GTA CCC TTG CCG CAT CCA CGA AAC TTT/5'-GCT CTA GAG ACC CAG GTT TTA-3'; for survivin region 1, 5'-CCA TGT AAG TCT TCT CTG GCC-3'/5'-GTG GAA AGA CAG ATA CCA TCC G-3'; and for survivin region 2, 5'-ATT AAC CGC CAG ATT TGA ATC GCG-3'/5'-AGG GTA ATT TTT GTG CTG TGT AGG-3'. The PCR conditions were 94°C for 5 min, 57°C for 30 s, 75°C for 1 min, one cycle; 94°C for 30 s, 57°C for 30 s, 75°C for 1 min, 29 cycles; and 75°C for 6 min using Pfu polymerase. The gene fragments were cloned into the pCR-Blunt II-TOPO vector according to the manufacturer's instructions (Invitrogen, Carlsbad, CA)
For SSCP, bacterial colonies were picked and lysed in water. For a 10 µl reaction, the PCR mixtures were as follows: 1 µl of Taq polymerase buffer, 1 µl of 2 mM dNTP, 1 µl of 5' primer (5 µM), 1 µl of 3' primer (5 µM), 0.1 µl of Taq polymerase, 3.8 µl of water, 2 µl of lysed bacteria and 0.1 µl of [32P]dCTP (1°C). The PCR conditions were as above. PCR products were digested with NlaIII for TBP, and AluI for c-Myc, survivin region 1 and survivin region 2 respectively. The digested fragments were then run on acrylamide gel at 6 V for 18 h. For 100 ml of the gel mixture, 10 ml of glycerol, 10 ml of 10xTBE buffer, 15 ml of acrylamide (40% at 19:1), 64.2 ml of water, 0.8 ml of 10% ammonium persulfate and 36 µl of TEMED were added. The gels were exposed to X-ray film at 80°C. DNA clones that showed an aberrant SSCP pattern were sequenced. The maps of the non-Ig genes are shown in Fig. 1
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Fig. 1. Maps of the human BCL6, c-Myc, survivin and TBP genes (12,2022). White boxes, translated exons; grey boxes, untranslated exons. The bent arrows indicate the transcription start sites. Regions amplified and sequenced are indicated by converging arrows.
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For the Ig gene analysis of donor A, RT-PCR was carried out on the total PBL RNA using reverse transcriptase (Stratagene) and Pfu DNA polymerase. The RT primer was either oligo(dT), or the respective short or long primers shown below (9). The PCR reaction was carried out with the VH4 primer and one of the four C region primers shown below: VH4 (5'-ACTAGTCGACCCTGTCCCTCACCTGC(A/G)CTGTC-3'), CµRT (long) (5'-GCCAGCTGTGTCGGACATGAC-3'), Cµb (short) (5'-GGGGAATTCTCACAGGAGACGAGGGGGAA-3'), C
RT (long) (5'-TCTTGTCCACCTTGGTGTTGCT-3') and C
b (short) (5'-ATCAGTCGACAAGACCGATGGGCCCTTGGTGGA-3'). The
primers are homologous to the CH1 domains of all human
genes, except
3. The PCR products were cloned and sequenced.
Expression of BCL6, TBP, c-myc and survivin
A human tonsil was taken from a 14-year-old patient during routine tonsillectomy and minced. CD19+ B cells were obtained from the cell suspension by using Dynal beads (Dynal, Lake Success, NY). The cells were then stained with FITCgoat anti-human IgD (Southern Biotechnology, Birmingham, AL) and phycoerythrin (PE)mouse anti-human CD38 (PharMingen, San Diego, CA). IgDCD38+ B cells and IgD+CD38 B cells were separated on a flow cytometer (Epics Elite ESP; Coulter, Hialeah, FL).
RNA was isolated by using RNA STAT-60 (Tel-Test, Friendswood, TX). Oligo(dT) was used to generate fist-strand cDNA, and PCR was carried out in the presence of following primers: for G3PDH, G3PDH.1 (sense): TGC ACC ACC AAC TGC TTA GC and G3PDH.2 (antisense): TTT CTA GAC GGC AGG TCA GG; for BCL6, BCL6.1 (sense): GCA AGA AGT TTC TAG GAA AGG CCG and BCL6.2 (antisense): TGC AGG TAC ATA GCC GTG GC; for c-Myc, c-Myc.1 (sense): GCT TCT CTG AAA GGC TCT CC and c-Myc.2 (antisense): TTG ATG AAG GTC TCG TCG TCC; for TBP, TBP-44 (sense): TTT AAC TTC GCT TCC GCT GGC and TBP-330 (antisense): GGA CTA AAG ATA GGG ATT CCG; for survivin, Surv.1 (sense): TCA AGA ACT GGC CCT TCT TGG and Surv.2 (antisense): GCA CTT TCT TCG CAG TTT CCT C. PCR cycles were 30 for G3PDH, 32 for BCL6, and 35 for c-Myc, TBP and survivin. In Fig. 3
, the G3PDH sample was diluted 1.5 times.

