Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

Beatriz Goyenechea; Norman Klix; José Yélamos; Gareth T. Williams; Andrew Riddell; Michael S. Neuberger; César Milstein

doi:10.1093/emboj/16.13.3987

Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

Goyenechea, Beatriz; Klix, Norman; Yélamos, José; Williams, Gareth T.; Riddell, Andrew; Neuberger, Michael S.; Milstein, César 1997-07-01 00:00:00 The EMBO Journal Vol.16 No.13 pp.3987–3994, 1997 Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers and Milstein, 1995; Storb, 1996; Weill and Reynaud, Beatriz Goyenechea, Norman Klix, 1996). Jose´ Ye´lamos, Gareth T.Williams, Experiments to date looking at the requirement for cis- Andrew Riddell, Michael S.Neuberger and 1 acting elements have focused largely on the overall extent Ce´sar Milstein of mutation rather than differentiating between the fre- Medical Research Council Laboratory of Molecular Biology, quency with which a gene is targeted for mutation and Hills Road, Cambridge CB2 2QH, UK the extent of mutant accumulation once so targeted. We were therefore moved to extend our previous work on the Corresponding authors immunoglobulin κ locus (Betz et al., 1994), dissecting B.Goyenechea and N.Klix contributed equally to this work the contributions of the two κ enhancer regions and asking whether the portions necessary for transcription The V regions of immunoglobulin κ transgenes are enhancement corresponded to the portions essential for targets for hypermutation in germinal centre B cells. hypermutation, paying special attention to the distinction We show by use of modiﬁed transgenes that the between mutational targeting and mutation accumulation. recruitment of hypermutation is substantially impaired by deletion of the nuclear matrix attachment region (MAR) which ﬂanks the intron-enhancer (Ei). Results Decreased mutation is also obtained if Ei, the core region of the κ3-enhancer (E3) or the E3-ﬂank are Removal of either MAR or Ei diminishes removed individually. A broad correlation between hypermutation expression and mutation is indicated not only by the In previous work, by comparing V region mutation in Lκ fact that the deletions affecting mutation also give and LκΔ[Ei/MAR] transgenes, we showed that removal reduced transgene expression, but especially by the of a 688 bp region from the J –C intron leads to a very κ κ ﬁnding that, within a single mouse, transgene mutation substantial reduction in hypermutation (Betz et al., 1994). was considerably reduced in germinal centre B cells This deletion takes out both the intron-enhancer (Ei), that poorly expressed the transgene as compared with which has been shown in transfection assays to confer strongly expressing cells. We also observed that the lymphoid-speciﬁc transcriptional enhancement (Picard and diminished mutation in transgenes carrying regulatory Schaffner, 1984; Queen and Stafford, 1984), and the element deletions was manifested by an increased MAR, which was identiﬁed as a nuclear matrix attachment proportion of B cells in which the transgene had not region (Cockerill and Garrard, 1986) but which does not been targeted at all for mutation rather than in the exhibit cell type speciﬁcity and which has not been shown extent of mutation accumulation once targeted. Since to have any major role in transcriptional activation. mutations appear to be incorporated stepwise, the To determine whether it was the removal of Ei or of results point to a connection between transcription MAR that was responsible for the reduction in hypermut- initiation and the clonal recruitment of hypermutation, ation, we prepared Lκ derivatives that were separately with hypermutation being more fastidious than tran- depleted of either element. Two constructs (differing in scription in requiring the presence of a full complement the precise location of the deletion endpoints) were made of regulatory elements. for each type of deletion (Figure 1) and multiple transgenic Keywords: diversity/enhancers/hypermutation/ mouse lines established. immunoglobulin/MAR/somatic mutation Hypermutation was assessed by PCR cloning and sequencing of the transgenic V genes from sorted germinal centre B cells obtained from the mouse Peyer’s patches. Whilst there is signiﬁcant founder-to-founder variation, it Introduction is notable that, compared with the Lκ controls, deletion Somatic hypermutation is a major contributor to antibody of either Ei or MAR on their own is sufﬁcient to give a afﬁnity maturation. In germinal centre B cells, nucleotide dramatic drop in hypermutation (Table I). The effect of substitutions are introduced into a region of several the MAR deletion, which in all four lines examined leads kilobases of DNA that includes the rearranged V gene to mutation rates that are severely diminished or even segments of the immunoglobulin heavy and light chain undetectable above background, is more severe than that loci. The mechanism of hypermutation is unknown, of Ei. although several lines of evidence point to a linkage to The diminished mutation of the LκΔ[Ei] and LκΔ[MAR] transcription. Thus, the process reveals strand polarity, it transgenes is largely attributable to a large decrease in the is inhibited by the removal of cis-acting transcription proportion of transgenic V gene clones that carry any enhancer elements and the mutation domain is located mutations at all. This ﬁgure drops from a range of 65– downstream of the promoter (see reviews by Neuberger 75% in the Lκ control mice to 5–33% in the LκΔ[Ei] and © Oxford University Press 3987 B.Goyenechea et al. Fig. 1. The transgenes. The transgenes are all based on the ancestral constructs Lκ or LκΔB (both of which are good hypermutation targets) as described in Materials and methods; in LκΔB, the region between the BamHI (B) sites downstream of C is deleted as indicated. Lκ is of mouse origin except for a small region (in white) including most of C , which is of rat origin. The extent of the internal deletions in the different constructs is indicated with the numbering following Max et al. (1981) in the J –C intron and Meyer and Neuberger (1989) around the 3-enhancer. Other κ κ restriction sites are abbreviated H, HindIII; Hf, HinfI (not all sites depicted); Hp, HpaI; R, EcoRI; N, a NotI linker that has been inserted into an EcoRI site; S, SacI; and X, XhoI. Various factor-binding sites within the enhancers and MAR are indicated. LκΔ[MAR] animals. Furthermore, the few LκΔ[Ei] and 1994). Transgenic mice were established to discriminate LκΔ[MAR] transgene copies that do get targeted for the relative roles of the core and ﬂank in mutational mutation are still able to accumulate multiple nucleotide targeting. Whilst again the different founder lines mutate substitutions (Figure 2). If we restrict our analysis to to different extents, it is clear that deletion of either mutated sequences, then the average number of substitu- the core or the ﬂank results in diminished targeting of tions accumulated in the mutated LκΔ[Ei] and LκΔ[MAR] hypermutation (Table I and Figure 2). As with the Ei and V genes is only reduced ~2-fold compared with the MAR deletions, whilst there is a decrease in the number number of substitutions accumulated on the parental Lκ of transgene copies that are targeted for mutation, those transgene (Table I). These effects are speciﬁc to the that are so targeted can still accumulate multiple nucleotide transgenes themselves since they are observed in cells substitutions. that otherwise retain their normal mutational capacity. Thus, sequence analysis of PCR-ampliﬁed clones derived Deletions affect κ expression from the 3-ﬂank of rearrangements of V J558 family The expression of the various transgenes was monitored members to J 4 reveals that LκΔ[Ei], LκΔ[MAR] and by ﬂow cytometric analysis of splenic B cells using an control mice are similar with respect to the accumulation anti-rat κ antibody, exploiting the fact that the C exon of mutations in their endogenous heavy chain loci (8.7, (but not the remainder) of the various transgenic constructs 14 and 9.1 substitutions/kb respectively determined as is of rat origin. Even amongst the B cells of a single described in Materials and methods). transgenic mouse, there is heterogeneity with respect to Thus, both the Ei and, particularly, the MAR deletions transgene expression on the B-cell surface, with the effect a severe inhibition of the targeting of the transgene transgenic κ being in competition with endogenous mouse for hypermutation; it remains possible that, in addition, κ in those cells in which κ gene expression is not allelically these same deletions also effect a small inhibition of excluded (Figure 3). Whereas the Lκ construct expresses mutation accumulation once the transgenic V gene has well in four independent