Eur. J. Immunol. 1991. 21: 1499-1504
Piona Dariavachv, Gareth T. Williams, Kathryn Campbell, Sven Pettersson. and Michael S. Neuberger MRC Laboratory of Molecular Biology, Cambridge and Center for Biotechnology., Karolinska Institute, Huddinge
Mouse IgH 3’-enhancer
1499
The mouse IgH 3’-enhancer A lymphoid-specific transcription enhancer element has recently been identified at the far 3’ end of the rat immunoglobulin heavy chain (IgH) locus. Sequence analysis presented here reveals that this enhancer is flanked by a 350-bp invert repeat, giving a structure reminiscent of a transposable element. We therefore screened for the equivalent enhancer in the mouse to determine whether its presence was conserved during evolution. A mouse homologue was indeed identified and is located 16 kb downstream of the C,1 exon. It is also flanked by invert repeats and these are not repeated throughout the genome.The mouse and rat enhancers retain high sequence homology. As regard activity, the IgH 3’-enhancer is lymphoid specific. However, this activity was detected in two plasmacytoma lines tested but not in two B cell lymphomas nor in HeLa cells suggesting that the enhancer may only play a stage-specific role during lymphocyte differentiation. As regards function within the IgH locus, we found that inclusion of the mouse IgH 3’-enhancer (in addition to the intron-enhancer) on p gene expression plasmids effected a small increase in mRNA levels in stable plasmacytoma transfectants.
1 Introduction The expression of immunoglobulin genes is controlled by lymphoid-specificenhancer elements acting in concert with lymphoid-specific promoters. In the case of the x locus, a moderately weak enhancer was initially identified in the J,-C, intron [ l , 21; subsequently, a second, stronger enhancer was discovered 3‘ of the C, exon [3]. It transpires that it is likely to be the x3’-enhancer which plays a critical role in ensuring the high level expression of rearranged x genes [4, 51. The function of the intron-enhancer is less clear; it could, for example, be involved in the regulation of V,-J, joining. In the case of the H chain locus, there is also a lymphoidspecific intron-enhancer and lymphoid-specific VH gene promoters, although the IgH intron-enhancer appears considerably stronger than the x intron-enhancer. Nevertheless, several lines of evidence led us to look for additional enhancer(s) in the IgH locus. First, rearranged mouse IgH genes which have deleted their IgH intronenhancer can nevertheless retain full transcriptional activity [6-91. Second, many IgH transgenesare not expressed at the same high level as the endogenous IgH gene ([lo] and references therein). Third, the c-myc proto-oncogene can become activated on translocation into the IgH locus without necessarily becoming linked to the IgH intronenhancer [ 111. Since we had available to us a set of cosmids covering the rat IgH locus, it wasin the rat that we originally identified the IgH 3’-enhancer [12]. However, unlike the situation in the mous x locus, it has not yet been ascertained whether the IgH 3‘-enhancer does in fact play a role in IgH gene transcription. Here we show that the rat IgH 3’-enhancer is flanked by a nearly perfectly matched invert repeat sequence. The
similarity of such a structure to that of a transposon led us to ask whether the IgH 3’-enhancer identified in the rat has been conserved during evolution. We have, therefore, now isolated the mouse IgH 3’-enhancer and show here that it is homologous to that of the rat both as regards sequence and organization although the enhancer is present in the two species in opposite orientations. We further show that its inclusion stimulates expression of rearranged p genes that have been stably transfected into a plasmacytoma host. Interestingly, however, while the enhancer is also active in two plasmacytoma lines when tested by transient transfection assays, we found no such activity in two B cell lymphomas tested. This suggests that the enhancer may only play a role during the later stages of lymphocyte development.
2 Materials and methods 2.1 Phage and cosmid libraries and DNA sequence determination A phage library generated by cloning a partial Sau3AI digest of BALB/c mouse liver DNA into the vector A2001 was a gift from Franco Calabi. Filters of a cosmid library (Clontech, Palo Alto, CA) generated by cloning sheared BALB/c mouse liver DNA into the Bam HI site of cosmid pWE15 were a gift from Lai-chu Wu. DNA of cosmid Cos37-1-1 which includes rat C, and the rat IgH 3’enhancer [13] was a gift from M. Briiggemann (AFRC, Babraham, Cambridge). DNA sequencing was carried out by the chain termination method using Sequenase (USB, Cleveland, OH). 2.2 Cell l i e s and transfections and enhancer assays
MPC 11 3U4 (obtained from B. Wasylyk, LGME, Stras[I 92221’ bourg), NSO [14], HeLa (from our laboratory collection), Permanent address: Laboratoire d’ImmunogenCtique Mol15cu- HOPC-1 [15], A20 and M12 cells [16] were cultured in laire, URA CNRS 1191, Universitk Montpellier 11, Place E. DMEM/10% FCS containing 50 p~ 2-ME. Bataillon, F-34095 Montpellier Cedex 5, France Correspondence: Michael S. Neuberger, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, GB
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Transient transfection of MPCll BU4 plasmacytoma or HeLa cells was performed by calcium phosphate coprecip0014-2980/91/0606-1499$3.50+ .25/0
P. Dariavach. G. T. Williams. K. Campbell et al.
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Eur. J. Irnmunol. 1991. 21: 1499-1504
3 Results
itation [17] using 20 pg of test plasmid and 5 pg of internal reference. For HOPC-1, A20and M12, DEAE-dextran was used to mediate transfection followed by a shock with 10% DMSO as previously described [ 18J.The test plasmids were constructed from pUC12 derivatives that contained the human P-globin gene from 128 nucleotides upstream of the transcription start to the Pst I site downstream of the coding sequence cloned between the Xba I and Pst I sites of the polylinker. Enhancer fragments to be assayed were introduced between the Sac1 and XbaI sites upstream of the gene. The internal reference was provided by plasmid nSVHPa2 [19] which contains the human a*-globin gene. Total cytoplasmic RNA was prepared from cells 30-36 h after transfection and assayed by ribonuclease protection assays as previously described [3].
