Tandem kappa immunoglobulin promoters are equally active in the presence of the kappa enhancer: Implications for models of enhancer function

Cell, Vol. 46, 253-262,

July 16, 1966, Copyright

0 1966 by Cell Press

Tandem Kappa lmmunoglobulin Promoters Are Equally Active in the Presence of the Kappa Enhancer: Implications for Models of Enhancer Function Michael L. Atchison and Robert P. Perry Institute for Cancer Research Fox Chase Cancer Center 7701 Burholme Avenue Philadelphia, Pennsylvania 19111

Summary Transcription of immunoglobulin K genes is regulated by enhancer and promoter elements, both of which function in a tissue-specific fashion. We have studied the interaction of these elements by transfecting plasmacytoma cells with genes that have tandem K promoters located next to a single K enhancer and assaying these genes for transient or stable transcription. We find that the promoters located proximal and distal to the enhancer function identically whether they are separated by 440 bp or by 2.7 kb or whether they are located 1.7 or 7.7 kb away from the enhancer. Our reK enhancer sults indicate that the immunoglobulin does not operate as a bidirectional entry site for RNA polymerase or for other factors associated with the transcription complex. Rather, they suggest that the enhancer exerts its influence uniformly over large distances and independently of the presence of intervening promoters. Introduction Production of a functional immunoglobulin K gene requires the somatic recombination of one of several hundred germ-line V, genes to one of four joining (J) segments (Max et al., 1979; Sakano et al., 1979). Unrearranged V, genes are transcriptionally silent (Mather and Perry, 1981) are in a DNAase l-resistant conformation, and are methylated (Storb et al., 1981; Weischet et al., 1982; Mather and Perry, 1983). Upon rearrangement to the C, locus, they acquire a DNAase l-sensitive conformation, become hypomethylated, and are transcriptionally active (Mather and Perry, 1983). Since there are no significant differences in DNA sequence between germ-line and rearranged V, promoters (Nishioka and Leder, 1980; Pech et al., 1981) somatic recombination to the C, locus appears to be required for their transcriptional competence. Recently, sequences required for transcription from V, promoters have been identified within the large intron separating the J region from C, (Queen and Baltimore, 1983; Picard and Schaffner, 1984; Bergman et al., 1984; Potter et al., 1984). These DNA sequences are similar to viral enhancers (see Khoury and Gruss, 1983) in that they activate transcription from eukaryotic promoters in cis, function in either orientation, operate over large distances, and can activate some heterologous promoters. Similar enhancers are also associated with the immunoglobulin heavy chain genes (Gillies et al., 1983; Banerji et al., 1983; Neuberger, 1983; Mercola et al., 1983;

Mason et al., 1985). lmmunoglobulin K enhancers have been found to function only in cells of lymphoid origin and thus operate in a tissue-specific manner (Queen and Baltimore, 1983; Stafford and Queen, 1983; Picard and Schaffner, 1984; Potter et al., 1984). Competition experiments have suggested the existence in lymphoid cells of titratable, trans-acting factors that interact with the heavy chain enhancer (Mercola et al., 1985) an interpretation corroborated by DNAase I footprinting studies (Ephrussi et al., 1985; Church et al., 1985). Presumably, such factors are also required for the function of K enhancers. Other recent studies have shown that DNA sequences in the region of the V, promoters are also important for efficient expression of immunoglobulin genes in lymphoid cells. Deletion analyses of immunoglobulin K promoters have determined that sequences approximately 90 bp 5’ of the cap site in K chain genes are required for transcription (Bergman et al., 1984; Falkner and Zachau, 1984). Interestingly, inspection of the sequence in this region has revealed the existence of an octanucleotide sequence (ATTTGCAT), which is highly conserved in all K chains (see Parslow et al., 1984) and which appears to serve as a binding site for a nuclear factor (Singh et al., 1988). Heavy chain genes contain the same octanucleotide sequence in the reverse complement orientation at a similar relative position. It is now known that immunoglobulin K promoters, in addition to the K enhancer, operate in a tissue-specific manner. Thus, when the K enhancer is replaced with a viral enhancer that functions in lymphoid as well as nonlymphoid cells, transcription originating from the K promoter is still observed only in lymphoid cells. (Foster et al., 1985; Picard and Schaffner, 1985; Gopal et al., 1985). While the tissue-specific activities of the K promoter and enhancer sequences are clearly both required for transcription, their mode of interaction is presently unclear. One model of enhancer function suggests that enhancers provide a bidirectional entry site for RNA polymerase molecules or for other factors associated with the transcription complex (Wasylyck et al., 1983). This model was proposed on the basis of promoter dampening effects in which expression from promoters located distal to the SV40 enhancer were thought to be inhibited by enhancerproximal promoters (Wasylyck et al., 1983; Moreau et al., 1981; de Villiers et al., 1982; Kadesch and Berg, 1983). Subsequently, however, it has been suggested that this dampening effect may be largely due to the distance of the promoter from the SV40 enhancer regardless of whether a proximal promoter is present (Wasylyck et al., 1984). Other models of enhancer function involve a general opening of surrounding chromatin, interactions between proteins bound at enhancers and promoters, or the ability of enhancers to target promoters to active regions of the nuclear matrix (see Khoury and Gruss, 1983). Presently, it is unclear which of these models applies to the immunoglobulin enhancers. These enhancers are lo-