lines with few spleen cells been targeted. expressing endogenous mouse κ, this same dominance is not observed with the Lκ derivatives in which Ei, parts Removal of either E3-core or E3-ﬂank diminishes of E3 or MAR have been removed (Table II). The level hypermutation of transgene expression broadly correlates with the level The E3, which has also been shown to be important for of hypermutation, although the correlation is not a straight- mutational targeting (Betz et al., 1994), is composed of a forward one. core enhancer region surrounded by a conserved ﬂank, In contrast to these ﬂow cytometric results, however, which can suppress the activity of the core in pre-B cell no decrease in transgene expression effected by the Ei lines (Pongubala and Atchison, 1991; Meyer and Ireland, deletion is evident if expression is estimated by the 3988 Somatic mutation of the Igκ locus Table I. Mutation of the transgenes Mouse line Clones Mutations Mutations/10 bp total All Mutated All Mutated clones clones Lκ Line 3 76 53 267 12.4 20.0 Line 6 73 54 262 12.7 17.2 Line NG 59 43 197 11.8 16.2 Line WTM7 88 52 184 7.4 12.5 LκΔ[Ei/MAR] 75 10 16 0.8 LκΔ[3E] 37 13 23 2.2 6.3 LκΔ[MAR]S Line 1 42 2 2 0.2 LκΔ[MAR]L Line 1 61 10 23 1.3 8.1 Line 2 63 14 34 1.9 8.6 Line 3 42 2 5 0.4 LκΔ[Ei]S Line 1 75 19 48 2.3 9.0 Line 2 60 20 49 3.0 8.7 LκΔ[Ei]L Line 1 225 43 161 2.5 13.3 LκΔ[E3-Flank] Line 1 63 6 9 0.5 Line 2 101 27 70 2.3 9.1 LκΔ[E3-Core] Line 1 98 25 41 1.5 5.8 Line 2 42 13 19 1.7 Too few mutated clones for meaningful calculation. The Vκ segment of each transgene was cloned following PCR ampliﬁcation from sorted germinal centre B cells. For each transgene, the table gives the total number of PCR clones sequenced, the number of those that carried one or more mutations within the V segment Fig. 2. Frequency distribution of clones with respect to the number of (282 bp), the total number of mutations identiﬁed and the mutation mutations they carry. In each pie chart, the size of each segment is a frequency. This frequency (point mutations per 10 bp) is computed measure of the proportion of clones that carry the indicated number of both with respect to all clones analysed and with respect to only those mutations. For each type of transgenic construct (grouped simply as clones that carry mutations. For several transgenic lines, mutation was Lκ, Δ[MAR], Δ[Ei], Δ[E3-Core] and Δ[E3-Flank]), the results of the analysed in multiple individual animals. The variation in mutation analysis of the individual animals presented in Table I are pooled rates between animals was lower than that found when comparing together and presented as a single pie chart. different mouse lines that carried the same transgene. The data for the Lκ,LκΔ[Ei/MAR] and LκΔ[3E]transgenes are taken from Betz et al. (1994), Gonza´lez-Ferna´ndez and Milstein (1993), Ye´lamos et al. (1995) and Goyenechea and Milstein (1996). Only the unmodiﬁed Lκ transgenes in the NG and WTM7 mice are used in the computation. mouse was more efﬁcient in those cells which expressed the transgene highly than in those of the transgene-dull population. Fractionation of Peyer’s patch B cells from abundance of transgene mRNA in transgene-positive LκΔ[Ei] and LκΔ[MAR] mice into populations that were hi splenic B cell hybridomas (data not shown). This parallels either bright or dull for transgenic κ expression (TGκ lo previous observations with the LκΔ[Ei/MAR] transgene and TGκ ) revealed that most mutated transgenes were where the simultaneous removal of both Ei and MAR had found amongst the more brightly expressing cells (Figure little effect on the abundance of κ mRNA in hybridomas 4). Although transgene hypermutation was much dimin- but led to a signiﬁcant drop in transgene expression, as ished in the transgene-dull population, the endogenous well as exclusion of endogenous κ expression as judged heavy chain locus was still at least as well mutated; the by ﬂow-cytometric analysis of splenic B cells (Betz et al., mutation frequency in the 3-ﬂank of V J558 family 1994; Meyer et al., 1996). Thus, the ﬂow-cytometric member–J 4 integrations was six substitutions/kb in the monitor of expression on the B-cell surface is a better transgene-bright LκΔ[MAR] germinal centre B cell sub- correlate of hypermutability. population and 16/kb in the transgene-dull subpopulation. Thus, the decreased efﬁciency of mutational targeting of Most transgene mutations are found in those the transgene in the transgene-dull subpopulation does not B cells strongly expressing the transgene reﬂect any impairment of the hypermutation capability of Given this correlation between hypermutation and cell the cells themselves; the somewhat increased mutation of surface expression of the transgene, we wondered whether the endogenous loci in these cells is a topic for further the targeting of transgene hypermutation in a particular investigation. 3989 B.Goyenechea et al. Table II. Transgenic and endogenous κ chain expression Mouse line Rat-κ Mouse-κ Mutations/ 10 bp Non-transgenic 0 100 – Lκ Line 6 100 0 12.7 Line NG 95 5 11.8 Line WTM7 94 11 7.4 tLκΔ[Ei/MAR] 55 41 0.8 LκΔ[E3] 4 72 2.2 LκΔ[MAR]S Line 1 8 91 0.2 LκΔ[MAR]L Line 1 25 54 1.3 Line 2 25 61 1.9 Line 3 70 14 0.4 LκΔ[Ei]S Line 1 59 42 2.3 Line 2 70 29 3.0 LκΔ[Ei]L Line 1 45 53 2.5 LκΔ[E3-Flank] Line 1 17 69 0.5 LκΔ[E3-Core] Line 1 28 42 1.5 Line 2 4 96 1.7 Igκ expression on the surface of splenic B cells was determined by cytoﬂuorimetry on at least two animals for each transgenic line. The values (mean κ ﬂuorescence on B220 cells) are normalized with respect to Lκ6 transgenic and non-transgenic litter mates, giving these control lines values of 100 and 0 for transgenic/endogenous κ expression as indicated. Hypermutation frequencies, taken from Table I, are shown for comparison. The critical importance of the MAR was not anticipated. Whilst originally deﬁned by an in vitro nuclear matrix binding assay (Cockerill and Garrard, 1986), no major functional importance for the MAR is apparent from a deletion analysis of the κ intronic enhancer performed using transfection into lymphoid cell lines (Queen and Stafford, 1984). This might, in part, reﬂect an inadequacy of transfection assays in revealing all sequences that support enhancer activity. Thus, the region ﬂanking the core of the IgH intronic enhancer [which also possesses MAR activity (Cockerill et al., 1987)] was found to be Fig. 3. Transgene expression. Expression was analysed by cytoﬂuorimetric analysis staining of splenic B cells with critical for the expression of IgH transgenes although phycoerythrin-conjugated anti-CD45R(B220) and either mouse dispensable for the activity of the enhancer in cell line (endogenous) or rat (transgenic) κ biotinylated mAbs and transfection assays (Forrester et al., 1994). An ability of FITC-conjugated streptavidin. Ei/MAR and E3 to partially cross-substitute for each other in some functions, as suggested by in vivo gene Discussion targeting experiments (Gorman et al., 1996; Xu et al., MAR and E3-ﬂank are essential for full 1996), might explain the fact that we can detect consider- hypermutation able residual κ transgene expression after deletion of the The results described here further link hypermutation and intronic MAR. expression. Previous results have shown the importance With regard to the requirement for the E3 ﬂanking of the Ei/MAR and E3 regions for immunoglobulin κ region, this region (like that ﬂanking the IgH intronic hypermutation (Betz et al., 1994). The dissection of the enhancer), whilst having no enhancer activity of its own, contributions of the core enhancers and their ﬂanks reveals has been shown to be able to regulate the activity of a that the ﬂanks are also essential for the proper recruitment linked enhancer in appropriate cell types (Imler et al., of hypermutation. Indeed, the effect of the MAR deletion 1987; Scheuermann and Chen, 1989; Pongubala and is even more pronounced than that of Ei removal. Atchison, 1991; Meyer and Ireland, 1994). It will 3990 Somatic mutation of the Igκ locus expression. Signiﬁcantly more of the mutated transgenes were found in the brightly staining subpopulation than in the transgene-dull subpopulation, even though the hypermutation mechanism was fully effective (as judged by mutations at the endogenous IgH locus). How might hypermutation be linked to transcription? The results throw new light on the link between hypermut- ation and transcription. The data suggest that the reduction in hypermutation is more in the targeting of the defective transgene copies for mutation rather than in the extent of mutation accumulation once targeted. Indeed, what seems Fig. 4. Transgene hypermutation in germinal centre B cells that are to be affected is the probability of a particular transgene bright or dull for