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Fig. 3. Comparison of transcripts of G3PDH, BCL6, c-Myc, TBP and survivin in IgD+CD38 and IgDCD38+ human tonsillar B cells. G3PDH was amplified for 30 cycles and the product diluted 1:1.5 before electrophoresis. BCL6 was amplified for 32 cycles, the rest for 35 cycles.
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Determination of mutability indices (MI)
Studies by Wysocki et al. (10,11) of large databases of somatically hypermutated mouse and human Ig genes have determined the relative mutabilities of the 64 nucleotide triplets. These were carried out with non-selectable sequences. In the case of the human VH genes, they were derived from non-productively rearranged H chain alleles in B cells that had undergone somatic hypermutation. The values of the MI range from 0.0 to 2.62. An index value of 1.0 is assigned to a triplet that is found to be mutated at a frequency that would be expected if hypermutation is a random process. A value of >1.0 indicates that the triplet is a favored target of somatic hypermutation, a `hot spot'; the larger the value, the greater the likelihood of mutation. Likewise, values of <1.0 predict `cold spots', unfavored targets of hypermutation. Based on their findings, we developed a computer-based analysis program that scans a given sequence and records the MI of each triplet in single nucleotide steps (N. M. Shen et al., unpublished).
The `intrinsic mutability' (Table 4
) of a sequence was derived for each sequence and used to determine if the Ig or non-Ig gene primary sequence was skewed towards hot or cold spots. Intrinsic mutability was defined by us as an average of the (percentage occurrence of each triplet in a sequence)x(MI of that triplet) (11; G. Shapiro and L. Wysocki, pers. commun.). It is a weighted average of the MI of all triplets in the sequence.
For the determination of the MI of the mutated nucleotides in Tables 2 and 3
the following procedure was used. The quintuplet of nucleotides with the mutated nucleotide in the center was broken up into three triplets in which the mutated nucleotide is at the third, second and first position respectively. For each of the triplets, the mutation index (MI) for the mutated nucleotide was taken from the analysis of Wysocki et al. (11), Table III (G. Shapiro and L. Wysocki, pers. commun.). Example: Table 3
, top sequence (TBP, donor C, the C* of GGC*CC is mutated): MI of the C* in GGC*, GC*C, and C*CC are 0.74, 1.33, and 0.67 respectively, giving the mean of 0.91 which is recorded as the MI of that C*. The highest values in Tables 2 and 3
are higher than in Table 4
, because the MI of an individual nucleotide can be higher than the MI of any triplet [cf. Tables II and III in (11)].
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Results
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Mutations in Ig genes in memory B cells
In general, the VH genes of B cells that have switched to the expression of an Ig
constant region show higher levels of somatic hypermutation than IgM genes. Rearranged and switched Ig genes of the VH4 family were amplified by PCR using a 5' primer for VH4 and a 3' primer for µ or
. The memory B cells of donor A (Ig genes of donors C and D were not tested) showed high levels of somatic hypermutation in the VH sequence (Fig. 2
). The average MI of the mutated nucleotides in the sequenced regions is 1.28, i.e. >1.0, as expected for mutations that were targeted by the Ig gene somatic hypermutation mechanism. The constant regions were not mutated above background levels of Pfu error (not shown).
Mutations in non-Ig genes
As described before, the BCL6 gene was found to be highly mutated in memory B cells, but not virgin B cells of donor A, as well as in memory B cells of donors C and D (Table 1
). Another donor, B, did not have significant numbers of IgMIgD B cells and thus IgMloIgD B cells were used. With more extensive analysis, BCL6 was also found to be mutated in donor B, although at a lower frequency than in donors A, C and D (A. Sibley et al., unpublished). Possibly, the scarcity of B cells with a true memory B cell surface marker phenotype was due to a recent asthma attack suffered by this donor. The IgG genes of this donor were highly mutated (not shown), but they are of course selected as genes that have undergone Ig switch recombination. The BCL6 gene sample, on the other hand, comes from the total B cell sample that is not enriched for IgG expression.