Cell 254

cated much farther from their promoters (2.5 to 3.5 kb for K) than are viral enhancers (e.g., SV40 is only 107-251 bp 5’of the early transcription cap site). If a promoter dampening effect is largely due to the distance of the promoter from the enhancer sequence, as suggested by Wasylyck et al. (1984) there may be no dampening effect of tandem V, promoters driven off of the K enhancer. On the contrary, if cellular enhancers do indeed serve as bidirectional entry sites for RNA polymerase or other factors associated with the transcription complex, one would predict that a promoter proximal to the enhancer should dampen the activity of a more distal promoter by acting as a sink for such molecules and preventing them from reaching the second promoter site. Recently, Wang and Calame (1985) have reported that a germ-line Vu promoter 17.5 kb upstream of the heavy chain enhancer is active in an IgG-secreting plasmacytoma, even though there is a rearranged VH promoter between it and the enhancer. However, a rigorous assessment of enhancer influence on the transcriptional activity of adjacent promoters requires that the enhancer be operational at the time of assay, and this may not be the case for these Vu promoters. The finding of cell mutants in which endogenous heavy chain genes remain active even though the enhancer has been completely deleted suggests that the heavy chain enhancer may not be continually required for immunoglobulin transcription (Wabl and Burrows, 1984; Klein et al., 1984; Eckhardt and Birshtein, 1985). In this view, the enhancer function is presumably needed for the establishment of transcriptional activity at the immunoglobulin locus, but not for the maintenance of the transcriptionally active state. Indeed, Zaller and Eckhardt (1985) have observed that a heavy chain gene that is still transcriptionally active at its endogenous location despite the lack of an enhancer nevertheless requires the enhancer in order to be transcribed when introduced into plasmacytoma cells by transfection. Thus, it appears that transcriptional activation of Vu promoters requires enhancer function, but at later times the enhancer may not be functional or may be subordinate to other regulatory elements. For a mechanistic evaluation, it is imperative to study enhancer function during the initiation of transcriptional activity, when the enhancer is known to be required. This condition is satisfied in experiments involving the transfection of exogenous DNA into cells, but might not be satisfied when measuring the activity of endogenous genes in plasmacytomas. To settle the question of whether an intervening promoter would influence the functional interaction between the K enhancer and promoter elements, we have constructed plasmids carrying tandem V, promoters linked to the immunoglobulin K enhancer sequence and studied the activity of each promoter in transient and stable transfection assays. In both assay systems, we find no evidence for promoter dampening effects by a proximal promoter on a distal one, regardless of whether the promoter sequences are separated by 440 bp or by 2.7 kb. This finding is inconsistent with the bidirectional entry model. Moreover, we find that the K enhancer can activate tandem promoters with equal efficiency irrespective of whether