3.1 The rat IgH 3'-enhancer is flanked by invert repeats Preliminary evidence that the rat IgH 3'-enhancer is flanked by an invert repeat came from analysis of an M13 subclone of the enhancer; digestion of the single-stranded DNAwith nuclease S1 yielded a resistant fragment of about 310 bp (data not shown).The sequence of the Stu I-Eco RV fragment of the rat 3'-enhancer has been determined previously [ 121.We therefore determined the sequence of a more extensive region and the new sequence (see Fig. 3) does indeed confirm the existence of flanking invert repeats (Fig. 1). These repeats show only 16 mismatches over a 357-bp stretch (> 95% identity).
Transfectants of NSO that carry a long or short transfected x gene in a pSV2neo-based vector (Lx or S x ; constructs 1 and 4) have been described previously [ 5 ] ; these cells were super-transfected with pSV2gpt-based plasmids that contained a rearranged mouse p gene (pSV-V,1; [20]) or a derivative of pSV-V,1 which includes the Stu I fragment of the mouse IgH 3'-enhancer cloned into the plasmid's unique Xho I site.
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3.2 Isolation and sequence of the mouse IgH 3'-enhancer This unusual structure, which is reminiscent of that of a transposon, led us to ask whether the location of the enhancer was conserved in rat and mouse. A mouse liver library in bacteriophage h was, therefore, screened using as
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Figure 1. (A) Alignment of the 5'-repeat with the invert of the 3'-repeat of the rat enhancer. Nuclcotide positions are numbered as in Fig. 3 and positions of identity arc indicated by an asterisk. (B) Invert repeats in the rat IgH 3'-enhancer. Sequences of at least 12 bases of invert identity are joined by a line. The enhancer core lies within the Stu I-Eco RV fragment (indicated by a line) whose sequence was determined previously [ S ] .(C) Invert repeats in the mouse IgH 3'-enhancer. (D) Southern blot of BamHI-digested genomic mouse DNA hybridized with a probe that includes the repeat flanking the mouse IgH3'-enhancer (the EcoRI-Apa LI fragment depicted asprobe A in Fig. 4; nucleotides 55-748 in the mouse IgH 3'-enhancer sequence). DNA was either from mouse liver or from thc MOPC315 or J558L plasmacytornas. The filter was washed at 65 "C in 2 x SSC.
Eur. J. lmmunol. 1991. 21: 1490-1504 lOKb
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Figure 2. Map of the mouse IgH locus and of regions present in cosmid and phage clones.
a probe an Acc I-Bgl I subfragment from the core of the rat 3’-enhancer (nucleotides 432-748 in the rat sequence: see Fig. 4). Two overlapping groups of clones (AM2 and h M 3 ) were isolated. The restriction map of hM2 is depicted in Fig. 2; phage k M 3 extends 3‘ of hM2. The XbaI fragment of hM2 that contained the enhancer homology was subcloned into plasmid pIC20H; a processive set of deletions was made using exonuclease 111 and subcloned into M13. The sequence of the mouse IgH 3’-enhancer was determined and is shown in Fig. 3 where it is aligned with that of the rat. The enhancer of the two species show good homology although the [GT],[(G or C)A]g, repeat of the rat is not observed in the mouse. Furthermore, while the invert repeats that flank the rat IgH 3‘-enhancer show a 96% identity, the internal 250 nucleotides of the equivalent mouse repeats manifest only 89% identity (Fig. 1C). The octanucleotide element ATTTGCAT is conserved in rat and mouse and it is also notable that the mouse enhancer (position 1346) contains the hexamer motif CACGTG which has been identified as a binding site for the product of the c-myc gene [21]. We used a fragment spanning one of the flanking repeats to probe the mouse genome to see if the repeats are part of a larger family of repetitive sequences. The results (Fig. 1D) show that probe A hybridizes strongly to a 2-kb EcoRI fragment (from which it is derived) and more weakly to a 12-kb fragment (which includes the homologous flanking repeat) and a third band of 4.8 kb. However, the absence of further bands suggests that the repeats flanking the mouse 3’-enhancer are not part of a larger family of highly conserved repeats. 3.3 The mouse IgH 3‘-enhancer is located 16 kb downstream of C, Neither hM2 nor AM3 hybridized to an immunoglobulin a, H chain cDNA probe.Therefore, to determine whether the mouse enhancer was indeed linked to the IgH locus, we needed to establish an overlap between phage hM2 and IgH locus DNA. We screened a mouse cosmid library for clones containing the C, region; the probe was an 1100-nt fragment generated by polymerase chain reaction that spans the coding region of the a membrane exon. The restriction map of the cosmid isolated (CosMlOB) is depicted in Fig. 2. Cross-hybridization of subfragments of CosM10B with those of AM2 as well as restriction mapping allowed the overlap to be established. Phage Ch28.M.Iga27 (described in [22]) was also found to overlap
Figure 3. Alignement of the sequences of the mouse and rat IgH 3‘-enhancers. The invert repeats and octanucleotide element are boxed.
