Vol. 10, No. 3

MOLECULAR AND CELLULAR BIOLOGY, Mar. 1990, p. 1076-1083 0270-7306/90/0301076-08$02.00/0 Copyright C 1990, American Society for Microbiology

Immunoglobulin Heavy-Chain Enhancer Is Required To Maintain Transfected y2A Gene Expression in a Pre-B-Cell Line BARBARA PORTON, DENNIS M. ZALLER,t REBECCA LIEBERSON, AND LAUREL A. ECKHARDT*

Department

of Biological

Sciences, Columbia University, New York, New York 10021

Received 3 October 1989/Accepted 22 November 1989

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a y2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of y2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

should prove to be enhancer independent after activation in the presence of an enhancer. To test this model, we generated several pre-B cell lines carrying a transfected y2a heavy-chain gene in which the IgH enhancer was located between DH and JH recombination signals. Independent, stable transformants expressing this gene were placed under conditions that favored the outgrowth of subclones that had undergone D-to-J joining within the transfected gene, thus deleting the IgH enhancer. The levels of heavy-chain mRNA and protein made by the primary transformants and the enhancer-deficient subclones were then measured. The results of these experiments indicate that a transfected IgH gene remains enhancer dependent even after its initial activation in a pre-B cell. We discuss these findings in relation to the "establishmentonly" and the "establishment-plus-maintenance" models of enhancer function.

The efficient expression of a cloned immunoglobulin heavy-chain (IgH) gene, when introduced into myeloma cells, requires the presence of IgH enhancer sequences (5, 10, 25). The role of this enhancer in the expression of endogenous IgH genes, however, remains undefined. We (37) and others (1, 14, 34) have reported IgH enhancerindependent expression of endogenous IgH genes. For example, such expression was seen in an immunoglobulin G2a (IgG2a)-producing mouse myeloma cell line (9921) that arose as a class switch variant of MPC11, an IgG2b producer (15). The class switch rearrangement that resulted in a switch from -y2b to -y2a heavy-chain production included deletion of the IgH enhancer (7, 37). A possible explanation for the continued expression of this gene in the absence of the IgH enhancer was that an alternate enhancer lay elsewhere within or around the newly formed y2a transcription unit of 9921. In a previous study (37) we showed that no such enhancer lay within the boundaries of the cloned 9921 gene. In the present study, we have extended our search for an enhancer within the cloned 9921 y2a gene-flanking sequences to an area of approximately 32 kilobase pairs (kb). Again, no region with enhancer function has been found. As an alternate explanation for the disparate behavior of the 9921 cloned and endogenous IgH genes, we proposed (37) a model of enhancer function in which the enhancer serves only to establish high-level transcription of the heavychain gene and is not required to maintain it. The transfected and the endogenous genes might differ with respect to enhancer dependence only because they differ with respect to their state of activation. If the establishment of high-level expression is qualitatively the same for both an endogenous and a transfected gene, expression of a transfected IgH gene

MATERIALS AND METHODS Plasmid DNA constructions. The construction of p99y2a has been described previously (37). Briefly, the IgH gene sequences in this plasmid cover a 12.3-kb region including the entire y2a transcription unit with 1.3-kb 3'-flanking sequences and 0.5-kb 5'-flanking sequences. This 12.3-kb region was ligated to pSV2gpt (23, 24) to allow replication in bacterial cells and selection of mammalian cell transformants (selection for xanthine, guanine phosphoribosyltransferase [xgpt] expression). p99-y2a3' consists of p99y2a to which has been ligated an additional 7.3 kb of C-y2a 3'-flanking sequences (Fig. 1A) (33, 36). pvcDEJ consists of a y2a gene (V region gene of MPC11 and 9921) with the 1-kb XbaI IgH enhancer fragment placed at a site outside of the y2a transcription unit (Fig. 1B). The -y2a gene within this construction includes sequences from nucleotide position -153 (with respect to transcription) to ca. 1.2 kb 3' of C-y2a; ca. 6 kb of V-C intron sequences (between the BamHI site near

Corresponding author. t Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125. *