they are located 1.7 or 7.7 kb away, suggesting that the domain of influence of this enhancer encompasses a rather broad zone. Results Transcriptional Activity of Tandem V, Promoters Adjacent to the K Enhancer To study the activity of the K enhancer on tandem V, promoters, plasmids were constructed containing a single K enhancer and two functional variable region genes. The V, genes chosen for study were the V,19A gene expressed in plasmacytoma MPCll (Mather and Perry, 1983) and the V,21E gene expressed in plasmacytoma PC8884 (Heinrich et al., 1984). The promoter regions of both of these genes have been sequenced in our laboratory, and their transcriptional start sites can easily be monitored by Sl nuclease analysis. The predominant V,19A cap site has been mapped by direct chemical analysis of K messenger RNA to a G residue three nucleotides upstream of the translational start site (Kelley et al., 1982). A similar analysis indicated that transcripts emanating from the V,21C gene, a close relative of V,21E, initiate at several closely spaced residues approximately 30 bp upstream of the translational start site (Kelley et al., 1982; unpublished results). As will be seen below, this heterogeneity in cap sites is reflected in a multiplicity of Sl-protected fragments for both V,21E and V,21C transcripts. RNA isolated from MPCll cells serves as a positive control for Sl assays of both V,19 and V,21 transcripts because these cells contain a transcriptionally active, but aberrantly rearranged V,21C gene in addition to a productively rearranged V,19 gene. The V,21C transcripts are spliced in such a way that the leader exon is joined directly to C, rather than to the V region, resulting in a mature mRNA of approximately 800 bp (Seidman and Leder, 1980; Perry et al., 1980; Choi et al., 1980). Several of the constructs used are shown schematically in Figure 1. V21 is the V,21E gene, including 1.8 kb of sequence 5’ of the promoter and 1.1 kb of sequence 3’ of C,, inserted at the BamHI-Xbal sites of plasmid pUC18. For one series of experiments, a 3.6 kb BamHl fragment containing the early region of polyoma virus was inserted at the vector BamHl or Smal sites to allow replication of the plasmid in mouse cells. When constructs lacking the polyoma replicon (those prefixed by X) were used, the transfection efficiency was monitored by a Southern blot analysis of DNA in the Hirt supernatant fraction (Hirt, 1967). In plasmid V21V19, a 2.3 kb Xbal fragment, which contains the V,19A gene and 400 bp of 5’-flanking sequence and extends 3’to the Xbal site in the J-C, intron, was inserted at the intronic Xbal site in V21. In this construct, the V,21E and V,19A promoters are approximately 2.8 kb apart and are 6.2 and 3.4 kb from the enhancer, respectively. In plasmid V19V21, the same V,19A Xbal segment was inserted 1.8 kb upstream of the V,21E gene such that the promoters were 3.7 kb apart and 7.7 and 3.9 kb from the K enhancer. To determine whether any pOtential transcriptional dampening of the upstream promoter may be caused merely by the insertion of a relatively large

Interaction

between K Enhancer

and Promoter

255

-

108

-36

I23456 Figure 1. Map of lmmunoglobulin Tandem V, Promoters

K Constructs

Containing

Single or

Segments from the mouse immunoglobulin V,21E gene are represented by open bars; those from the V,19A gene are stippled. Filled regions indicate exons. The position of the K enhancer (Queen and Stafford, 1984) is denoted by cross-hatching, and plasmid pUC18 DNA is shown as a thin line. In plasmid V21L19, T indicates the position of the TATA box and 0 indicates the location of the conserved octanucleotide sequence (Parslow et al., 1984). Constructs denoted by two names were inserted into vectors containing or lacking polyoma sequences; those prefaced with the letter X lack polyoma sequences. The symbols Py and the arrow indicate the position and transcriptional orientation of a 3.6 kb BamHl DNA fragment containing the polyoma early region, which was inserted into the polyoma-containing plasmids. The parentheses encompassing the enhancer element denote the boundaries of the deletions in constructs XE-V21 and XE-V21V19. L: leader exon; V: variable region exon; C,: K constant region exon; E: enhancer sequence; Ah: Ahalll: Av: Avall; 8: BamHI; Bg: 88111; Bn: Banll; H: Hindlll; N: Ncol; S: Sall; X: Xbal.

(2.3 kb) segment of DNA between the upstream promoter and the enhancer, plasmid V21L19 was constructed. In this plasmid, only 320 bp of DNA containing the V,19A leader exon, 198 bp of 5’-flanking sequence, and 48 bp of intron were inserted into the intronic Xbal site of plasmid V21 such that the promoters were 2.6 kb apart and 4.2 and 1.7 kb from the K enhancer. Thus, the V,21E promoter was moved only 320 bp farther upstream from the K enhancer than in its normal position. The above constructs were transfected into S194 plasmacytoma cells by the DEAE-dextran/chloroquine diphosphate method; and 36-40 hr later, total cytoplasmic RNA was assayed for transcripts initiating at the proper start sites by quantitative Sl nuclease treatment and electrophoresis of the Sl-protected fragments on denaturing polyacrylamide gels. The RNA from cells transfected with plasmid V21 protected fragments of 53 to 60 nucleotides (Figure 2A), identical to those observed in the control MPCll RNA, indicating accurate transcriptional initiation approximately 30 bp upstream of the translational start site. RNA from cells transfected with constructs V21V19, V19V21, and V21L19 yielded identically sized V,21E fragments with no apparent difference in promoter activity, regardless of which promoter was proximal to the enhancer or whether the distance between one of the promoters and the enhancer was only 1.7 kb or as much