transgene expression. Peyer’s patches germinal copy becoming committed to the hypermutation pathway. hi centre B-cells [(CD45R)B220 , PNA ] were fractionated further into Reduced mutational targeting not only correlates broadly hi lo subpopulations staining brightly (TG(rat)κ ) and dully (TG(rat)κ ) for with reduced overall expression but, more signiﬁcantly, rat κ; transgene hypermutation was analysed by PCR. Endogenous those B cells within the germinal centres of a single mouse mutation of the region ﬂanking the 3 side of V J558 family member– J 4 integrations was determined as described by Jolly et al. (1997) that stain brightly for transgene expression are more likely using DNA from the fractionated LκΔ[Ei] cell populations (see text). to have targeted the transgene for mutation. Ampliﬁcation of the transgenic V gene was performed using Taq Nevertheless, the correlation is not straightforward. In polymerase for LκΔ[MAR] and Pfu polymerase for LκΔ[Ei]. Pie some transgenic mice (such as LκΔ[MAR] line L3), cis- charts are included to demonstrate the background error rate due to element removal has relatively little effect on expression PCR alone, using these two polymerases as determined using cloned hybridoma controls. (possibly owing to integration position effects) but there is a substantial reduction in hypermutation. This, taken obviously be interesting to ascertain whether the E3 together with the fact that it is mutational targeting (rather ﬂanking region also displays MAR activity. than the accumulation of mutations once targeted) that is affected by cis-element removal, leads us to put forward Correlation between hypermutation and a model in which, whilst mutational targeting is linked to transcription transcription initiation, the recruitment of hypermutation Tinkering with enhancer elements and their ﬂanks gave is critically dependent on the precise nature and quality rise to transgenes which showed signiﬁcant founder-to- of the transcription initiation complex. Thus, removal of a founder variation in behaviour; this is presumably due to cis-element is likely to affect the nature of the transcription position effects which are minimized when the transgenes initiation complex; the change in this complex could then contain their full complement of cis-regulatory elements. differentially affect the recruitment of the hypermutation Nevertheless, the experiments reveal a broad correlation machinery as opposed to the recruitment of RNA poly- between the elements required for transgene hypermutation merase II. and those required for expression, with the removal of a The dependence of hypermutation on the immuno- single cis-element usually having a more dramatic effect globulin enhancers as well as the fact that the mutation on hypermutation than transcription. domain is located downstream of the promoter has led us This broad correlation between expression and mutation and others to propose that some form of transcription- is particularly evident when expression is monitored by coupled repair (Hanawalt, 1995) could form the basis of ﬂow cytometric analysis of transgene expression on the hypermutation (Neuberger and Milstein, 1995; Peters and surface of splenic B cells rather than by Northern blot Storb, 1996). To account for the results presented here, analysis of mRNA accumulation in splenic hybridomas. we propose a model (Figure 5) in which a hypermutating Thus, whereas removal of Ei and/or MAR from the priming factor (HPF), speciﬁc to hypermutating B cells, transgene signiﬁcantly diminished κ expression in the can be recruited during assembly of the immunoglobulin ﬂow cytometric assay, removal of Ei/MAR or MAR gene transcription initiation complex. HPF recruitment is alone had relatively little effect on the accumulation of viewed as being effected by the particular constellation immunoglobulin κ mRNA in terminally differentiated of transcription factors forming the initiation complex but plasma cells (Xu et al., 1989; Meyer et al., 1990, 1996; is not an all or nothing event. Most complete initiation or Betz et al., 1994; data not shown). This contrast probably modiﬁed complexes will recruit HPF, but a small propor- reﬂects that whereas Ei/MAR may regulate κ expression tion may not. In contrast, incomplete initation complexes at the surface Ig B cell stage of development, κ expression (which will form as a consequence of cis-regulatory in terminally differentiated antibody-secreting cells may element disruption and/or positional effects), whilst often be mainly under the control of E3 (Meyer et al., 1990, able to bring in RNA polymerase, will be impaired 1996; Roque et al., 1996). though not wholly incompetent at HPF recruitment. The A quite distinct pointer to the correlation between recruitment of HPF then leads to hypermutation on poly- transcription and hypermutation came from analysing Lκ- merase halting, possibly through the involvement of a derived transgenes which did not fully suppress endo- speciﬁc error-prone repair, as discussed in the legend to genous κ rearrangement. Here we found that germinal Figure 5. centre B cells could be fractionated into a subpopulation Many studies have indicated that, when V genes are that stained brightly and one that stained dully for transgene subjected to hypermutation, relatively small numbers (e.g. 3991 B.Goyenechea et al. Fig. 5. Model for immunoglobulin gene hypermutation. Hypermutation is envisaged as being effected by the recruitment of hypermutating primer factor (HPF) to the RNA polymerase II transcription initiation complex. HPF recruitment is a stochastic event (illustrated with thick and thin arrows) which can be clonally maintained. Thus, a transgene retaining the full complement of transcription regulatory elements is likely (but not certain) to form a complete transcription initiation complex that will recruit HPF; in contrast, a transgene lacking Ei/MAR is more likely to form a partial transcription initiation complex which, whilst able to recruit RNA polymerase II, has a diminished probability of recruiting HPF. We envisage that HPF either accompanies the polymerase during transcription elongation or modiﬁes the elongation complex with hypermutation ensuing, either because there is increased gratuitous polymerase stalling and subsequent transcription-coupled repair (as proposed by Peters and Storb, 1996) or (as illustrated here) because, on stalling, a speciﬁc error-prone repair complex is recruited. one to four) mutations are introduced in a single cell cycle The analysis suggests that there may be stochastically [see, for example, Clarke et al. (1985) as well as papers determined heterogeneity in the extent to which the cited by Kepler and Perelson (1993)]; V genes that have immunoglobulin genes in different B-cell clones [or, accumulated large numbers of nucleotide substitutions indeed, different genes or transgene copies within the have achieved this through being subjected to mutation in same cell (Rogerson et al., 1991; Ye´lamos et al., 1995)] sequential cell cycles. Bearing this in mind, then the fact are targeted for mutation. In both the Peyer’s patch that the mice transgenic for the poorly targeted Lκ deletion germinal centre B-cell population and the antigen-selected derivatives nevertheless contain transgenic V genes carry- B cells from hyperimmune mice, a signiﬁcant proportion ing large numbers of nucleotide substitutions implies that, of clones (~20%) have not targeted their endogenous once a particular transgene is targeted for mutation, it immunoglobulin genes for mutation. This could reﬂect a might well remain marked as hypermutation-accessible strategy for maintaining a B-cell population in which one through subsequent cell divisions. For these reasons, we immunoglobulin locus has been targeted, but not the other, propose that HPF recruitment manifests some clonal or for preserving an unmutated memory B-cell pool. stability. The mechanism of maintaining such stability of Whilst these proposals are clearly speculative, our data association is, of course, a matter for speculation; however, do suggest that the recruitment of mutation is intimately it may be signiﬁcant that there is substantial overlap linked to the recruitment of polymerase II transcription between the elements that we ﬁnd here to be required for complexes, but with hypermutation being more fastidious full mutation and those that have been identiﬁed as playing in its requirement for the recruitment of a complete a role in demethylation (Lichtenstein et al., 1994). complex. 