Eur. J. Immunol. 1991. 22: 1499-1504
P. Dariavach, G. T. Williams, K . Campbell et al.
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both CosMlOB and hM2. The restriction map reveals that the mouse IgH 3’-enhancer is located 16 kb downstream of the C,1 exon. 3.4 The mouse IgH 3’-enhancer is inverted with respect t o the rat 3’-enhancer Hybridization of subclones of the mouse IgH 3’-enhancer to phage hM2 suggested that the 3‘-enhancers of mouse and rat were present in the genome in opposite orientations with respect to the IgH C-regions.To confirm that this was not a cloning artefact, we carried out a Southern blot analysis of genomic mouse DNA. The core region of the enhancer in rat and mouse contains a conserved EcoRI site. In genomic mouse plots, probes F and C hybridize to a 12-kb Eco RI fragment whereas probes B and E hybridize to a 2-kb Eco RI fragment (Fig. 4A). All these probes light up the same sized fragments in phage AM2 and, moreover, probe F hybridizes to a region just 3’ of C, in cosmid MlOB (not shown). Thus, probe E lights up the C,-distal side of the EcoRI site in the mouse 3’-enhancer. However, probe E is an Acc I-Eco RI subclone of the rat 3’-enhancer and derives from a region (nucleotides 432-565) which is located on the C,-proximal side of the Eco RI site within
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3.5 The mouse IgH 3’-enhancer is lymphoid specific To confirm that the putative mouse 3’-enhancer does indeed exhibit enhancer activity, the 908-bp Stu 1 fragment was linked to an enhancerless human P-globin gene and introduced into MPCll mouse plasmacytoma cells along with an a*-globin gene as internal reference. As shown in Fig. 5, the IgH 3‘-enhancer (like the SV40 and IgH intron-enhancers) does indeed potentiate P-globin expression in the transfected myeloma cells.Furthermore, like the IgH intron-enhancer but in contrast to the SV40 enhancer, the IgH 3’-enhancer is inactive in HeLa cells. Thus, the mouse IgH 3’-enhancer appears lymphoid-specific.Within the lymphoid lineage, activity is detected in another mouse
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the rat 3’-enhancer [ 12].This is confirmed in Fig. 4B where we show that, in the rat C, cosmid 37-1-1, probe E and a C,-membrane probe light up a common 19-kb EcoRI fragment whereas probe D hybridizes to the C,-distal side of the rat IgH 3’-enhancer. Similar results are obtained when probing a rat liver genomic DNA blot (not shown). Thus, the rat and mouse enhancers are in opposite orientations but the exact extent of the inversion is not yet identified.
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Figure 4. The mouse and rat IgH 3‘-enhancers are in opposite orientations. (A) A restriction map of the mouse CJgH 3’-enhancer region is used to show the derivation of the probes used for probing an Eco RI (R) digest of BALB/c mouse liver DNA. Probes A , B, C and Fare Eco RI-Apa LI, Stu I-Xba I , Xba I-Pst I and Sac I subfragmentsof phage hM2 whereas probes D and E were obtained as Eco RI-Bgl I (nucleotides 565-740) and Acc I-Eco RI (nucleotides 431-565) subfragments of the rat IgH 3’-enhancer over a region where the mouse and rat enhancers are highly homologous. (B) Map of the rat C&H 3’-enhancer region. Cosmid 37-1-1 DNA was digested with various restriction endonucleases and hybridized as indicated. Restriction sites are abbreviated C, ClaI; N. NruI: R, EcoRI; S, SacI; X, XhoI.
Mouse IgH 3’-enhancer
Eur. J. Immunol. 1991. 21: 1499-1504
Figure 5. The mouse IgH 3’-enhancer exhibits lymphoid-specific transcriptional activity. Cells were transfected with a test plasmid containing the human P-globin gene linked to the SV40, rat IgH-intron, mouse IgH-intron, mouse IgH-3‘ or no enhancer as indicated along with an arglobin plasmid as internal reference. Markers (M) were provided by a Hpa I1 digest of pBR322.
plasmacytoma (HOPC1) but not in the B cell lymphomas M12 or A20.This raises the possibility that the 3’-enhancer may only be activated at a late stage in lymphocyte differentiation.
3.6 Effect of including the 3‘-enhancer on the expression of transfected Ig genes One of the reasons for looking for the IgH 3’-enhancer was the fact that while IgH genes introduced into plasmacyto-
1503
mas or into the mouse germ line are usually well expressed, the level of this expression often falls short of that of the endogenous IgH gene. Could the absence of the 3‘enhancer on the transfected gene be responsible for this shortfall ? To test this, we transfected mouse plasmacytoma cells with plasmid pSV-V,l, which contains a rearranged p gene including the IgH intron-enhancer, and a derivative, pSV-V,1[3’E], which also includes the rat IgH 3’-enhancer cloned downstream of the transcription unit. As a plasmacytoma host we used NSO cells that had been previously transfected with one of two plasmids encoding a mouse x chain. The NSO[Sx] cells carry a transfected x gene that includes the x-intron but not the x3’-enhancer whereas the transfected gene in the NSO[Lx] cells includes both the x-intron and the x3’-enhancers [5]. Northern blot analysis of pools of transfected cells (Fig. 6) reveals that inclusion of the IgH 3’-enhancer on plasmid pSV-V,1 has a small effect (about twofold as judged by densitometry) on the level of p gene expression in the transfected cells. In keeping with our previous results [5], inclusion of the x3’-enhancer causes a roughly fivefold increase in the level of expression of the x gene transfected into NSO cells.
4 Discussion Thus, like the rat, the mouse also contains a lymphoidspecific enhancer located downstream of the CH regions. The enhancer in both species is flanked by an invert repeat. Interestingly, certain differences between rat and mouse in the sequences of the upstream copy of their repeats are also found in the downstream copy. Thus, nucleotides 574-578 (CATGA) in the mouse upstream repeat are not present in the rat; similarly, the invert of this sequence (TCATG) is retained in the downstream copy of the mouse repeat but is nevertheless still absent in the rat. Thus, the flanking repeats appear to have co-evolved with information transfer between the two such that some changes that occur in the upstream repeat also become fixed in the downstream COPY. The significance of these repeats flanking the enhancer is unknown. They do not appear to be present elsewhere in the genome and, despite the similarity of the sequence organization to that of a transposon, there is no reason to believe that the enhancer has transposed during evolution. It is, however, clear that the enhancer has inverted during evolution in that the enhancer is present in rat and mouse in opposite orientations. Inspection of the sequences presented in Fig. 3 suggest that this inversion has not occurred by recombination between the 350 nucleotide repeats. However, the enhancer could nevertheless be flanked by more distant repeats that could have acted as a focus for such inversion. Clearly, it will be interesting to sequence a more extensive region around the enhancer as well as identify the location and structure of the IgH 3’-enhancer in other species.