12 Rsa I

RSOI

I

345

Hinf I

I

Hinf

l-.-A7GiE+

d 53-60NT K

21E

l08NT

*

-e 36NT KiSP,

Figure 2. Tandem V, Promoters Are Activated by the K Enhancer S194 cells were transfected with the polyoma sequence-containing constructs shown in Figure 1, and total cytoplasmic RNA was isolated 36-40 hr later. One hundred ug of RNA from each transfection was assayed by quantitative Sl nuclease analyses with single-stranded probes specific for V,21 (A) or V,19 (6) transcription. The Sl probes are diagrammed in the bottom panels with the length of the undigested probe and the distance from the 32P-labeled nucleotide to the cap site indicated. MPCll DNA was used as a positive control for both probes since it contains transcripts initiating from genes of both V, families. Ten and 3.3 ug of total cytoplasmic MPCll RNA were used with the V,21E and V,19A probes, respectively. After hybridization and Sl nuclease digestion, the protected DNA fragments were electrophoresed on either 6% (A) or 10% (B) polyacrylamide-urea gels, and the bands were visualized by autoradiography. Above each lane is indicated the cell from which the RNA was isolated, followed (in parenthesis) by the construct used for transfection. Arrows show the position of the full-length, protected probe (due to reannealing of incompletely separated strands and protection by RNA transcripts initiated upstream of the probe) and the DNA fragments protected by correctly initiated transcripts. The numbers adjacent to each arrow represent the size of each band in nucleotides. The dotted arrow shows the position of a second V,21C initiation site used in MPCll cells, which is observed to a much lesser extent in the V,21E gene used in our constructs (see Figure 6).

as 7.7 kb (Figure 2A). Analysis of transcripts initiated by the V,19A promoter yielded identical results: the V,19A gene was expressed at the same rate irrespective of whether it was located proximal or distal to the K enhancer (Figure 28). In MPCll cells, a minor fraction of V,21 transcripts are initiated at a secondary site about 18-25 nucleotides upstream of the major start sites, yielding a 78 nucleotide Sl-protected fragment. Although transcripts initiating from this site are not detected in transient expression experiments, they are clearly observed in stably transformed cells (see Figure 5). Identical results were obtained with constructs lacking the polyoma replicon (see Figure 4A, lanes 3-5), demonstrating that the equivalent expression of the tandem K promoters is not caused by the presence of this element.

Cell 256

&A.

L,19 . ... / ‘. -\ / puCl8 ’ ’

XL19

OT

E w

CK

m

I-

OT

{14c21E [ iLKI Mr........ i.C....‘....i.... c -. -\ -. /- _/’

-..

XL21L19

/

f OT

T

i kb Figure 3. Map of K Constructs Proximity

Containing

V, Promoters

in Close

Leader region segments from the mouse immunoglobulin V,21E gene are represented by open bars; those from the V,19A gene are stippled. Filled regions indicate exons. The position of the K enhancer is denoted by cross-hatching, and plasmid pUC18 DNA is shown as a thin line. T and 0 indicate the positions of the TATA box and the conserved octanucleotide sequence, respectively. L: leader exon; C,: K constant region: E: enhancer sequence.