3992 Somatic mutation of the Igκ locus Acknowledgements Materials and methods We thank John Jarvis and Sarah Davies for derivation and testing of the Transgenes and transgenic mice transgenic mice. N.K. is a graduate student of the Institute for Biology The transgenes are all directly based on Lκ (Sharpe et al., 1991) except II, University of Freiburg and supported by an EC training grant. We for LκΔ[Ei]Line L, LκΔ[MAR]Line S, LκΔ[E3-Flank] and LκΔ[E3- are grateful to the National Foundation for Cancer Research and the Core], which are based on the Lκ derivative LκΔB(Ye´lamos et al., Association for International Cancer Research (for grant support to 1995). The long (L) and short (S) deletions of both Ei and MAR were C.M.) and the Howard Hughes Medical Institute (for an International created individually working with either (i) a HindIII–HpaI subclone of Research Scholar’s award to M.S.N.). Lκ in Bluescript and using PCR to create the speciﬁc deletions or (ii) SacII–HpaI subclones of LκΔ[Ei/MAR] in which the Ei/MAR has been deleted and replaced by a HindIII site (Betz et al., 1994), and using PCR to reintroduce either Ei or MAR alone as HindIII fragments. References The variant Ei/MAR subclones were then resected back into Lκ or ´ ´ Betz,A.G., Milstein,C., Gonzalez-Fernandez,A.F., Pannell,R., Larson,T. LκΔB as indicated in the legend to Figure 1. Using the numbering of and Neuberger,M.S. (1994) Elements regulating somatic hyper- Max et al. (1981), which places the ﬁrst A of the intronic HindIII site mutation of an immunoglobulin κ gene: critical role for the intron at position 3395, the sequences across the deletion borders are: ΔEiLineL 3757 4136 enhancer/matrix attachment region. Cell, 77, 239–248. [CTACTT // AAGGCC], using primers MAR1 (5-TATTAAAAG- Clarke,S., Huppi,K., Ruezinsky,D., Staudt,L., Gerhard,W. and CTTAATGTATATTAATC-3) and MAR2 (5-TGACTCTTAAGTAGTT- 3818 4089 Weigert,M.G. (1985) Inter- and intraclonal diversity in the antibody TCAAGAGTT-3); ΔEiLineS [CAATTC /GAAGCT/ GAATTG], response to inﬂuenza hemagglutinin. J. Exp. Med., 161, 6704–6887. using the primers LuLu7 (5-CCCTTGCTCCGCGGGAACCACTTT- Cockerill,P.N. and Garrard,W.T. (1986) Chromosomal loop anchorage CCTGAG-3) and MJS195 (5-CGGAAGCTTCGAATTGACATCATT- 3400 3692 of the κ immunoglobulin gene occurs next to the enhancer in a region TTAAATTAAAAG-3); ΔMARLineS [AAGCTT // TTGTGT], containing topoisomerase II sites. Cell, 44, 273–282. ampliﬁed with IE1 (5-TTTATAAGCTTTTGTGTTTGACCC-3), and Cockerill,P.N., Yuen,M.-H. and Garrard,W.T. (1987) The enhancer of RCKN127 (5-IIIIIIGCGGCCGCGACTGWGGCACCTCCAG-3) and 3400 3842 the immunoglobulin heavy chain locus is ﬂanked by presumptive ΔMARLineL [AAGCTT /C/ GAAAGG], ampliﬁed with MSN196 chromosomal loop anchorage elements. J. Biol. Chem., 262, 5394– (5-CGGAAGCTTCGAAAGGCTGCTCATAATTCTA-3); and MSN197 (5-CGGAAGCTTAAGCCAGGGTCTGTATTTG-3). This last con- Forrester,W.C., van Genderen,C., Jenuwein,T. and Grosschedl,R. (1994) struct also contains a direct tandem duplication of nucleotides 4089– Dependence of enhancer-mediated transcription of the immunoglobulin 4140 as a consequence of the insertion of the PCR fragment into μ gene on nuclear matrix attachment regions. Science, 265, LκΔ[Ei/MAR]; underlined letters represent nucleotides inserted at the 1221–1225. deletion borders. Go´nzalez-Fernandez,A. and Milstein,C. (1993) Analysis of somatic For LκΔ[E3-Flank], the core region of E3 (extending from GAG- hypermutation in mouse Peyer’s patches using immunoglobulin κ TGT to GCCTGG , nucleotide numbering according to the sequence light chain transgenes. Proc. Natl Acad. Sci. USA, 90, 9862–9866 in Meyer and Neuberger, 1989) was PCR ampliﬁed with the oligonucleo- Gorman,J.R., van der Stoep,N., Monroe,R., Cogne´,M., Davidson,L. and tides Dino1 (5-TTATCTCGAGTGTCCCAGTGACCAA-3) and Dino2 Alt,F.W. (1996) The Igκ 3 enhancer inﬂuences the ratio of Igκ versus (5-GGAGTGCGGCCGCCAGGCTGTTGGAGG-3)asan XhoI–NotI Igλ B lymphocytes. Immunity, 5, 241–252. fragment and substituted between the unique XhoI and NotI sites of Goyenechea,B. and Milstein,C. (1996) Modifying the sequence of an LκΔB. For LκΔ[E3-Core], the deletion was created on an XhoI–NotI immunoglobulin V-gene alters the resulting pattern of hypermutation. subclone by religating between blunted NcoI and BspMI sites with the 390 568 Proc. Natl Acad. Sci. USA, 93, 13979–13984. sequence across the deletion border being CCCATG // TACCCC. Hanawalt,P.C. (1995) DNA repair comes of age. Mutat. Res., 336, Transgenic mice were established by microinjection to (C57BL/ 101–113. 6CBA)F1 zygotes. Founders (identiﬁed by tail blotting and serum Imler,J.-L., Lemaire,C., Wasylyk,C. and Wasylyk,B. (1987) Negative enzyme-linked immunosorbent assay) were bred against F1s, and trans- regulation contributes to tissue speciﬁcity of the immunoglobulin gene copy numbers estimated by Southern blotting (10 copies, heavy-chain enhancer. Mol. Cell. Biol., 7, 2558–2567. Δ[MAR]S1, Δ[E3-Flank]2 and Δ[E3-Core]1; eight copies, Δ[MAR]L2 Jolly,C.J., Klix,N. and Neuberger,M.S. (1997) Rapid methods for the and Δ[Ei]L1; six copies, Δ[MAR]L1 and Δ[E3-Core]2; four copies, analysis of immunoglobulin gene hypermutation: application to Δ[Ei]S1; three copies, Δ[Ei]S2; two copies, Δ[MAR]L3 and Δ[E3- transgenic and gene targeted mice. Nucleic Acids Res., 25, 1913–1919. Flank]1). Kepler,T.B. and Perelson,A.S. (1993) Cyclic re-entry of germinal centre B cells and the efﬁciency of afﬁnity maturation. Immunol. Today, 14, Monitoring hypermutation 412–415. Hypermutation was assessed by sequencing the transgenic V Ox-1 after Lichtenstein,M., Keini,G., Cedar,H. and Bergman,Y. (1994) B cell- hi PCR ampliﬁcation from PNA B220 germinal centre B cells that had speciﬁc demethylation: a novel role for the intronic κ chain enhancer been sorted from pooled Peyer’s patches of 4- to 6-month-old animals sequence. Cell, 76, 913–923. as previously described (Go´nzalez-Ferna´ndez and Milstein, 1993). Max,E.E., Maizel,J.V. and Leder,P. (1981) Nucleotide sequence of a 5.5- Hybridization with a transgene-speciﬁc oligonucleotide (Betz et al., kilobase DNA segment containing the mouse κ immunoglobulin J 1994) was used to conﬁrm the transgenic origin of the cloned V Ox-1 and C region genes. J. Biol. Chem., 256, 5116–5120. genes. Mutation of the endogenous IgH locus was monitored by Meyer,K.B. and Ireland,J. (1994) Activation of the immunoglobulin κ3- determining the sequence of the region ﬂanking the 3 border of J 4 enhancer in pre-B cells correlates with the suppression of a nuclear rearrangements of V J558 family members following PCR ampliﬁcation factor binding to a sequence ﬂanking the active core. Nucleic Acids as descibed elsewhere (Jolly et al., 1997). Sorting of germinal centre B Res., 22, 1576–1582. cells of LκΔ[Ei] and LκΔ[MAR] into subpopulations staining brightly Meyer,K.B. and Neuberger,M.S. (1989) The immunoglobulin κ locus and dully for transgenic (rat) κ was performed by pre-incubation with contains a second, stronger B-cell-speciﬁc enhancer which is located biotinylated anti-rat κ monoclonal antibody (R6.7/9.1; Springer et al., downstream of the constant region. EMBO J., 8, 1959–1964. 1982) in the presence of 2% mouse serum prior to washing and staining Meyer,K.B., Sharpe,M.J., Surani,M.A. and Neuberger,M.S. (1990) The for peanut agglutinin (PNA) and CD45R(B220) as previously described importance of the 3-enhancer region in immunoglobulin κ gene (Go´nzalez-Ferna´ndez and Milstein, 1993) but in the presence of Red670- expression. Nucleic Acids Res., 18, 5609–5615. conjugated streptavidin (Gibco). Meyer,K.B., Teh,Y.-M. and Neuberger,M.S. (1996) The Igκ 3-enhancer triggers gene expression in early B lymphocytes but its activity is Monitoring expression enhanced on B cell activation. Int. Immunol., 8, 1561–1568. Flow cytometric assay of transgenic and endogenous mouse κ expression Neuberger,M.S. and Milstein,C. (1995) Somatic hypermutation. Curr. on splenic B cells was performed by staining with a biotinylated Opin. Immunol., 7, 248–254. monoclonal antibody against either mouse (187.1; Yelton et al., 1981) Peters,A. and Storb,U. (1996) Somatic hypermutation of immunoglobulin or rat κ chains followed by washing and incubation with phycoerythrin- genes is linked to transcription initiation. Immunity, 4, 57–65. conjugated RA3-6B2(Gibco) anti-CD45R(B220) antibody and ﬂuores- Picard,D. and Schaffner,W. (1984) A lymphocyte speciﬁc enhancer in cein isothiocyanate (FITC)–streptavidin. the mouse immunoglobulin κ gene. Nature, 307, 80–82. 