Figure6. Effect of the 3’-enhancer on the expression of stably transfected kHgenes* RNA was extracted from Pools Of NSO transfectantscarrying either a short ( S x ) or long x ( L x )gene which had been supertransfected with either pSV-V,l (denoted V,,), pSV-V,1[3,E] (denoted V,[3fE]) or no (-) plasmid. A Northern blot of this RNA was then hybridized with either C , or V,-Ox-1 probes.The transfected Sx and Lx genes contain aV,-Ox-1 variable segment [ 5 ] .
to discussed in the introduction, there are believe that there must be more than one enhancer in the IgH locus. There are also grounds for believing that at least one of these extra enhancer(s) is located at the 3‘-end of the locus. Not only is the c-myc proto-oncogene found translocated to the c, switch region in many mouse plasmacytomas, but a mouse plasmacytoma variant has been
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P. Dariavach, G . T. Williams, K. Campbell et al.
described which has decreased levels of a mRNA and which harbors a deletion starting 3' of C, and extending downstream [23]. Furthermore, the region 3' of C, has also been found to be involved in DNA rearrangement and inversion events in certain plasmacytomas [24]. However, there is as yet no direct evidence of an important role for the IgH 3'-enhancer in IgH gene expression. An interesting fact to come out of this work was the fact that while transfection assays reveal that the IgH 3'enhancer is strongly active in the MPCll and HOPC-1 plasmacytomas, no such strong activity was detected in the A20 or M12 B cell lymphomas. Clearly, the difference could simply be quantitative and we cannot rule out the possibility that the enhancer is active in both B cells and plasma cells but that the B cell activity is simply below the level of detection of our transfection assays. Alternatively, the IgH 3'-enhancer may only play a role during the late stages of B cell differentiation. This is particularly intriguing in view of the fact that one of the derivatives of the 18-81 pre-B cell line described by Wabl and Burrows [7] that has deleted the IgH intron-enhancer is only found to express this enhancerdeleted allele following fusion with a plasmacytoma. This suggests that at least in the case of this particular intronenhancer deletion, the remaining element responsible for activating p gene expression is active in plasma but not pre-B cells. Furthermore, analysis of a variant of an IgA-secreting plasmacytoma with decreased levels of a mRNA also points to the existence of sequences which are located 3' of C, and which are involved in IgH expression [23]. Finally, chromosomal translocations which activate c-myc by linking it to the IgH 3'-region are also only characteristic of tumors of the later stages of B cell differentiation in rodents.Thus it is possible that the mouse IgH 3'-enhancer does indeed play a role in activating translocated c-myc alleles as well as in potentiating transcription from IgH genes that harbor deletions of their intron-enhancer. However, we certainly cannot rule the possible existence and involvement of other as yet unidentified enhancers of the IgH locus. Furthermore, it will also be important to delineate the cell-type specificity of the 3'-enhancer more precisely,presumably by use of transgenic mice. We are grateful to Franco Calahi, Lai-chu Wu and iY Honjo forgifts of libraries or DNA clones and to Kerstin Meyer for comments on the manuscript. Received January 21. 1991.
Eur. J. Immunol. 1991. 21: 1499-1504
5 References Picard, D. and Schaffner, W., Nature 1984. 307: 80. Queen. C. and Stafford, J., Mol. Cell Biol. 1984. 4: 1042. Meyer, K. B. and Neuberger. M. S., EMBO J . 1989.8: 1959. x u , M . . Hammer, R. E . , Plasquez,V. c., Jones. S. L. and Garrard, W. T., J. Biol. Chem. 1989. 264: 21 190. 5 Meyer, K. B.. Sharpe. M. J., Surani, M. A . and Neuberger, M. S., Nucleic Acids Res. 1990. 18: 5609. 6 Klein, S.. Sablitzky. F. and Radbruch, A . . EMBO J. 1984. 3:
1 2 3 4
2473. 7 Wabl. M. and Burrows. I? D., Proc. Natl. Acad. Sci. USA 1984. 81: 2452. 8 Aguilera, R. J., Hope,T. J. and Sakano, H . . EMBO J. 1985. 4: 3689. 9 Eckhardt, L. A . and Birshtein, B. K . , Mol. Cell. Biol. 1985.5: 856. 10 Pettersson, S., Sharpe. M. J., Gilmore, D. R.. Surani. M. A . and Neuberger, M. S., Int. Immunol. 1989. 1: 509. 11 Neuberger. M. S. and Calabi, F., Nature 1983. 305: 240. 12 Pettersson, S., Cook. G . P., Bruggemann. M.,Williams, G.T. and Neuberger, M. S.. Nature 1990. 344: 6262. 13 Bruggemann, M . , Free, J.. Diamond, A . , Howard, J., Cobbold, s. and Waldmann. H., Proc. Natl. Acad. Sci. USA 1986. 83: 6075. 14 Clark, M. R. and Milstein. C.. Somatic Cell Genet. 1981. 7: 657. 15 Weigert. M. G . , Cesari, 1. M..Yonkovitch. S.J. and Cohn. M.. Nature 1970. 228: 1045. 16 Kim, K. J., Kanellopoulos-Langevin, C., Mervin, R.W., Sachs. D. H . and Asofsky. R.. J. Irnmunol. 1979. 122: 549. 17 Graham, R. and Van der Eb, A . , Virology 1973. 52: 456. 18 Mason. J. O..Williams, G. T. and Neuberger. M. S.. Cell 1985. 41: 479. 19 Treisman. R . H . , Cell 1985. 42: 889. 20 Neuberger. M. S.. EMBO J. 1983. 2: 1373. 21 Blackwel1.T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N. and Weintraub. H . , Science 1990. 25: 1149. 22 Nishida.Y., Katoka, T., Ishida, N . , Nakai, S., Kishimoto. T., Bottcher. I . and Honjo,T., Proc. Natl. Acad. Sci. USA 1981. 78: 1581. 23 Gregor. P. D. and Morrison, S. L., Mol . Cell Biol. 1986. 6: 1903. 24 Giannini, S. L.. Calvo, C. F., Martinez, N.. Ding, G.-F. and Birshtein. B. K., J. Cell. Biochem. 1990. Suppl. 1 4 0 : Abstr. M219.