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ROLE OF IgH ENHANCER IN TRANSFECTED GENES

VOL. 10, 1990

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,

JH2 and the EcoRI site immediately upstream of Cy2a) has been deleted with respect to the endogenous 9921 gene (7). The IgH enhancer in pvcDEJ lies adjacent to the xgpt gene with eucaryotic promoter and enhancer, and both of these sequences are flanked by DQ52 and JH4 (the D and J gene segments were isolated from pJT28 [6], kindly supplied by F. Alt, and are the only D and J segments present in the pvcDEJ and pvcDJ constructions). The remainder of pvcDEJ consists of a BamHI-SalI fragment containing most of the plasmid pSV2neo (32). The only difference between pvcDEJ and pvcDJ is that the latter lacks the IgH enhancer. Genomic DNA blot analysis. Agarose gel electrophoresis, transfer to nitrocellulose, and DNA hybridizations were performed as previously described (8). A 15-,ug sample of restriction enzyme-digested DNA (estimated from the opti-

1077

cal density of the DNA solutions at 260 nm) was run in each lane. DNA probes included Jil (probe 1 in Fig. 1B [18]) Cy2a-CH3 (probe 3 in Fig. 1B [33]), and a 600-base-pair fragment adjacent to DQ52 (probe 2 in Fig. 1B). Enzyme-linked immunosorbent assays. Enzyme-linked immunosorbent assays were performed essentially as described previously (37). Immulon 2 plates (Dynatech Laboratories, Inc.) were coated with affinity-purified Fc fragmentspecific rabbit anti-mouse IgG (Pel-Freeze). Nonidet P-40 cytoplasmic lysate (150 ,il) made from 3 x 106 cells was serially diluted, and 50-,ul portions of each dilution were then incubated with the coated wells. y2a heavy chains were detected with alkaline phosphatase-conjugated rabbit antimouse IgG2a, y2a specific (Zymed Laboratories, Inc.). Transfection and cell culture. The mouse myeloma cell line J558L (26) was transfected by protoplast fusion as previously described (26, 29), and transformants were isolated by growth in Dulbecco modified Eagle medium supplemented with 15% equine serum and mycophenolic acid (6 jig/ml), hypoxanthine (15 p.g/ml), and xanthine (250 ,ug/ml) (MHX medium). 16C1.1 cells (1 x 107) were washed with phosphatebuffered saline (0.15 M NaCl, 0.15 M NaPO4 [pH 7.2]) and electroporated with linearized plasmid DNA at 250 V and 960 ,uF essentially as described previously (27). Immediately after electroporation, the cells were dispensed into 96-well microdilution plates at 105 cells per well. At 48 h after transfection, the cells were transferred into medium containing 1 mg of geneticin G418 (GIBCO Laboratories) per ml to select for cells expressing the Neor gene. Colonies were visible 14 to 21 days after transfection. In most transfection experiments, transformants arose in 50 to 70% of the culture wells, indicating that each transformant line was probably not derived from a single cell. A total of 140 pvcDEJ transformants and 30 pvcDJ transformants were subcloned in 96-well microdilution plates by limiting dilution. Single colonies arose in 10% of the wells in 40 of the pvcDEJ and 10 of the pvcDJ subcloning plates. An average of three subclones were picked from each plate and expanded in medium containing G418 or MHX. To select against expression of the bacterial xgpt gene, we grew cells for 2 weeks in G418 medium containing 6thioguanine (10-4 M) until 95 to 99% of the cells had died and the surviving population of cells was expanded. RNA blot analyses. RNA blots were performed essentially as described by Maniatis et al. (17). Approximately 10 jig of poly(A)+ RNA isolated from ca. 108 cells was applied to formaldehyde-agarose gels, blotted to nitrocellulose, and hybridized with a probe for the CH3 domain of the y2a constant region (-y2a-CH3, probe 3 in Fig. 1B [33]). The blot was subsequently erased and rehybridized with an actin cDNA probe, which was used to quantify the amount of poly(A)+ RNA in each gel lane. RESULTS We previously showed that despite the high levels of IgH production in a cell line (9921) expressing an enhancerless y2a gene, the same y2a gene could not be expressed efficiently when cloned and reintroduced into J558L myeloma cells (37). Within the limits of the 9921 gene construction, therefore, there was no functional equivalent of the deleted IgH enhancer. To test sequences farther 3' of the 9921 y2a gene, we constructed the plasmid p99y2a3' (Fig. 1A; see Materials and Methods). This plasmid and a control con-

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MOL. CELL. BIOL.