3993 B.Goyenechea et al. Pongubala,J.M.R. and Atchison,M.L. (1991) Functional characterization of the developmentally controlled kappa 3 enhancer: regulation by Id, a repressor of helix–loop–helix transcription factors. Mol. Cell Biol., 11, 1040–1047. Queen,C. and Stafford,J. (1984) Fine mapping of an immunoglobulin gene activator. Mol. Cell. Biol., 4, 1042–1049. Rogerson,B., Hackett,J., Peters,A., Haasch,D. and Storb,U. (1991) Mutation pattern of immunglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication. EMBO J., 10, 4331–4341. Roque,M.C., Smith,P.A. and Blasquez,V.C. (1996) A developmentally regulated chromatin structure at the mouse immunoglobulin κ 3 enhancer. Mol. Cell. Biol., 16, 3138–3155. Scheuermann,R.H. and Chen,U. (1989) A developmental-speciﬁc factor binds to suppressor sites ﬂanking the immunoglobulin heavy-chain enhancer. Genes Dev., 3, 1255–1266. Sharpe,M.J., Milstein,C., Jarvis,J.M. and Neuberger,M.S. (1991) Somatic hypermutation of immunoglobulin kappa may depend on sequences 3 of Cκ and occurs on passenger transgenes. EMBO J., 10, 2139–2145. Springer,T.A., Battacharya,A., Cardoza,J.T. and Sa´nchez-Madrid,F. (1982) Monoclonal antibodies speciﬁc for rat IgG1, IgG2a and IgG2b subclasses and κ light chain monotypic and allotyopic determinants: reagents for use with rat monoclonal antibodies. Hybridoma, 1, 257–264. Storb,U. (1996) Molecular mechanism of somatic hypermutation of immunoglobulin genes. Curr. Opin. Immunol., 8, 206–214. Weill,J.-C. and Reynaud,C.-A. (1996) Rearrangement/hypermutation/ gene conversion: when, where, why? Immunol. Today, 17, 92–96. Xu,M., Hammer,R.E., Blasquez,V.C., Jones,S.L. and Garrard,W.T. (1989) Immunoglobulin gene expression after stable integration. II. Role of the intronic MAR and enhancer in transgenic mice. J. Biol. Chem., 264, 21190–21195. Xu,Y., Davidson,L., Alt,F.W. and Baltimore,D. (1996) Deletion of the Igκ light chain intronic enhancer/matrix attachment region impairs but does not abolish VκJκ rearrangement. Immunity, 4, 377–385. Ye´lamos,J., Klix,N., Goyenechea,B., Lozano,F., Chui,Y.L., Gonza´lez- Ferna´ndez,A., Pannel,R., Neuberger,M.S. and Milstein,C. (1995) Targeting of non-Ig sequences in place of the V segment by somatic hypermutation. Nature, 376, 225–229. Yelton,D.E., Desaymard,C. and Scharff,M.D. (1981) Use of monoclonal anti-mouse immunoglobulin to detect mouse antibodies. Hybridoma, 1, 5–14. Received on February 24, 1997; revised on March 26, 1997 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals http://www.deepdyve.com/lp/springer-journals/cells-strongly-expressing-ig-transgenes-show-clonal-recruitment-of-Q5h4anQ7HV

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Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1997
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/16.13.3987
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.16 No.13 pp.3987–3994, 1997 Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers and Milstein, 1995; Storb, 1996; Weill and Reynaud, Beatriz Goyenechea, Norman Klix, 1996). Jose´ Ye´lamos, Gareth T.Williams, Experiments to date looking at the requirement for cis- Andrew Riddell, Michael S.Neuberger and 1 acting elements have focused largely on the overall extent Ce´sar Milstein of mutation rather than differentiating between the fre- Medical Research Council Laboratory of Molecular Biology, quency with which a gene is targeted for mutation and Hills Road, Cambridge CB2 2QH, UK the extent of mutant accumulation once so targeted. We were therefore moved to extend our previous work on the Corresponding authors immunoglobulin κ locus (Betz et al., 1994), dissecting B.Goyenechea and N.Klix contributed equally to this work the contributions of the two κ enhancer regions and asking whether the portions necessary for transcription The V regions of immunoglobulin κ transgenes are enhancement corresponded to the portions essential for targets for hypermutation in germinal centre B cells. hypermutation, paying special attention to the distinction We show by use of modiﬁed transgenes that the between mutational targeting and mutation accumulation. recruitment of hypermutation is substantially impaired by deletion of the nuclear matrix attachment region (MAR) which ﬂanks the intron-enhancer (Ei). Results Decreased mutation is also obtained if Ei, the core region of the κ3-enhancer (E3) or the E3-ﬂank are Removal of either MAR or Ei diminishes removed individually. A broad correlation between hypermutation expression and mutation is indicated not only by the In previous work, by comparing V region mutation in Lκ fact that the deletions affecting mutation also give and LκΔ[Ei/MAR] transgenes, we showed that removal reduced transgene expression, but especially by the of a 688 bp region from the J –C intron leads to a very κ κ ﬁnding that, within a single mouse, transgene mutation substantial reduction in hypermutation (Betz et al., 1994). was considerably reduced in germinal centre B cells This deletion takes out both the intron-enhancer (Ei), that poorly expressed the transgene as compared with which has been shown in transfection assays to confer strongly expressing cells. We also observed that the lymphoid-speciﬁc transcriptional enhancement (Picard and diminished mutation in transgenes carrying regulatory Schaffner, 1984; Queen and Stafford, 1984), and the element deletions was manifested by an increased MAR, which was identiﬁed as a nuclear matrix attachment proportion of B cells in which the transgene had not region (Cockerill and Garrard, 1986) but which does not been targeted at all for mutation rather than in the exhibit cell type speciﬁcity and which has not been shown extent of mutation accumulation once targeted. Since to have any major role in transcriptional activation. mutations appear to be incorporated stepwise, the To determine whether it was the removal of Ei or of results point to a connection between transcription MAR that was responsible for the reduction in hypermut- initiation and the clonal recruitment of hypermutation, ation, we prepared Lκ derivatives that were separately with hypermutation being more fastidious than tran- depleted of either element. Two constructs (differing in scription in requiring the presence of a full complement the precise location of the deletion endpoints) were made of regulatory elements. for each type of deletion (Figure 1) and multiple transgenic Keywords: diversity/enhancers/hypermutation/ mouse lines established. immunoglobulin/MAR/somatic mutation Hypermutation was assessed by PCR cloning and sequencing of the transgenic V genes from sorted germinal centre B cells obtained from the mouse Peyer’s patches. Whilst there is signiﬁcant founder-to-founder variation, it Introduction is notable that, compared with the Lκ controls, deletion Somatic hypermutation is a major contributor to antibody of either Ei or MAR on their own is sufﬁcient to give a afﬁnity maturation. In germinal centre B cells, nucleotide dramatic drop in hypermutation (Table I). The effect of substitutions are introduced into a region of several the MAR deletion, which in all four lines examined leads kilobases of DNA that includes the rearranged V gene to mutation rates that are severely diminished or even segments of the immunoglobulin heavy and light chain undetectable above background, is more severe than that loci. The mechanism of hypermutation is unknown, of Ei. although several lines of evidence point to a linkage to The diminished mutation of the LκΔ[Ei] and LκΔ[MAR] transcription. Thus, the process reveals strand polarity, it transgenes is largely attributable to a large decrease in the is inhibited by the removal of cis-acting transcription proportion of transgenic V gene clones that carry any enhancer elements and the mutation domain is located mutations at all. This ﬁgure drops from a range of 65– downstream of the promoter (see reviews by Neuberger 75% in the Lκ control mice to 5–33% in the LκΔ[Ei] and © Oxford University Press 3987 B.Goyenechea et al. Fig. 1. The transgenes. The transgenes are all based on the ancestral constructs Lκ or LκΔB (both of which are good hypermutation targets) as described in Materials and methods; in LκΔB, the region between the BamHI (B) sites downstream of C is deleted as indicated. Lκ is of mouse origin except for a small region (in white) including most of C , which is of rat origin. The extent of the internal deletions in the different constructs is indicated with the numbering following Max et al. (1981) in the J –C intron and Meyer and Neuberger (1989) around the 3-enhancer. Other κ κ restriction sites are abbreviated H, HindIII; Hf, HinfI (not all sites depicted); Hp, HpaI; R, EcoRI; N, a NotI linker that has been inserted into an EcoRI site; S, SacI; and X, XhoI. Various factor-binding sites within the enhancers and MAR are indicated. LκΔ[MAR] animals. Furthermore, the few LκΔ[Ei] and 1994). Transgenic mice were established to discriminate LκΔ[MAR] transgene copies that do get targeted for the relative roles of the core and ﬂank in mutational mutation are still able to accumulate multiple nucleotide targeting. Whilst again the different founder lines mutate substitutions (Figure 2). If we restrict our analysis to to different extents, it is clear that deletion of either mutated sequences, then