Piona Dariavachv, Gareth T. Williams, Kathryn Campbell, Sven Pettersson. and Michael S. Neuberger MRC Laboratory of Molecular Biology, Cambridge and Center for Biotechnology., Karolinska Institute, Huddinge
Mouse IgH 3’-enhancer
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The mouse IgH 3’-enhancer A lymphoid-specific transcription enhancer element has recently been identified at the far 3’ end of the rat immunoglobulin heavy chain (IgH) locus. Sequence analysis presented here reveals that this enhancer is flanked by a 350-bp invert repeat, giving a structure reminiscent of a transposable element. We therefore screened for the equivalent enhancer in the mouse to determine whether its presence was conserved during evolution. A mouse homologue was indeed identified and is located 16 kb downstream of the C,1 exon. It is also flanked by invert repeats and these are not repeated throughout the genome.The mouse and rat enhancers retain high sequence homology. As regard activity, the IgH 3’-enhancer is lymphoid specific. However, this activity was detected in two plasmacytoma lines tested but not in two B cell lymphomas nor in HeLa cells suggesting that the enhancer may only play a stage-specific role during lymphocyte differentiation. As regards function within the IgH locus, we found that inclusion of the mouse IgH 3’-enhancer (in addition to the intron-enhancer) on p gene expression plasmids effected a small increase in mRNA levels in stable plasmacytoma transfectants.
1 Introduction The expression of immunoglobulin genes is controlled by lymphoid-specificenhancer elements acting in concert with lymphoid-specific promoters. In the case of the x locus, a moderately weak enhancer was initially identified in the J,-C, intron [ l , 21; subsequently, a second, stronger enhancer was discovered 3‘ of the C, exon [3]. It transpires that it is likely to be the x3’-enhancer which plays a critical role in ensuring the high level expression of rearranged x genes [4, 51. The function of the intron-enhancer is less clear; it could, for example, be involved in the regulation of V,-J, joining. In the case of the H chain locus, there is also a lymphoidspecific intron-enhancer and lymphoid-specific VH gene promoters, although the IgH intron-enhancer appears considerably stronger than the x intron-enhancer. Nevertheless, several lines of evidence led us to look for additional enhancer(s) in the IgH locus. First, rearranged mouse IgH genes which have deleted their IgH intronenhancer can nevertheless retain full transcriptional activity [6-91. Second, many IgH transgenesare not expressed at the same high level as the endogenous IgH gene ([lo] and references therein). Third, the c-myc proto-oncogene can become activated on translocation into the IgH locus without necessarily becoming linked to the IgH intronenhancer [ 111. Since we had available to us a set of cosmids covering the rat IgH locus, it wasin the rat that we originally identified the IgH 3’-enhancer [12]. However, unlike the situation in the mous x locus, it has not yet been ascertained whether the IgH 3‘-enhancer does in fact play a role in IgH gene transcription. Here we show that the rat IgH 3’-enhancer is flanked by a nearly perfectly matched invert repeat sequence. The
similarity of such a structure to that of a transposon led us to ask whether the IgH 3’-enhancer identified in the rat has been conserved during evolution. We have, therefore, now isolated the mouse IgH 3’-enhancer and show here that it is homologous to that of the rat both as regards sequence and organization although the enhancer is present in the two species in opposite orientations. We further show that its inclusion stimulates expression of rearranged p genes that have been stably transfected into a plasmacytoma host. Interestingly, however, while the enhancer is also active in two plasmacytoma lines when tested by transient transfection assays, we found no such activity in two B cell lymphomas tested. This suggests that the enhancer may only play a role during the later stages of lymphocyte development.
2 Materials and methods 2.1 Phage and cosmid libraries and DNA sequence determination A phage library generated by cloning a partial Sau3AI digest of BALB/c mouse liver DNA into the vector A2001 was a gift from Franco Calabi. Filters of a cosmid library (Clontech, Palo Alto, CA) generated by cloning sheared BALB/c mouse liver DNA into the Bam HI site of cosmid pWE15 were a gift from Lai-chu Wu. DNA of cosmid Cos37-1-1 which includes rat C, and the rat IgH 3’enhancer [13] was a gift from M. Briiggemann (AFRC, Babraham, Cambridge). DNA sequencing was carried out by the chain termination method using Sequenase (USB, Cleveland, OH). 2.2 Cell l i e s and transfections and enhancer assays
MPC 11 3U4 (obtained from B. Wasylyk, LGME, Stras[I 92221’ bourg), NSO [14], HeLa (from our laboratory collection), Permanent address: Laboratoire d’ImmunogenCtique Mol15cu- HOPC-1 [15], A20 and M12 cells [16] were cultured in laire, URA CNRS 1191, Universitk Montpellier 11, Place E. DMEM/10% FCS containing 50 p~ 2-ME. Bataillon, F-34095 Montpellier Cedex 5, France Correspondence: Michael S. Neuberger, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, GB
0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
Transient transfection of MPCll BU4 plasmacytoma or HeLa cells was performed by calcium phosphate coprecip0014-2980/91/0606-1499$3.50+ .25/0
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3 Results
itation [17] using 20 pg of test plasmid and 5 pg of internal reference. For HOPC-1, A20and M12, DEAE-dextran was used to mediate transfection followed by a shock with 10% DMSO as previously described [ 18J.The test plasmids were constructed from pUC12 derivatives that contained the human P-globin gene from 128 nucleotides upstream of the transcription start to the Pst I site downstream of the coding sequence cloned between the Xba I and Pst I sites of the polylinker. Enhancer fragments to be assayed were introduced between the Sac1 and XbaI sites upstream of the gene. The internal reference was provided by plasmid nSVHPa2 [19] which contains the human a*-globin gene. Total cytoplasmic RNA was prepared from cells 30-36 h after transfection and assayed by ribonuclease protection assays as previously described [3].