PORTON ET AL.

struction that included the IgH enhancer (p99Enhy2a3') were transfected into the X light chain-producing myeloma line, J558L. Six of six transformants obtained by transfection with the IgH enhancer-containing plasmid had accumulated high levels of IgG in 48-h culture supernatants. In contrast, six of six transformants containing p99-y2a3' secreted much reduced levels of IgG (1/80 to 1/100 that secreted by p99Enh-y2a transformants; data not shown). The plasmid constructions of this and our previous study include, collectively, all of the DNA lying within 32 kb 3' of the 9921 heavy-chain gene promoter (with the exception of an approximately 1.6-kb region [see the legend to Fig. 1A]). Our results demonstrate that no functional enhancer lies within these sequences. Two recombinant plasmids (pvcDEJ and pvcDJ) were constructed to test an alternate hypothesis proposed to explain the continued high-level expression of the enhancerless y2a gene in 9921. According to this hypothesis, an enhancer is required only to establish high-level gene expression, not to maintain it. Both the pvcDEJ and pvcDJ constructions contain a y2a heavy-chain gene, a neomycin resistance gene, and the bacterial xgpt gene flanked by DH and JH recombination signals (Fig. 1B). The variable- and constant-region sequences of the -y2a heavy-chain gene are those of the 9921 myeloma. The pvcDEJ and pvcDJ plasmids differ with respect to the IgH enhancer: pvcDEJ contains a 1-kb XbaI fragment including the IgH enhancer at a site adjacent to the xgpt gene, whereas pvcDJ lacks this fragment. pvcDEJ was designed to permit the establishment of IgH gene expression under enhancer control and then allow for subsequent loss of the enhancer; pvcDJ was used to demonstrate that establishment of high-level IgH expression in the pvcDEJ transformants is enhancer dependent. It has been shown that when DH and JH sequences, with their adjoining recombination signal sequences, are introduced into pre-B cells, DH-to-JH joining occurs (6). In our constructions, joining would also lead to deletion of the IgH enhancer and the selectable marker xgpt. Both constructions were introduced into a hypoxanthine, guanine phosphoribosyltransferase (hgprt)deficient derivative (16C1.1) of the pre-B cell line 18-81 (28). As a result, transformants that undergo D-J joining and thereby lose the bacterial xgpt gene can be isolated by growth in 6-thioguanine (a toxic substrate analog of both the mammalian hgprt and bacterial xgpt enzymes). pvcDEJ and pvcDJ were linearized with NdeI and transfected into 16C1.1 cells, and transformants were isolated in G418 medium which requires Neor expression. Many independent transformants were analyzed to ensure that the effect of enhancer deletion, rather than the effects of any particular chromosomal position on transfected gene expression, would be measured in these experiments. Because D-J joining occurs spontaneously in the 16C1.1 cell line, enhancer sequences are continuously being lost in pvcDEJ transformants. To assess -y2a expression in IgH enhancer-positive transformants, we grew pvcDEJ transformants in medium containing mycophenolic acid, hypoxanthine, and xanthine (MHX medium). This medium requires expression of the xgpt gene lying between the DH and JH sequences and therefore allows for the selective growth of cells that have not undergone D-J joining. To select for growth of cells that have undergone D-J joining, we grew pvcDEJ subclones in G418 medium supplemented with 6-thioguanine. Approximately 1 to 10% of the cells in each transformant subclone survived in this selective medium and were expanded for further analysis. To deter-