Three other non-Ig genes were analyzed from memory and virgin B cells of donors C and D (Table 1
). These donors had the highest mutation frequencies of the four donors in the BCL6 gene and therefore the greatest likelihood of having other non-Ig genes mutated if they were mutable by the Ig hypermutation process. The TBP (basal transcription factor) and c-Myc (replication regulator) genes are expected to be well expressed in mutating B cells which are transcriptionally active and proliferate highly. The survivin gene has been shown to be expressed in lymphomas derived from germinal center blasts (12) as well as in tonsillar germinal center B cells (13). We have compared the expression of these genes and of BCL6 in germinal center B cells and virgin B cells from human tonsil (Fig. 3
). All four genes are up-regulated in IgD, CD38+ germinal center B cells, compared with the IgD+, CD38 virgin B cells (14). The up-regulation is apparent compared with the G3PDH gene (Fig. 3
). Since somatic hypermutation takes place in germinal center B cells and these cells give rise to memory B cells, we can assume, that all four non-Ig genes tested were well expressed during the mutation process.
Between 29,250 and 101,280 nucleotides of the non-Ig, non-BCL6 genes were assayed for mutation by SSCP in memory B cells. Very few mutations were found and confirmed by sequencing. With three exceptions (Table 1
), each of the mutated DNA clones had only one single point mutation within the 759888 nucleotides sequenced. The average mutation frequencies ranged from 3x105 to 1.4x104 in the memory B cells. Mutations were also found in the virgin B cells. In donor D, the mutation frequencies were somewhat higher in the memory B cells compared with virgin B cells (memory/virgin = 2, 2, 5 and 1.7 times). In donor C, in two of the sequenced regions (TBP and survivin 1) the memory B cells showed 2 and 3 times higher mutation values than the virgin B cells; in the other two gene regions (c-Myc and survivin 2) the virgin B cells showed higher mutation values (1.3 and 1.7 times). The slightly higher values in memory B cells may be due to the fact that in these 44- and 47-year-old individuals the memory B cells had undergone many more cell cycles than the virgin B cells by repeated entries into the rapidly dividing germinal center compartment (15). A similar suggestion of chronic stimulation has been made for mutations in the untranslocated c-Myc gene in mucosa-associated lymphomas (16). Thus, the small differences between memory and virgin B cell mutations may be real, but not due to the process of somatic hypermutation of Ig genes (see below). However, the mutation frequencies in the memory B cells in all eight tests are not significantly different from the mutation frequencies in virgin B cells (P = 0.063 for mutations per total nucleotides analyzed or P = 0.152 for mutated DNA clones per total clones, using the MantelHaenszel test).
Thus we conclude that these genes are not mutated by the Ig hypermutation process. This conclusion is further supported by comparing the MI of the triplets containing mutated nucleotides in the Ig/BCL6 genes with those in the other three non-Ig genes (Fig. 2
, legend; Tables 2 and 3
). The MI in memory B cells for Ig and BCL6 (1.28 and 1.34) are well above 1.0, whereas those for the other three genes are below 1.0 (0.520.97). This suggests that the latter mutations did not arise as a consequence of the somatic hypermutation process, but were created by other processes. This is further borne out by inspecting the individual triplets in which the mutations occurred. For BCL6, 25 of 70 mutations in memory B cells were in hot spots with a MI > 1.5. For the other three non-Ig genes, one of 34 mutations in memory B cells had a MI > 1.5 (1.64), but one of 20 mutations of virgin B cells also had a high index (2.04). Finally, in the non-BCL6 genes, a number of deletions were found (Table 3
), whereas none were found in BCL6 (Table 2
). Deletions are extremely rare in somatic hypermutation. They are present as ~1% of somatic mutations in Ig genes. In fact, none were seen among the 418 Ig mutations found in this study (Fig. 2
). Taking all of these findings together strongly suggests that the TBP, c-Myc and survivin genes are not targeted by the Ig somatic hypermutation mechanism.
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Discussion
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Somatic hypermutation of Ig genes of human memory B cells
High levels of somatic hypermutation were seen in rearranged IgVH genes of the memory B cells of donors A and B (Fig. 2
and not shown). In donor A, where rearranged µ and
genes of total PBL were studied, every
gene had point mutations with an average of 12.6% of the sequenced nucleotides mutated (Fig. 2
). The highest frequency of mutations in a single VH sequence was 20.2%. The µ sequences were mutated to a lesser degree, with an average of 2.9% of the sequenced nucleotides mutated. These numbers make an interesting comparison with the analysis of somatic hypermutation in the same VH4 genes of peripheral blood B cells of a 4-year-old child (17). There, 2.6% of the VH4 nucleotides of the µ sequences were mutated and 3.2% of the
sequences. The mutation frequencies in µ genes are very similar in the child and the adult, probably indicating that most of the µ-expressing B cells are recent emigrants from the bone marrow that have undergone a first round of somatic hypermutation. In contrast, the mutation frequencies in the
genes of the older donors are ~4-fold higher than those of the child. This may support the notion that memory B cells reenter the germinal centers multiple times during their lifespan and accumulate additional mutations. This possibility appears more likely than the alternative, in which during life the more mutated genes become more and more dominant, although this possibility cannot be ruled out.