3.1 The rat IgH 3'-enhancer is flanked by invert repeats Preliminary evidence that the rat IgH 3'-enhancer is flanked by an invert repeat came from analysis of an M13 subclone of the enhancer; digestion of the single-stranded DNAwith nuclease S1 yielded a resistant fragment of about 310 bp (data not shown).The sequence of the Stu I-Eco RV fragment of the rat 3'-enhancer has been determined previously [ 121.We therefore determined the sequence of a more extensive region and the new sequence (see Fig. 3) does indeed confirm the existence of flanking invert repeats (Fig. 1). These repeats show only 16 mismatches over a 357-bp stretch (> 95% identity).
Transfectants of NSO that carry a long or short transfected x gene in a pSV2neo-based vector (Lx or S x ; constructs 1 and 4) have been described previously [ 5 ] ; these cells were super-transfected with pSV2gpt-based plasmids that contained a rearranged mouse p gene (pSV-V,1; [20]) or a derivative of pSV-V,1 which includes the Stu I fragment of the mouse IgH 3'-enhancer cloned into the plasmid's unique Xho I site.
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3.2 Isolation and sequence of the mouse IgH 3'-enhancer This unusual structure, which is reminiscent of that of a transposon, led us to ask whether the location of the enhancer was conserved in rat and mouse. A mouse liver library in bacteriophage h was, therefore, screened using as
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Figure 1. (A) Alignment of the 5'-repeat with the invert of the 3'-repeat of the rat enhancer. Nuclcotide positions are numbered as in Fig. 3 and positions of identity arc indicated by an asterisk. (B) Invert repeats in the rat IgH 3'-enhancer. Sequences of at least 12 bases of invert identity are joined by a line. The enhancer core lies within the Stu I-Eco RV fragment (indicated by a line) whose sequence was determined previously [ S ] .(C) Invert repeats in the mouse IgH 3'-enhancer. (D) Southern blot of BamHI-digested genomic mouse DNA hybridized with a probe that includes the repeat flanking the mouse IgH3'-enhancer (the EcoRI-Apa LI fragment depicted asprobe A in Fig. 4; nucleotides 55-748 in the mouse IgH 3'-enhancer sequence). DNA was either from mouse liver or from thc MOPC315 or J558L plasmacytornas. The filter was washed at 65 "C in 2 x SSC.
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a probe an Acc I-Bgl I subfragment from the core of the rat 3’-enhancer (nucleotides 432-748 in the rat sequence: see Fig. 4). Two overlapping groups of clones (AM2 and h M 3 ) were isolated. The restriction map of hM2 is depicted in Fig. 2; phage k M 3 extends 3‘ of hM2. The XbaI fragment of hM2 that contained the enhancer homology was subcloned into plasmid pIC20H; a processive set of deletions was made using exonuclease 111 and subcloned into M13. The sequence of the mouse IgH 3’-enhancer was determined and is shown in Fig. 3 where it is aligned with that of the rat. The enhancer of the two species show good homology although the [GT],[(G or C)A]g, repeat of the rat is not observed in the mouse. Furthermore, while the invert repeats that flank the rat IgH 3‘-enhancer show a 96% identity, the internal 250 nucleotides of the equivalent mouse repeats manifest only 89% identity (Fig. 1C). The octanucleotide element ATTTGCAT is conserved in rat and mouse and it is also notable that the mouse enhancer (position 1346) contains the hexamer motif CACGTG which has been identified as a binding site for the product of the c-myc gene [21]. We used a fragment spanning one of the flanking repeats to probe the mouse genome to see if the repeats are part of a larger family of repetitive sequences. The results (Fig. 1D) show that probe A hybridizes strongly to a 2-kb EcoRI fragment (from which it is derived) and more weakly to a 12-kb fragment (which includes the homologous flanking repeat) and a third band of 4.8 kb. However, the absence of further bands suggests that the repeats flanking the mouse 3’-enhancer are not part of a larger family of highly conserved repeats. 3.3 The mouse IgH 3‘-enhancer is located 16 kb downstream of C, Neither hM2 nor AM3 hybridized to an immunoglobulin a, H chain cDNA probe.Therefore, to determine whether the mouse enhancer was indeed linked to the IgH locus, we needed to establish an overlap between phage hM2 and IgH locus DNA. We screened a mouse cosmid library for clones containing the C, region; the probe was an 1100-nt fragment generated by polymerase chain reaction that spans the coding region of the a membrane exon. The restriction map of the cosmid isolated (CosMlOB) is depicted in Fig. 2. Cross-hybridization of subfragments of CosM10B with those of AM2 as well as restriction mapping allowed the overlap to be established. Phage Ch28.M.Iga27 (described in [22]) was also found to overlap
Figure 3. Alignement of the sequences of the mouse and rat IgH 3‘-enhancers. The invert repeats and octanucleotide element are boxed.