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FIG. 2. Genomic DNA blot analysis demonstrating loss of IgH enhancer. DNA from pvcDEJ (lanes DEJ) and pvcDAEJ (lanes DAEJ) transformants was digested with BamHI, electrophoresed on an agarose gel, blotted to nitrocellulose, and then hybridized with Jll (probe 1 in Fig. 1B). DNA analyses were done on more than 40 pvcDEJ transformants and their 6-Thior derivatives. Representative data from six transformants of each type are shown. The data shown are from several independent gels, so that the migration distance for the 1-kb enhancer fragment differs among gel tracks. Molecular size markers indicated adjacent to DEJ-45 are Hindlll fragments from lambda bacteriophage (2.3 to 23 kb) and HaeIII fragments from bacteriophage 4X174 (1.0 kb; 1.3 kb) and provide a reference for the other gel tracks. A bracket with arrow denotes the 1-kb enhancer fragment in DEJ transformants and its absence in DAEJ transformants. No fragment was seen in the pvcDAEJ transformant blots even after a longer exposure. The two fragments detected in 16C1.1 (lane 16C1) and all of the transformants are derived from the endogenous IgH loci in this cell line. Additional high-molecularweight fragments are the result of unique integration events for individual transformants and are maintained between each DEJ transformant and its DAEJ derivative.

mine whether this population of cells was IgH enhancer deficient, we isolated genomic DNA from 6-thioguanineresistant (6-Thior) clones arising from 40 independent pvcDEJ transformants. The DNA was digested with BamHI and probed with Jll (probe 1 in Fig. 1B [18]), which hybridizes to the 1-kb IgH enhancer fragment within the pvcDEJ construction. This fragment was present in DNA from all of the pvcDEJ transformants before growth in 6-thioguanine (Fig. 2). After growth in 6-thioguanine, 15 6-Thior subclones derived from independent pvcDEJ transformants had lost this 1-kb fragment and were thus IgH enhancer deficient (Fig. 2). We refer to these enhancerdeficient subclones as pvcDAEJ cell lines. The remainder of the 6-thioguanine-resistant clones retained the 1-kb IgH enhancer fragment; presumably they had lost xgpt activity through a mechanism other than gene deletion by D-J joining. Many of these clones were included in the quantitative immunoassay described below, but were not analyzed further. DNA from the pvcDEJ and pvcDAEJ transformants was digested with EcoRI and hybridized with a fragment from the Ampr region of pBR322 as a means of determining the copy number. From this analysis, it was estimated that each transformant carried one to three copies of the transfected plasmid (data not shown). To verify that the transfected -y2a heavy-chain transcription unit remained intact in the pvcDAEJ and pvcDJ cell

ROLE OF IgH ENHANCER IN TRANSFECTED GENES

VOL. 10, 1990

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lines, genomic DNA from the 15 pvcDAEJ cell lines, 1 pvcDEJ transformant, and 2 pvcDJ transformants was digested with both KpnI and PvuI. The plasmid constructions cut with these enzymes generate an 8-kb fragment containing the entire -y2a heavy-chain transcription unit (Fig. 1B). A DNA probe containing the CH3 domain of Cy2a (probe 3 in Fig. 1B [3]) was used to detect this fragment in genomic DNA blots. Representative data are shown in Fig. 3. Of the 15 pvcDAEJ transformants, 11 retained this fragment. The remaining four were excluded from further analysis. The region of the pvcDEJ construction involved in D-J joining was analyzed by blot analysis of XbaI-digested genomic DNA before and after joining. Before D-J joining, a 5.4-kb fragment should be detectable with the Jll probe (Fig. 1B). After D-J joining, this fragment should be replaced by a 1.7-kb fragment. The 5.4-kb fragment, as expected, was present in the pvcDEJ transformants (see Fig. 4). In six of the pvcDAEJ transformants, the 5.4-kb fragment had been replaced by a 1.7-kb fragment, suggesting that the proper D-J joins had occurred in these enhancer-deficient cell lines (data for these transformants are shown in Fig. 4). The same results were seen when XbaI-digested DNA and a probe for Dh flanking sequences were used (probe 2 in Fig. 1B; data not shown). In the remaining 5 of the 11 pvcDAEJ transformants that retained the transfected y2a transcription unit, the IgH enhancer deletion appeared to have resulted from a more complex DNA rearrangement (data not shown). A quantitative enzyme-linked immunosorbent assay was used to compare cytoplasmic -y2a heavy-chain protein levels in the pvcDEJ, pvcDJ, and pvcDAEJ cell lines. pvcDEJ transformants used for this assay were maintained in MHX medium and therefore should not include any D-J joined cells missing either the xgpt gene or the IgH enhancer. All of the pvcDEJ transformants expressed the transfected y2a heavy-chain gene, whereas y2a heavy chain was undetectable in the pvcDJ and pvcDAEJ transformants (representative results are shown in Fig. 5). 6-Thioguanine-resistant pvcDEJ subclones that had not deleted the IgH enhancer fragment continued to express y2a protein (data not shown). Although no expression of the pvcDJ construc-