the average number of substitu- the core or the ﬂank results in diminished targeting of tions accumulated in the mutated LκΔ[Ei] and LκΔ[MAR] hypermutation (Table I and Figure 2). As with the Ei and V genes is only reduced ~2-fold compared with the MAR deletions, whilst there is a decrease in the number number of substitutions accumulated on the parental Lκ of transgene copies that are targeted for mutation, those transgene (Table I). These effects are speciﬁc to the that are so targeted can still accumulate multiple nucleotide transgenes themselves since they are observed in cells substitutions. that otherwise retain their normal mutational capacity. Thus, sequence analysis of PCR-ampliﬁed clones derived Deletions affect κ expression from the 3-ﬂank of rearrangements of V J558 family The expression of the various transgenes was monitored members to J 4 reveals that LκΔ[Ei], LκΔ[MAR] and by ﬂow cytometric analysis of splenic B cells using an control mice are similar with respect to the accumulation anti-rat κ antibody, exploiting the fact that the C exon of mutations in their endogenous heavy chain loci (8.7, (but not the remainder) of the various transgenic constructs 14 and 9.1 substitutions/kb respectively determined as is of rat origin. Even amongst the B cells of a single described in Materials and methods). transgenic mouse, there is heterogeneity with respect to Thus, both the Ei and, particularly, the MAR deletions transgene expression on the B-cell surface, with the effect a severe inhibition of the targeting of the transgene transgenic κ being in competition with endogenous mouse for hypermutation; it remains possible that, in addition, κ in those cells in which κ gene expression is not allelically these same deletions also effect a small inhibition of excluded (Figure 3). Whereas the Lκ construct expresses mutation accumulation once the transgenic V gene has well in four independent lines with few spleen cells been targeted. expressing endogenous mouse κ, this same dominance is not observed with the Lκ derivatives in which Ei, parts Removal of either E3-core or E3-ﬂank diminishes of E3 or MAR have been removed (Table II). The level hypermutation of transgene expression broadly correlates with the level The E3, which has also been shown to be important for of hypermutation, although the correlation is not a straight- mutational targeting (Betz et al., 1994), is composed of a forward one. core enhancer region surrounded by a conserved ﬂank, In contrast to these ﬂow cytometric results, however, which can suppress the activity of the core in pre-B cell no decrease in transgene expression effected by the Ei lines (Pongubala and Atchison, 1991; Meyer and Ireland, deletion is evident if expression is estimated by the 3988 Somatic mutation of the Igκ locus Table I. Mutation of the transgenes Mouse line Clones Mutations Mutations/10 bp total All Mutated All Mutated clones clones Lκ Line 3 76 53 267 12.4 20.0 Line 6 73 54 262 12.7 17.2 Line NG 59 43 197 11.8 16.2 Line WTM7 88 52 184 7.4 12.5 LκΔ[Ei/MAR] 75 10 16 0.8 LκΔ[3E] 37 13 23 2.2 6.3 LκΔ[MAR]S Line 1 42 2 2 0.2 LκΔ[MAR]L Line 1 61 10 23 1.3 8.1 Line 2 63 14 34 1.9 8.6 Line 3 42 2 5 0.4 LκΔ[Ei]S Line 1 75 19 48 2.3 9.0 Line 2 60 20 49 3.0 8.7 LκΔ[Ei]L Line 1 225 43 161 2.5 13.3 LκΔ[E3-Flank] Line 1 63 6 9 0.5 Line 2 101 27 70 2.3 9.1 LκΔ[E3-Core] Line 1 98 25 41 1.5 5.8 Line 2 42 13 19 1.7 Too few mutated clones for meaningful calculation. The Vκ segment of each transgene was cloned following PCR ampliﬁcation from sorted germinal centre B cells. For each transgene, the table gives the total number of PCR clones sequenced, the number of those that carried one or more mutations within the V segment Fig. 2. Frequency distribution of clones with respect to the number of (282 bp), the total number of mutations identiﬁed and the mutation mutations they carry. In each pie chart, the size of each segment is a frequency. This frequency (point mutations per 10 bp) is computed measure of the proportion of clones that carry the indicated number of both with respect to all clones analysed and with respect to only those mutations. For each type of transgenic construct (grouped simply as clones that carry mutations. For several transgenic lines, mutation was Lκ, Δ[MAR], Δ[Ei], Δ[E3-Core] and Δ[E3-Flank]), the results of the analysed in multiple individual animals. The variation in mutation analysis of the individual animals presented in Table I are pooled rates between animals was lower than that found when comparing together and presented as a single pie chart. different mouse lines that carried the same transgene. The data for the Lκ,LκΔ[Ei/MAR] and LκΔ[3E]transgenes are taken from Betz et al. (1994), Gonza´lez-Ferna´ndez and Milstein (1993), Ye´lamos et al. (1995) and Goyenechea and Milstein (1996). Only the unmodiﬁed Lκ transgenes in the NG and WTM7 mice are used in the computation. mouse was more efﬁcient in those cells which expressed the transgene highly than in those of the transgene-dull population. Fractionation of Peyer’s patch B cells from abundance of transgene mRNA in transgene-positive LκΔ[Ei] and LκΔ[MAR] mice into populations that were hi splenic B cell hybridomas (data not shown). This parallels either bright or dull for transgenic κ expression (TGκ lo previous observations with the LκΔ[Ei/MAR] transgene and TGκ ) revealed that most mutated transgenes were where the simultaneous removal of both Ei and MAR had found amongst the more brightly expressing cells (Figure little effect on the abundance of κ mRNA in hybridomas 4). Although transgene hypermutation was much dimin- but led to a signiﬁcant drop in transgene expression, as ished in the transgene-dull population, the endogenous well as exclusion of endogenous κ expression as judged heavy chain locus was still at least as well mutated; the by ﬂow-cytometric analysis of splenic B cells (Betz et al., mutation frequency in the 3-ﬂank of V J558 family 1994; Meyer et al., 1996). Thus, the ﬂow-cytometric member–J 4 integrations was six substitutions/kb in the monitor of expression on the B-cell surface is a better transgene-bright LκΔ[MAR] germinal centre B cell sub- correlate of hypermutability. population and 16/kb in the transgene-dull subpopulation. Thus, the decreased efﬁciency of mutational targeting of Most transgene mutations are found in those the transgene in the transgene-dull subpopulation does not B cells strongly expressing the transgene reﬂect any impairment of the hypermutation capability of Given this correlation between hypermutation and cell the cells themselves; the somewhat increased mutation of surface expression of the transgene, we wondered whether the endogenous loci in these cells is a topic for further the targeting of transgene hypermutation in a particular investigation. 3989 B.Goyenechea et al. Table II. Transgenic and endogenous κ chain expression Mouse line Rat-κ Mouse-κ Mutations/ 10 bp Non-transgenic 0 100 – Lκ Line 6 100 0 12.7 Line NG 95 5 11.8 Line WTM7 94 11 7.4 tLκΔ[Ei/MAR] 55 41 0.8 LκΔ[E3] 4 72 2.2 LκΔ[MAR]S Line 1 8 91 0.2 LκΔ[MAR]L Line 1 25 54 1.3 Line 2 25 61 1.9 Line 3 70 14 0.4 LκΔ[Ei]S Line 1 59 42 2.3 Line 2 70 29 3.0 LκΔ[Ei]L Line 1 45 53 2.5 LκΔ[E3-Flank] Line 1 17 69 0.5 LκΔ[E3-Core] Line 1 28 42 1.5 Line 2 4 96 1.7 Igκ expression on the surface of splenic B cells was determined by cytoﬂuorimetry on at least two animals for each transgenic line. The values (mean κ ﬂuorescence on B220 cells) are normalized with respect to Lκ6 transgenic and non-transgenic litter mates, giving these control lines values of 100 and 0 for transgenic/endogenous κ expression as indicated. Hypermutation frequencies, taken from Table I, are shown for comparison. The critical importance of the MAR was not anticipated. Whilst originally deﬁned by an in vitro nuclear matrix binding assay (Cockerill and Garrard, 1986), no major functional importance for the MAR is apparent from a deletion analysis of the κ intronic enhancer performed using transfection into lymphoid cell lines (Queen and Stafford, 1984). This might, in part, reﬂect an inadequacy of transfection assays in revealing all sequences that support enhancer activity. Thus, the region ﬂanking the core of the IgH intronic enhancer [which also possesses MAR activity (Cockerill et al., 1987)] was found to be Fig. 3. Transgene expression. Expression was analysed by cytoﬂuorimetric analysis staining of splenic B cells with critical for the expression of IgH transgenes although phycoerythrin-conjugated anti-CD45R(B220) and either mouse dispensable for the activity of the enhancer in cell line (endogenous) or rat (transgenic) κ biotinylated mAbs and transfection assays (Forrester et al., 1994). An ability of FITC-conjugated streptavidin. Ei/MAR and E3 to partially cross-substitute