Eur. J. Immunol. 1991. 22: 1499-1504
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both CosMlOB and hM2. The restriction map reveals that the mouse IgH 3’-enhancer is located 16 kb downstream of the C,1 exon. 3.4 The mouse IgH 3’-enhancer is inverted with respect t o the rat 3’-enhancer Hybridization of subclones of the mouse IgH 3’-enhancer to phage hM2 suggested that the 3‘-enhancers of mouse and rat were present in the genome in opposite orientations with respect to the IgH C-regions.To confirm that this was not a cloning artefact, we carried out a Southern blot analysis of genomic mouse DNA. The core region of the enhancer in rat and mouse contains a conserved EcoRI site. In genomic mouse plots, probes F and C hybridize to a 12-kb Eco RI fragment whereas probes B and E hybridize to a 2-kb Eco RI fragment (Fig. 4A). All these probes light up the same sized fragments in phage AM2 and, moreover, probe F hybridizes to a region just 3’ of C, in cosmid MlOB (not shown). Thus, probe E lights up the C,-distal side of the EcoRI site in the mouse 3’-enhancer. However, probe E is an Acc I-Eco RI subclone of the rat 3’-enhancer and derives from a region (nucleotides 432-565) which is located on the C,-proximal side of the Eco RI site within
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3.5 The mouse IgH 3’-enhancer is lymphoid specific To confirm that the putative mouse 3’-enhancer does indeed exhibit enhancer activity, the 908-bp Stu 1 fragment was linked to an enhancerless human P-globin gene and introduced into MPCll mouse plasmacytoma cells along with an a*-globin gene as internal reference. As shown in Fig. 5, the IgH 3‘-enhancer (like the SV40 and IgH intron-enhancers) does indeed potentiate P-globin expression in the transfected myeloma cells.Furthermore, like the IgH intron-enhancer but in contrast to the SV40 enhancer, the IgH 3’-enhancer is inactive in HeLa cells. Thus, the mouse IgH 3’-enhancer appears lymphoid-specific.Within the lymphoid lineage, activity is detected in another mouse
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the rat 3’-enhancer [ 12].This is confirmed in Fig. 4B where we show that, in the rat C, cosmid 37-1-1, probe E and a C,-membrane probe light up a common 19-kb EcoRI fragment whereas probe D hybridizes to the C,-distal side of the rat IgH 3’-enhancer. Similar results are obtained when probing a rat liver genomic DNA blot (not shown). Thus, the rat and mouse enhancers are in opposite orientations but the exact extent of the inversion is not yet identified.
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Figure 4. The mouse and rat IgH 3‘-enhancers are in opposite orientations. (A) A restriction map of the mouse CJgH 3’-enhancer region is used to show the derivation of the probes used for probing an Eco RI (R) digest of BALB/c mouse liver DNA. Probes A , B, C and Fare Eco RI-Apa LI, Stu I-Xba I , Xba I-Pst I and Sac I subfragmentsof phage hM2 whereas probes D and E were obtained as Eco RI-Bgl I (nucleotides 565-740) and Acc I-Eco RI (nucleotides 431-565) subfragments of the rat IgH 3’-enhancer over a region where the mouse and rat enhancers are highly homologous. (B) Map of the rat C&H 3’-enhancer region. Cosmid 37-1-1 DNA was digested with various restriction endonucleases and hybridized as indicated. Restriction sites are abbreviated C, ClaI; N. NruI: R, EcoRI; S, SacI; X, XhoI.
Mouse IgH 3’-enhancer
Eur. J. Immunol. 1991. 21: 1499-1504
Figure 5. The mouse IgH 3’-enhancer exhibits lymphoid-specific transcriptional activity. Cells were transfected with a test plasmid containing the human P-globin gene linked to the SV40, rat IgH-intron, mouse IgH-intron, mouse IgH-3‘ or no enhancer as indicated along with an arglobin plasmid as internal reference. Markers (M) were provided by a Hpa I1 digest of pBR322.
plasmacytoma (HOPC1) but not in the B cell lymphomas M12 or A20.This raises the possibility that the 3’-enhancer may only be activated at a late stage in lymphocyte differentiation.
3.6 Effect of including the 3‘-enhancer on the expression of transfected Ig genes One of the reasons for looking for the IgH 3’-enhancer was the fact that while IgH genes introduced into plasmacyto-
1503
mas or into the mouse germ line are usually well expressed, the level of this expression often falls short of that of the endogenous IgH gene. Could the absence of the 3‘enhancer on the transfected gene be responsible for this shortfall ? To test this, we transfected mouse plasmacytoma cells with plasmid pSV-V,l, which contains a rearranged p gene including the IgH intron-enhancer, and a derivative, pSV-V,1[3’E], which also includes the rat IgH 3’-enhancer cloned downstream of the transcription unit. As a plasmacytoma host we used NSO cells that had been previously transfected with one of two plasmids encoding a mouse x chain. The NSO[Sx] cells carry a transfected x gene that includes the x-intron but not the x3’-enhancer whereas the transfected gene in the NSO[Lx] cells includes both the x-intron and the x3’-enhancers [5]. Northern blot analysis of pools of transfected cells (Fig. 6) reveals that inclusion of the IgH 3’-enhancer on plasmid pSV-V,1 has a small effect (about twofold as judged by densitometry) on the level of p gene expression in the transfected cells. In keeping with our previous results [5], inclusion of the x3’-enhancer causes a roughly fivefold increase in the level of expression of the x gene transfected into NSO cells.
4 Discussion Thus, like the rat, the mouse also contains a lymphoidspecific enhancer located downstream of the CH regions. The enhancer in both species is flanked by an invert repeat. Interestingly, certain differences between rat and mouse in the sequences of the upstream copy of their repeats are also found in the downstream copy. Thus, nucleotides 574-578 (CATGA) in the mouse upstream repeat are not present in the rat; similarly, the invert of this sequence (TCATG) is retained in the downstream copy of the mouse repeat but is nevertheless still absent in the rat. Thus, the flanking repeats appear to have co-evolved with information transfer between the two such that some changes that occur in the upstream repeat also become fixed in the downstream COPY. The significance of these repeats flanking the enhancer is unknown. They do not appear to be present elsewhere in the genome and, despite the similarity of the sequence organization to that of a transposon, there is no reason to believe that the enhancer has transposed during evolution. It is, however, clear that the enhancer has inverted during evolution in that the enhancer is present in rat and mouse in opposite orientations. Inspection of the sequences presented in Fig. 3 suggest that this inversion has not occurred by recombination between the 350 nucleotide repeats. However, the enhancer could nevertheless be flanked by more distant repeats that could have acted as a focus for such inversion. Clearly, it will be interesting to sequence a more extensive region around the enhancer as well as identify the location and structure of the IgH 3’-enhancer in other species.