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FIG. 4. Genomic DNA blot analysis demonstrating D-J rearrangements in some of the pvcDAEJ transformants. DNA was digested with XbaI and hybridized with Jll (probe 1 in Fig. IB). One arrow points to the 5.4-kb band expected before D-J joining, and the other points to a 1.7-kb band expected after joining. (As described in the legend to Fig. 2, data were from several gels so that migration distances vary.) The 5.4-kb band is found only in the pvcDEJ transformants (lanes DEJ), whereas the 1.7-kb band is found in all of the pvcDAEJ transformants shown (lanes DAEJ). It is also present in DEJ-72 and DEJ-90, indicating that some D-J joining has already occurred in these two populations of G418-resistant cells. The two fragments detected in lane 16C1 and all of the transformants are derived from endogenous IgH sequences.

tion was detectable in 16C1.1 cells, the same construction led to very low but detectable levels of -y2a when transfected into the myeloma J558L. This low level of expression, previously seen with enhancerless IgH genes in myeloma cells (37), demonstrates that the pvcDJ construction was not defective. We analyzed poly(A)+ RNA from several of the pvcDEJ, pvcDAEJ, and pvcDJ cell lines to learn whether the y2a protein levels in these cells correlated with steady-state concentrations of y2a mRNA (representative data are shown in Fig. 6). As expected, y2a mRNA was detected in the pvcDEJ transformants but not in their pvcDAEJ derivatives, nor in the pvcDJ transformants.

DISCUSSION We have previously shown that an endogenous IgH gene can continue expression after deletion of the IgH enhancer (37). In the cell line 9921, deletion of this enhancer accompanied a heavy-chain class switch from y2b to y2a expression. Endogenous IgH genes showing similar IgH enhancer independence have been described by others (1, 13, 14, 34). In the present study, we have shown that no sequence with enhancer activity lies within 32 kb 3' of the 9921 y2a gene promoter. With respect to 5'-flanking sequences to this gene, the high transcriptional activity of rearranged as opposed to unrearranged (germ line) variable-region genes in myeloma cells has been interpreted as an indication that transcriptional enhancer elements functional in myelomas do not lie 5' of variable-region coding sequences (20). In addition, in two studies, no enhancer activity was found in sequences up to 5.7 kb 5' of one germ line heavy-chain variable-region gene (35) or within 17.5 kb 5' of another (21). We previously proposed that the IgH enhancer independence of the endogenous 9921 gene might be explained if this enhancer were required only to establish high-level gene expression and not to maintain it (37). The findings of Wang and Calame (35) and Atchison and Perry (4) are consistent with this establishment-only model. In a competition exper-