for each other in some functions, as suggested by in vivo gene Discussion targeting experiments (Gorman et al., 1996; Xu et al., MAR and E3-ﬂank are essential for full 1996), might explain the fact that we can detect consider- hypermutation able residual κ transgene expression after deletion of the The results described here further link hypermutation and intronic MAR. expression. Previous results have shown the importance With regard to the requirement for the E3 ﬂanking of the Ei/MAR and E3 regions for immunoglobulin κ region, this region (like that ﬂanking the IgH intronic hypermutation (Betz et al., 1994). The dissection of the enhancer), whilst having no enhancer activity of its own, contributions of the core enhancers and their ﬂanks reveals has been shown to be able to regulate the activity of a that the ﬂanks are also essential for the proper recruitment linked enhancer in appropriate cell types (Imler et al., of hypermutation. Indeed, the effect of the MAR deletion 1987; Scheuermann and Chen, 1989; Pongubala and is even more pronounced than that of Ei removal. Atchison, 1991; Meyer and Ireland, 1994). It will 3990 Somatic mutation of the Igκ locus expression. Signiﬁcantly more of the mutated transgenes were found in the brightly staining subpopulation than in the transgene-dull subpopulation, even though the hypermutation mechanism was fully effective (as judged by mutations at the endogenous IgH locus). How might hypermutation be linked to transcription? The results throw new light on the link between hypermut- ation and transcription. The data suggest that the reduction in hypermutation is more in the targeting of the defective transgene copies for mutation rather than in the extent of mutation accumulation once targeted. Indeed, what seems Fig. 4. Transgene hypermutation in germinal centre B cells that are to be affected is the probability of a particular transgene bright or dull for transgene expression. Peyer’s patches germinal copy becoming committed to the hypermutation pathway. hi centre B-cells [(CD45R)B220 , PNA ] were fractionated further into Reduced mutational targeting not only correlates broadly hi lo subpopulations staining brightly (TG(rat)κ ) and dully (TG(rat)κ ) for with reduced overall expression but, more signiﬁcantly, rat κ; transgene hypermutation was analysed by PCR. Endogenous those B cells within the germinal centres of a single mouse mutation of the region ﬂanking the 3 side of V J558 family member– J 4 integrations was determined as described by Jolly et al. (1997) that stain brightly for transgene expression are more likely using DNA from the fractionated LκΔ[Ei] cell populations (see text). to have targeted the transgene for mutation. Ampliﬁcation of the transgenic V gene was performed using Taq Nevertheless, the correlation is not straightforward. In polymerase for LκΔ[MAR] and Pfu polymerase for LκΔ[Ei]. Pie some transgenic mice (such as LκΔ[MAR] line L3), cis- charts are included to demonstrate the background error rate due to element removal has relatively little effect on expression PCR alone, using these two polymerases as determined using cloned hybridoma controls. (possibly owing to integration position effects) but there is a substantial reduction in hypermutation. This, taken obviously be interesting to ascertain whether the E3 together with the fact that it is mutational targeting (rather ﬂanking region also displays MAR activity. than the accumulation of mutations once targeted) that is affected by cis-element removal, leads us to put forward Correlation between hypermutation and a model in which, whilst mutational targeting is linked to transcription transcription initiation, the recruitment of hypermutation Tinkering with enhancer elements and their ﬂanks gave is critically dependent on the precise nature and quality rise to transgenes which showed signiﬁcant founder-to- of the transcription initiation complex. Thus, removal of a founder variation in behaviour; this is presumably due to cis-element is likely to affect the nature of the transcription position effects which are minimized when the transgenes initiation complex; the change in this complex could then contain their full complement of cis-regulatory elements. differentially affect the recruitment of the hypermutation Nevertheless, the experiments reveal a broad correlation machinery as opposed to the recruitment of RNA poly- between the elements required for transgene hypermutation merase II. and those required for expression, with the removal of a The dependence of hypermutation on the immuno- single cis-element usually having a more dramatic effect globulin enhancers as well as the fact that the mutation on hypermutation than transcription. domain is located downstream of the promoter has led us This broad correlation between expression and mutation and others to propose that some form of transcription- is particularly evident when expression is monitored by coupled repair (Hanawalt, 1995) could form the basis of ﬂow cytometric analysis of transgene expression on the hypermutation (Neuberger and Milstein, 1995; Peters and surface of splenic B cells rather than by Northern blot Storb, 1996). To account for the results presented here, analysis of mRNA accumulation in splenic hybridomas. we propose a model (Figure 5) in which a hypermutating Thus, whereas removal of Ei and/or MAR from the priming factor (HPF), speciﬁc to hypermutating B cells, transgene signiﬁcantly diminished κ expression in the can be recruited during assembly of the immunoglobulin ﬂow cytometric assay, removal of Ei/MAR or MAR gene transcription initiation complex. HPF recruitment is alone had relatively little effect on the accumulation of viewed as being effected by the particular constellation immunoglobulin κ mRNA in terminally differentiated of transcription factors forming the initiation complex but plasma cells (Xu et al., 1989; Meyer et al., 1990, 1996; is not an all or nothing event. Most complete initiation or Betz et al., 1994; data not shown). This contrast probably modiﬁed complexes will recruit HPF, but a small propor- reﬂects that whereas Ei/MAR may regulate κ expression tion may not. In contrast, incomplete initation complexes at the surface Ig B cell stage of development, κ expression (which will form as a consequence of cis-regulatory in terminally differentiated antibody-secreting cells may element disruption and/or positional effects), whilst often be mainly under the control of E3 (Meyer et al., 1990, able to bring in RNA polymerase, will be impaired 1996; Roque et al., 1996). though not wholly incompetent at HPF recruitment. The A quite distinct pointer to the correlation between recruitment of HPF then leads to hypermutation on poly- transcription and hypermutation came from analysing Lκ- merase halting, possibly through the involvement of a derived transgenes which did not fully suppress endo- speciﬁc error-prone repair, as discussed in the legend to genous κ rearrangement. Here we found that germinal Figure 5. centre B cells could be fractionated into a subpopulation Many studies have indicated that, when V genes are that stained brightly and one that stained dully for transgene subjected to hypermutation, relatively small numbers (e.g. 3991 B.Goyenechea et al. Fig. 5. Model for immunoglobulin gene hypermutation. Hypermutation is envisaged as being effected by the recruitment of hypermutating primer factor (HPF) to the RNA polymerase II transcription initiation complex. HPF recruitment is a stochastic event (illustrated with thick and thin arrows) which can be clonally maintained. Thus, a transgene retaining the full complement of transcription regulatory elements is likely (but not certain) to form a complete transcription initiation complex that will recruit HPF; in contrast, a transgene lacking Ei/MAR is more likely to form a partial transcription initiation complex which, whilst able to recruit RNA polymerase II, has a diminished probability of recruiting HPF. We envisage that HPF either accompanies the polymerase during transcription elongation or modiﬁes the elongation complex with hypermutation ensuing, either because there is increased gratuitous polymerase stalling and subsequent transcription-coupled repair (as proposed by Peters and Storb, 1996) or (as illustrated here) because, on stalling, a speciﬁc error-prone repair complex is recruited. one to four) mutations are introduced in a single cell cycle The analysis suggests that there may be stochastically [see, for example, Clarke et al. (1985) as well as papers determined heterogeneity in the extent to which the cited by Kepler and Perelson (1993)]; V genes that have immunoglobulin genes in different B-cell clones [or, accumulated large numbers of nucleotide substitutions indeed, different genes