Figure6. Effect of the 3’-enhancer on the expression of stably transfected kHgenes* RNA was extracted from Pools Of NSO transfectantscarrying either a short ( S x ) or long x ( L x )gene which had been supertransfected with either pSV-V,l (denoted V,,), pSV-V,1[3,E] (denoted V,[3fE]) or no (-) plasmid. A Northern blot of this RNA was then hybridized with either C , or V,-Ox-1 probes.The transfected Sx and Lx genes contain aV,-Ox-1 variable segment [ 5 ] .
to discussed in the introduction, there are believe that there must be more than one enhancer in the IgH locus. There are also grounds for believing that at least one of these extra enhancer(s) is located at the 3‘-end of the locus. Not only is the c-myc proto-oncogene found translocated to the c, switch region in many mouse plasmacytomas, but a mouse plasmacytoma variant has been
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P. Dariavach, G . T. Williams, K. Campbell et al.
described which has decreased levels of a mRNA and which harbors a deletion starting 3' of C, and extending downstream [23]. Furthermore, the region 3' of C, has also been found to be involved in DNA rearrangement and inversion events in certain plasmacytomas [24]. However, there is as yet no direct evidence of an important role for the IgH 3'-enhancer in IgH gene expression. An interesting fact to come out of this work was the fact that while transfection assays reveal that the IgH 3'enhancer is strongly active in the MPCll and HOPC-1 plasmacytomas, no such strong activity was detected in the A20 or M12 B cell lymphomas. Clearly, the difference could simply be quantitative and we cannot rule out the possibility that the enhancer is active in both B cells and plasma cells but that the B cell activity is simply below the level of detection of our transfection assays. Alternatively, the IgH 3'-enhancer may only play a role during the late stages of B cell differentiation. This is particularly intriguing in view of the fact that one of the derivatives of the 18-81 pre-B cell line described by Wabl and Burrows [7] that has deleted the IgH intron-enhancer is only found to express this enhancerdeleted allele following fusion with a plasmacytoma. This suggests that at least in the case of this particular intronenhancer deletion, the remaining element responsible for activating p gene expression is active in plasma but not pre-B cells. Furthermore, analysis of a variant of an IgA-secreting plasmacytoma with decreased levels of a mRNA also points to the existence of sequences which are located 3' of C, and which are involved in IgH expression [23]. Finally, chromosomal translocations which activate c-myc by linking it to the IgH 3'-region are also only characteristic of tumors of the later stages of B cell differentiation in rodents.Thus it is possible that the mouse IgH 3'-enhancer does indeed play a role in activating translocated c-myc alleles as well as in potentiating transcription from IgH genes that harbor deletions of their intron-enhancer. However, we certainly cannot rule the possible existence and involvement of other as yet unidentified enhancers of the IgH locus. Furthermore, it will also be important to delineate the cell-type specificity of the 3'-enhancer more precisely,presumably by use of transgenic mice. We are grateful to Franco Calahi, Lai-chu Wu and iY Honjo forgifts of libraries or DNA clones and to Kerstin Meyer for comments on the manuscript. Received January 21. 1991.
Eur. J. Immunol. 1991. 21: 1499-1504
5 References Picard, D. and Schaffner, W., Nature 1984. 307: 80. Queen. C. and Stafford, J., Mol. Cell Biol. 1984. 4: 1042. Meyer, K. B. and Neuberger. M. S., EMBO J . 1989.8: 1959. x u , M . . Hammer, R. E . , Plasquez,V. c., Jones. S. L. and Garrard, W. T., J. Biol. Chem. 1989. 264: 21 190. 5 Meyer, K. B.. Sharpe. M. J., Surani, M. A . and Neuberger, M. S., Nucleic Acids Res. 1990. 18: 5609. 6 Klein, S.. Sablitzky. F. and Radbruch, A . . EMBO J. 1984. 3:
1 2 3 4
2473. 7 Wabl. M. and Burrows. I? D., Proc. Natl. Acad. Sci. USA 1984. 81: 2452. 8 Aguilera, R. J., Hope,T. J. and Sakano, H . . EMBO J. 1985. 4: 3689. 9 Eckhardt, L. A . and Birshtein, B. K . , Mol. Cell. Biol. 1985.5: 856. 10 Pettersson, S., Sharpe. M. J., Gilmore, D. R.. Surani. M. A . and Neuberger, M. S., Int. Immunol. 1989. 1: 509. 11 Neuberger. M. S. and Calabi, F., Nature 1983. 305: 240. 12 Pettersson, S., Cook. G . P., Bruggemann. M.,Williams, G.T. and Neuberger, M. S.. Nature 1990. 344: 6262. 13 Bruggemann, M . , Free, J.. Diamond, A . , Howard, J., Cobbold, s. and Waldmann. H., Proc. Natl. Acad. Sci. USA 1986. 83: 6075. 14 Clark, M. R. and Milstein. C.. Somatic Cell Genet. 1981. 7: 657. 15 Weigert. M. G . , Cesari, 1. M..Yonkovitch. S.J. and Cohn. M.. Nature 1970. 228: 1045. 16 Kim, K. J., Kanellopoulos-Langevin, C., Mervin, R.W., Sachs. D. H . and Asofsky. R.. J. Irnmunol. 1979. 122: 549. 17 Graham, R. and Van der Eb, A . , Virology 1973. 52: 456. 18 Mason. J. O..Williams, G. T. and Neuberger. M. S.. Cell 1985. 41: 479. 19 Treisman. R . H . , Cell 1985. 42: 889. 20 Neuberger. M. S.. EMBO J. 1983. 2: 1373. 21 Blackwel1.T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N. and Weintraub. H . , Science 1990. 25: 1149. 22 Nishida.Y., Katoka, T., Ishida, N . , Nakai, S., Kishimoto. T., Bottcher. I . and Honjo,T., Proc. Natl. Acad. Sci. USA 1981. 78: 1581. 23 Gregor. P. D. and Morrison, S. L., Mol . Cell Biol. 1986. 6: 1903. 24 Giannini, S. L.. Calvo, C. F., Martinez, N.. Ding, G.-F. and Birshtein. B. K., J. Cell. Biochem. 1990. Suppl. 1 4 0 : Abstr. M219.