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LOG (DIL) FIG. 5. Titration curves of immunoglobulin present in cytoplasmic lysates of 16C1.1 transformants. Nonidet P-40 cytoplasmic lysate (150 ,ul) made from 3 x 106 cells was serially diluted, and 50-pA portions of each dilution were incubated with the coated wells. The optical density at 405 nm (OD 405) is a measure of the enzyme-linked immunosorbent assay reaction product. pvcDEJ transformants were grown in MHX before use in this assay; their pvcDAEJ derivatives were grown in 6-thioguanine. (A) The titration curves of lysates from two independent pvcDEJ transformants (grown in MHX) are shown, as well as the curves of lysates from their 6-thioguanine-resistant (pvcDAEJ) derivatives. In this panel and the other panels of this figure, the curves for pvcDEJ and pvcDAEJ clone pairs are plotted similarly, e.g., the curves for pvcDEJ-80 (curve 80) and its pvcDAEJ derivative are both plotted as broken lines. The pvcDAEJ curves overlap that of 16C1.1, which is indicated by 16C1. In this, and in all enzyme-linked immunosorbent assays shown, a titration of purified y2a protein was run as a standard. In the assay shown, an optical density of 0.75 corresponded to a protein concentration of 0.50 p.g/ml. Transformants shown produce 0.7 (curve 90) to 5.8 (curve 80) p.g of -y2a per ml of cytoplasmic lysate. (B) Titration curves of lysates from pvcDEJ transformant 72 and its pvcDAEJ derivative. Curves are denoted as described in panel A. An optical density of 0.75 corresponds to a protein concentration of 0.27 pug/ml in this assay. (C) Titration curve of pvcDEJ-57 and its pvcDAEJ derivative. Curves are denoted as described in panel A. An optical density of 0.75 corresponds to a protein concentration of 0.38 p.g/ml. (D) Titration curve of pvcDEJ-52 (curve 52) and its pvcDA&EJ derivative, as well as titration curves for two pvcDJ transformants (DJ19 and DJ1). An optical density of 0.75 corresponds to a protein concentration of 0.09 p.g/ml.

iment, Wang and Calame showed that a simian virus 40 enhancer-activated gene, once activated, would continue expression after extensive depletion of enhancer-binding, trans-acting factors. Atchison and Perry have described a myeloma cell line in which the K immunoglobulin light chain is expressed in the absence of a trans-acting factor (NFKB) known to be required for intron enhancer function of that gene. In both of these cases, therefore, the trans-acting factors required for enhancer activity are absent and yet genes that were activated prior to the loss of these factors continue to be expressed. These results suggest that either the enhancer requires these factors only transiently for its activity or the enhancer itself is only transiently required by the gene it activates, consistent with the establishment-only model of enhancer function. Because a transfected gene requires activation upon its entry into the cell, it must be introduced into the cell in the presence of an enhancer. If enhancer-mediated activation of a transfected gene were qualitatively the same as for an

endogenous gene, however, the establishment-only model predicts that expression of a transfected gene would be enhancer independent after the initial activation of the gene. To test this model, we designed an experiment in which the IgH enhancer would be provided to initiate efficient expression of a transfected gene but subsequently would be removed. The IgH variable and constant regions of the transfected genes used (pvcDEJ and pvcDJ) were those of the gene we previously showed to be enhancer independent in its endogenous position (the 9921 gene [37]). These constructions were introduced into pre-B cells. Pre-B cells were used because deletion of the IgH enhancer after gene activation was achieved by D-J joining. The recombinase responsible for this joining is found in pre-B cells and thymocytes (2, 16), but not in myelomas. The activation of transfected immunoglobulin genes in pre-B cells is IgH enhancer dependent, as has been shown by others (9, 11, 19) and as is evident from the lack of -y2a gene expression in the pvcDJ transformants. The pvcDEJ transformants, which