Somatic hypermutation of non-Ig genes
As shown in transgenic mice, somatic hypermutation is linked to transcription (18). The genes that were tested for somatic hypermutation in this study are all well expressed in germinal center B cells (Fig. 2
) and should thus fulfill the transcription requirement. However, only Ig and BCL6 were found to be mutated. As shown now in three separate studies, the BCL6 gene is highly mutated in human memory B cells (68). So far, no other non-Ig genes expressed in germinal center B cells that are in their normal chromosomal location have been found mutable by the somatic hypermutation mechanism. If Ig type hypermutations occur in the TBP, c-Myc and survivin genes, they would most likely not interfere with gene function. The sequenced regions that comprise the position which is most mutable in Ig genes (within 0.41.0 kb from the promoter) reside in untranslated exons or introns (Fig. 1
). So far, of genes expressed in germinal center cells, besides BCL6, only TBP, c-Myc, survivin (this study) and S14 (6) have been investigated. It appears likely that some other non-Ig genes may be found to be mutated by the Ig somatic hypermutation mechanism.
The TBP, c-Myc and survivin genes were found to have very low levels of mutations that were not significantly higher in memory B cells than in virgin B cells. It is likely that the few mutations found in these genes are due to PCR errors and perhaps partly other mutation mechanisms. As shown in Fig. 2
(legend) and Tables 2 and 3
, the MI of mutations in the non-BCL6 genes are very different from those in BCL6 and Ig genes when one applies the criteria compiled by Wysocki et al. for non-selected Ig-specific somatic hypermutations (10,11). [Incidentally, the triplet rules are very similar for human and mouse genes (11).] The average MI were 1.35 for BCL6, 1.28 for Ig VH4-11, but well below 1.0 for the three other non-Ig genes in memory B cells. Thus, in the latter genes the Ig somatic mutation hot spots were not targeted. That is, the primary sequence context of the mutations observed in TBP, c-Myc and survivin does not match the sequence context of mutations in Ig and BCL6 genes. If sequence `hot spots' is a hallmark of somatic hypermutation, then the mutation pattern observed in TBP, c-Myc and survivin suggests that these mutations must have arisen by different rules than those governing somatic hypermutation of Ig genes.
The lack of targeting of the non-Ig genes TBP, c-Myc and survivin is not due to an intrinsic lack of Ig gene-type mutability of these three genes compared with BCL6 (Table 4
). Primary sequence hot spots seem to be important in targeting the mechanism of somatic hypermutation, once engaged on a particular gene. We hypothesize that the intrinsic mutability score (Table 4
) would predict the likelihood that a sequence will be a substrate for somatic hypermutation, if the primary sequence within the 5' end of the gene were the only targeting mechanism. The average MI of BCL6 is lower than those of the other three non-Ig genes. Furthermore, in BCL6 the percentage of the hottest hot spots is lower and the percentage of the coldest cold spots is higher than the average of the other three genes. Therefore, the fact that BCL6 is mutable by the Ig somatic hypermutation mechanism must be due to other cis-acting elements, besides the primary mutation target sequence. Presumably in Ig genes, these cis-acting elements lie within the Ig enhancers. Elimination of the Ig enhancers affects somatic mutation (3). We have postulated that the role of such cis elements is to target a mutator factor to RNA polymerase associated with an Ig promoter (19). Presumably, elements with the same function, but perhaps not necessarily the same sequence must be present within or near the BCL6 gene. It will be important to determine the identity of the cis elements and their trans-acting factors in Ig and BCL6 genes.
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Acknowledgments
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We are grateful to Phil Schumm for the statistical analysis and to Dr Fred Baroody for the surgical tonsil specimens. This work was supported by NIH grant GM38649. The FACS, sequencing and oligonucleotide synthesis facilities are partly supported by an NIH grant to the University of Chicago Cancer Research Center.
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Abbreviations
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MI mutation index |
PBL peripheral blood lymphocytes |
PE phycoerythrin |
SSCP single-stranded conformational polymorphism |
TBP TATA binding protein |
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Notes
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Transmitting editor: K. Knight
Received 20 December 1999,
accepted 31 March 2000.
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