or transgene copies within the have achieved this through being subjected to mutation in same cell (Rogerson et al., 1991; Ye´lamos et al., 1995)] sequential cell cycles. Bearing this in mind, then the fact are targeted for mutation. In both the Peyer’s patch that the mice transgenic for the poorly targeted Lκ deletion germinal centre B-cell population and the antigen-selected derivatives nevertheless contain transgenic V genes carry- B cells from hyperimmune mice, a signiﬁcant proportion ing large numbers of nucleotide substitutions implies that, of clones (~20%) have not targeted their endogenous once a particular transgene is targeted for mutation, it immunoglobulin genes for mutation. This could reﬂect a might well remain marked as hypermutation-accessible strategy for maintaining a B-cell population in which one through subsequent cell divisions. For these reasons, we immunoglobulin locus has been targeted, but not the other, propose that HPF recruitment manifests some clonal or for preserving an unmutated memory B-cell pool. stability. The mechanism of maintaining such stability of Whilst these proposals are clearly speculative, our data association is, of course, a matter for speculation; however, do suggest that the recruitment of mutation is intimately it may be signiﬁcant that there is substantial overlap linked to the recruitment of polymerase II transcription between the elements that we ﬁnd here to be required for complexes, but with hypermutation being more fastidious full mutation and those that have been identiﬁed as playing in its requirement for the recruitment of a complete a role in demethylation (Lichtenstein et al., 1994). complex. 3992 Somatic mutation of the Igκ locus Acknowledgements Materials and methods We thank John Jarvis and Sarah Davies for derivation and testing of the Transgenes and transgenic mice transgenic mice. N.K. is a graduate student of the Institute for Biology The transgenes are all directly based on Lκ (Sharpe et al., 1991) except II, University of Freiburg and supported by an EC training grant. We for LκΔ[Ei]Line L, LκΔ[MAR]Line S, LκΔ[E3-Flank] and LκΔ[E3- are grateful to the National Foundation for Cancer Research and the Core], which are based on the Lκ derivative LκΔB(Ye´lamos et al., Association for International Cancer Research (for grant support to 1995). The long (L) and short (S) deletions of both Ei and MAR were C.M.) and the Howard Hughes Medical Institute (for an International created individually working with either (i) a HindIII–HpaI subclone of Research Scholar’s award to M.S.N.). Lκ in Bluescript and using PCR to create the speciﬁc deletions or (ii) SacII–HpaI subclones of LκΔ[Ei/MAR] in which the Ei/MAR has been deleted and replaced by a HindIII site (Betz et al., 1994), and using PCR to reintroduce either Ei or MAR alone as HindIII fragments. References The variant Ei/MAR subclones were then resected back into Lκ or ´ ´ Betz,A.G., Milstein,C., Gonzalez-Fernandez,A.F., Pannell,R., Larson,T. LκΔB as indicated in the legend to Figure 1. Using the numbering of and Neuberger,M.S. (1994) Elements regulating somatic hyper- Max et al. (1981), which places the ﬁrst A of the intronic HindIII site mutation of an immunoglobulin κ gene: critical role for the intron at position 3395, the sequences across the deletion borders are: ΔEiLineL 3757 4136 enhancer/matrix attachment region. Cell, 77, 239–248. [CTACTT // AAGGCC], using primers MAR1 (5-TATTAAAAG- Clarke,S., Huppi,K., Ruezinsky,D., Staudt,L., Gerhard,W. and CTTAATGTATATTAATC-3) and MAR2 (5-TGACTCTTAAGTAGTT- 3818 4089 Weigert,M.G. (1985) Inter- and intraclonal diversity in the antibody TCAAGAGTT-3); ΔEiLineS [CAATTC /GAAGCT/ GAATTG], response to inﬂuenza hemagglutinin. J. Exp. Med., 161, 6704–6887. using the primers LuLu7 (5-CCCTTGCTCCGCGGGAACCACTTT- Cockerill,P.N. and Garrard,W.T. 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(1994) struct also contains a direct tandem duplication of nucleotides 4089– Dependence of enhancer-mediated transcription of the immunoglobulin 4140 as a consequence of the insertion of the PCR fragment into μ gene on nuclear matrix attachment regions. Science, 265, LκΔ[Ei/MAR]; underlined letters represent nucleotides inserted at the 1221–1225. deletion borders. Go´nzalez-Fernandez,A. and Milstein,C. (1993) Analysis of somatic For LκΔ[E3-Flank], the core region of E3 (extending from GAG- hypermutation in mouse Peyer’s patches using immunoglobulin κ TGT to GCCTGG , nucleotide numbering according to the sequence light chain transgenes. Proc. Natl Acad. Sci. USA, 90, 9862–9866 in Meyer and Neuberger, 1989) was PCR ampliﬁed with the oligonucleo- Gorman,J.R., van der Stoep,N., Monroe,R., Cogne´,M., Davidson,L. and tides Dino1 (5-TTATCTCGAGTGTCCCAGTGACCAA-3) and Dino2 Alt,F.W. (1996) The Igκ 3 enhancer inﬂuences the ratio of Igκ versus (5-GGAGTGCGGCCGCCAGGCTGTTGGAGG-3)asan XhoI–NotI Igλ B lymphocytes. Immunity, 5, 241–252. fragment and substituted between the unique XhoI and NotI sites of Goyenechea,B. and Milstein,C. (1996) Modifying the sequence of an LκΔB. For LκΔ[E3-Core], the deletion was created on an XhoI–NotI immunoglobulin V-gene alters the resulting pattern of hypermutation. subclone by religating between blunted NcoI and BspMI sites with the 390 568 Proc. Natl Acad. Sci. USA, 93, 13979–13984. sequence across the deletion border being CCCATG // TACCCC. Hanawalt,P.C. (1995) DNA repair comes of age. Mutat. Res., 336, Transgenic mice were established by microinjection to (C57BL/ 101–113. 6CBA)F1 zygotes. Founders (identiﬁed by tail blotting and serum Imler,J.-L., Lemaire,C., Wasylyk,C. and Wasylyk,B. (1987) Negative enzyme-linked immunosorbent assay) were bred against F1s, and trans- regulation contributes to tissue speciﬁcity of the immunoglobulin gene copy numbers estimated by Southern blotting (10 copies, heavy-chain enhancer. Mol. Cell. Biol., 7, 2558–2567. Δ[MAR]S1, Δ[E3-Flank]2 and Δ[E3-Core]1; eight copies, Δ[MAR]L2 Jolly,C.J., Klix,N. and Neuberger,M.S. (1997) Rapid methods for the and Δ[Ei]L1; six copies, Δ[MAR]L1 and Δ[E3-Core]2; four copies, analysis of immunoglobulin gene hypermutation: application to Δ[Ei]S1; three copies, Δ[Ei]S2; two copies, Δ[MAR]L3 and Δ[E3- transgenic and gene targeted mice. Nucleic Acids Res., 25, 1913–1919. Flank]1). Kepler,T.B. and Perelson,A.S. (1993) Cyclic re-entry of germinal centre B cells and the efﬁciency of afﬁnity maturation. Immunol. Today, 14, Monitoring hypermutation 412–415. Hypermutation was assessed by sequencing the transgenic V Ox-1 after Lichtenstein,M., Keini,G., Cedar,H. and Bergman,Y. (1994) B cell- hi PCR ampliﬁcation from PNA B220 germinal centre B cells that had speciﬁc demethylation: a novel role for the intronic κ chain enhancer been sorted from pooled Peyer’s patches of 4- to 6-month-old animals sequence. Cell, 76, 913–923. as previously described (Go´nzalez-Ferna´ndez and Milstein, 1993). Max,E.E., Maizel,J.V. and Leder,P. (1981) Nucleotide sequence of a 5.5- Hybridization with a transgene-speciﬁc oligonucleotide (Betz et al., kilobase DNA segment containing the mouse κ immunoglobulin J 1994) was used to conﬁrm the transgenic origin of the cloned V Ox-1 and C region genes. J. Biol. Chem., 256, 5116–5120. genes. Mutation of the endogenous IgH locus was monitored by Meyer,K.B. and Ireland,J. 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Immunol. Today, 17, 92–96. Xu,M., Hammer,R.E., Blasquez,V.C., Jones,S.L. and Garrard,W.T. (1989) Immunoglobulin gene expression after stable integration. II. Role of the intronic MAR and enhancer in transgenic mice. J. Biol. Chem., 264, 21190–21195. Xu,Y., Davidson,L., Alt,F.W. and Baltimore,D. (1996) Deletion of the Igκ light chain intronic enhancer/matrix attachment region impairs but does not abolish VκJκ rearrangement. Immunity, 4, 377–385. Ye´lamos,J., Klix,N., Goyenechea,B., Lozano,F., Chui,Y.L., Gonza´lez- Ferna´ndez,A., Pannel,R., Neuberger,M.S. and Milstein,C. (1995) Targeting of non-Ig sequences in place of the V segment by somatic hypermutation. Nature, 376, 225–229. Yelton,D.E., Desaymard,C. and Scharff,M.D. (1981) Use of monoclonal anti-mouse immunoglobulin to detect mouse antibodies. Hybridoma, 1, 5–14. Received on February 24, 1997; revised on March 26, 1997

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The EMBO Journal – Springer Journals

Published: Jul 1, 1997

Keywords: diversity; enhancers; hypermutation; immunoglobulin; MAR; somatic mutation

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Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

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Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

Cells strongly expressing Igκ transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers

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