ROLE OF IgH ENHANCER IN TRANSFECTED GENES

VOL. 10, 1990

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*

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FIG. 6. Poly(A)+ RNA blot analysis of transformed cell lines. 9921 (-y2a producer) poly(A)+ RNA served as a control for the expected ca. 2-kb transcript (the y2a gene in the DEJ and DAEJ constructions consists of the variable- and constant-region sequences of 9921 [see Materials and Methods]). The arrow points to y2a mRNA present in DEJ (lanes E) but not in DAEJ (lanes AE) transformants. Representative data shown are from a single gel blot and include three DEJ transformants and their DAEJ derivatives (lanes 57, 90, and 67), and one DJ transformant [lane DJ(19)]. The DAEJ-67 clone is one that deleted the IgH enhancer by a mechanism other than D-J joining (see text). However, it retains the y2a transcription unit (data not shown). Poly(A)+ RNA was electrophoresed on formaldehyde-agarose gels, blotted to nitrocellulose. and hybridized to y2a-CH3 (see Materials and Methods). The quantity of poly(A)+ RNA used in this analysis was approximately 10 ,ug (optical density at 260 nm) for most of the transformants and 2 to 3 ,ug for 9921. Poly(A)+ RNA for the pvcDEJ-90 (90E) transformant was obtained in low yield and was not quantitated. therefore, before gel analysis. A 20-min exposure of the 9921 gel lane is shown. The RNA gel lanes for the 90 (E and AE) transformants were exposed for 5 days; the remainder of th.z gel blot (57 and 67 transformants and the DJ transformant) was exposed for 3 days. This blot was stripped and rehybridized with an actin cDNA probe. Exposure of the actin hybridization was done for 3 h. Size markers are indicated to the right of the blots and correspond to 2.4- and 1.4-kb RNA fragments present in an RNA ladder standard (Bethesda Research Laboratories, Inc.). carry the y2a gene with an adjacent IgH enhancer, produced y2a protein. After deletion of the IgH enhancer, however, none of these transformants produced y2a protein or y2a mRNA although most (11 of them) retained an intact y2a transcription unit. These results indicate that the transfected -y2a gene is dependent upon the IgH enhancer for maintenance as well as for activation of its expression. In an independent study by Grosschedl and Marx (12), a strategy similar to the one described here was used to measure the effect of enhancer deletion upon a transfected gene that was being actively transcribed in pre-B cells. The gene used in that study encoded a ,. heavy chain and was composed, therefore, of a different set of immunoglobulin locus-derived sequences from those used in our -y2a gene construction. Nevertheless, as with the transfected -y2a gene, transfected ,u gene expression ceased upon loss of the IgH enhancer. It is noteworthy that the pre-B cell line used in the gene study and that used in our y2a gene study are unrelated and represent apparently different stages in pre-B cell develop-

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ment. PD31, used in the p. gene study, does not yet express endogenous IgH genes, whereas the pre-B line of our study (16C1.1 is a derivative of 18-81) does. It has been shown recently that these two pre-B cell lines have different profiles with respect to IgH enhancer-binding proteins (31). It was postulated that an enhancer-binding protein that is present in PD31 but absent in 18-81 (16C1.1) plays a negative role in the control of IgH enhancer function. The authors suggest that this factor may impede IgH enhancer function at the early stages in pre-B cell development and that its loss may be necessary before the IgH enhancer can assume full activity. It is significant, if not surprising, therefore, that transfected genes in PD31 and 18-81 behaved similarly with respect to enhancer function as measured in our study and that of Grosschedl and Marx (12). We have shown that IgH genes transfected into pre-B cells require the IgH enhancer to maintain their expression. Endogenous IgH genes that lack the IgH enhancer show no evidence of this requirement when expressed in myeloma and hybridoma cell lines. Grosschedl and Marx (12) and Schaffner (30) have proposed that additional enhancers (or equivalent genetic signals) lie in or near the IgH locus and can substitute for IgH enhancer function. A second enhancer, with B-cell-specific activity, has recently been found 9 kb 3' of the CK gene (22). The presence of this enhancer may explain the NFKB-independent expression of the endogenous K gene described by Atchison and Perry (4) and mentioned above, although the factor requirements of this second enhancer are not yet known (it includes an 8-of-10 match with the NFKB-binding site). In this and a previous study (37), we have searched for an analogous, "flanking'" IgH enhancer that might explain continued y2a expression in the IgH enhancer-deficient 9921 myeloma line. We have found no evidence of enhancer activity within the Cy2b-Cy2a region of the IgH gene cluster; our constructions cover most of the DNA lying between 2.5 kb 5' of Cy2b and 8.4 kb 3' of Cy2a. However, these results are not conclusive, since enhancers can act over long distances. If such a second IgH enhancer exists, it might be expected to have special features. The tissue specificity of

normal IgH expression would probably require that the enhancer be B cell specific. It might also be stage specific in its activity. Although IgH enhancer deletions in myeloma cells result in no change in IgH gene expression, instances have been reported in which similar deletions occurring in pre-B cells have resulted in the loss of IgH expression (3, 34). The latter findings are consistent with both the stablechange and the flanking-enhancer models, if it is assumed that the ability to induce a stable change or to activate the flanking enhancer(s) is acquired after the pre-B cell stage of B lymphocyte development. With respect to the flankingenhancer model, these findings suggest that the activity of such a flanking enhancer would be not only tissue specific but also limited to the later stages in B cell development. An alternative to the flanking-enhancer model is one in which the IgH enhancer induces a stable, activated state upon the gene it controls. Certainly, our results with the pvcDEJ constructions argue that the IgH enhancer does not function in this way under all circumstances. However, the results of the present study pertain to transfected genes in pre-B cells. It remains to be determined whether enhancer activation of a transfected gene is equivalent to that of an endogenous IgH gene and whether enhancers function identically in pre-B and myeloma cells. One way in which transfected and endogenous IgH genes differ is in their chromosomal position. Although it is known

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which DNA sequences (the IgH enhancer) are required to establish high-level transcription of an IgH gene, it is not known whether these alone are sufficient to establish the enhancer-independent state of IgH genes in myelomas. It is possible that IgH locus sequences not included in our constructions interact with the enhancer and/or promoter upon gene activation to establish a stable, active state. Such sequences need not be enhancers themselves. It can be concluded that if such sequences exist, they do not lie within the immunoglobulin coding or noncoding sequences of either our transfected gene construction or that of Grosschedl and Marx (12). Another reason why enhancer activation of a transfected gene might differ qualitatively from that of an endogenous gene is that they have very different developmental histories. That is, if the activation of an endogenous IgH gene and its subsequent progression toward constitutive expression involve an ordered series of events spanning several stages in the B-cell developmental program, it is unlikely that these events will be reenacted for the transfected gene in cell culture. It is also possible that the transfected genes used in the studies reported here require the enhancer even after activation, because the establishment of an enhancer-independent, active state does not occur at the pre-B cell stage of lymphocyte differentiation. These experiments were done with pre-B cells because of the requirement for D-J recombinase activity. However, the bulk of the evidence for enhancer-independent maintenance of gene transcription was found with cells that represent a later stage in B lymphocyte differentiation. Enhancer activation of an immunoglobulin gene in pre-B cells may be qualitatively different from that in myeloma cells. Indeed, B lymphocyte differentiation begins with the pre-B cell and ends in the plasmacyte. Irreversible commitment to immunoglobulin expression may not take place or be desirable at the earliest stages in differentiation-a time when mechanisms for eliminating or inactivating autoreactive cells are likely to operate. It might follow that this level of commitment to IgH expression can occur only later, perhaps after a cell has been induced by antigen and helper T cells to proliferate, differentiate, and form memory B cell and plasmacyte pools. These three explanations for the enhancer-dependent maintenance of transfected IgH gene expression are not mutually exclusive. For example, establishment of a stable, active state may require additional flanking sequences to those included in our transfected-gene constructions, may involve an ordered series of developmental steps, and may not take place at the early, pre-B cell stage of lymphocyte differentiation. Experiments like those described in this study but designed to allow for enhancer deletion in myelomas can be used to address aspects of all of these models. Such experiments are in progress. ACKNOWLEDGMENTS We thank Michael Kuehl for supplying the hgprt-deficient derivative of the 18-81 pre-B cell line. We thank Hanna Radomska for expert technical assistance and Stephanie Lieberman for computer renditions of the enzyme-linked immunosorbent assay data. We are grateful to Janet Kurjan and Michael Young for critical reading of the manuscript. These studies were supported in part by Public Health Service grant A121578 from the National Institutes of Health to L.A.E. B.P. and D.M.Z. were supported by National Institutes of Health training grant GM07216.

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ROLE OF IgH ENHANCER IN TRANSFECTED GENES

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