To determine whether there is any promoter dampening when K promoters are brought in Close proximity, we constructed the plasmids shown in Figure 3. Plasmid XL19 contains the 320 bp V,lQA promoter and leader exon DNA segment used in construct V21LlQ (see Figure 1) inserted at the intronic Xbal site 1.5 kb upstream of the K enhancer. This promoter thus lies approximately 1.7 kb upstream of the K enhancer. In plasmid XL21L19, a 470 bp DNA fragment containing the V,21E leader exon, 230 bp of 5’-flanking sequence, and 170 bp of intron, were inserted 5’ of the V,lQA promoter in XL19 such that the promoters were only about 440 bp apart (the distance between TATA boxes) and 2.1 and 1.7 kb from the enhancer. In addition, plasmid XL21LlQ.O- was constructed in which the proximal V,lQA promoter was replaced by a nonfunctional V,lQA promoter lacking critical sequences upstream of -42 (Falkner and Zachau, 1984; Bergman et al., 1984). When these constructs were transfected into S194 cells and assayed for accurately initiated V,lQ transcripts, identical activities were observed for LlQ and L21L19, whereas, as expected, no activity was detected for L21LlQ.O- (data not shown). When transcription from the V,21E promoter was assayed, constructs XL21LlQ and XL21LlQ.O- yielded Sl-protected fragments identical in size and frequency to those derived from constructs XV21VlQ and XVlQV21 (Figure 4A). Thus, transcription from the V,21E promoter is similar whether there is a functional or nonfunctional K promoter between it and the K enhancer. Furthermore, whether the K promoters are 3.8 kb or 440 bp apart, there is no dampening effect on a distal promoter by an enhancer-proximal promoter. To confirm that the transcriptional activity of these constructs is totally dependent on the function of the K enhancer, we examined the activity of two constructs,

XE-V21 and XE-V21V19, in which the enhancer region was deleted from plasmids XV21 and XV21V19, respectively (see Figure 1). These two constructs and plasmid XV21 were individually transfected into S194 cells, and RNA was analyzed by Sl nuclease protection of the V,21 probe (Figure 48). While construct XV21 produced readily detectable amounts of transcript initiating at the V,21 cap site, none was detected in RNA isolated from cells transfected with the two enhancer-lacking constructs. Analysis of Hirt supernatants of the transfected cells indicated that the absence of expression of the enhancer-lacking piasmids is not due to a lack of DNA entering the cell. Nor is it due to faulty RNA processing or decreased transcript stability, as evidenced by the fact that normal expression was observed when the K enhancer was reinserted upstream of the V, promoter(s) in these two constructs (data not shown). Lack of Promoter Dampening Effect in Stable Transformants Since circular plasmids are used in transient transcription assays, it is formally possible that the K enhancer is indeed operating as a bidirectional entry site for transcription factors and that the distal promoter is actually being driven by factors approaching it from the opposite direction on the circle. To control for this possibility, we examined the activity of one of the constructs when it was linearized and stably integrated into the mouse genome. For these experiments we used either a gel-purified 9.6 kb BamHl DNA fragment derived from plasmid XV21VlQ (see Figure 1) or plasmid XV21VlQ linearized at the unique Sal1 site in the vector polylinker region, which places all plasmid sequences on the 3’side of the immunoglobulin gene. These DNAs were individually cotransfected with BamHllinearized pSV2gpt into S194 cells by electroporation (Zimmerman and Vienken, 1982; Potter et al., 1984). Seven mycophenolic acid-resistant clones (Mulligan and Berg, 1981) were isolated, and their RNA was analyzed by Sl nuclease protection. As with the transient expression experiments, transcripts initiating accurately from both the V,21 and V,lQ promoters were detected in all of the stable transformants. As illustrated by the representative data of Figure 5, the relative proportions of V,21 and V,lQ transcripts are similar among the various transformants. In transformants that exhibit high levels of expression, e.g., V21/19-1, the amount of RNA initiated from the V,lQ and V,21 promoters is about 0.2 and 0.5 times that produced from the corresponding endogenous genes in MPCll cells. These conclusions from the Sl protection experiments were fully corroborated by a quantitative analysis of Northern blot data (see below). Copy Number and Context of Integrated K Genes The approximate number and organizational status of the K genes introduced into the S194 transformants was determined by Southern blot analysis. DNA from each of the transformants and from untransformed S194 cells was digested with Hindlll, and in some cases with EcoRI, and probed for the 5’and 3’portions of the introduced K gene, as well as for the cointegrated plasmid, pSV2gpt (Figure

Interaction 257

between

K Enhancer

and Promoter

Figure 4. The K Enhancer Activates V, Promoters at Various Distances

-V,2

I Probe

-vv,2i 12345678 DNA-

123 111’1

O-LSp)

-DNA

Sl nuclease analysis of RNA isolated from S194 cells transfected with the polyomalacking constructs shown in Figures 1 and 3 was performed as in Figure 2 with the V,21 single-stranded probe. After hybridization and Sl nuclease digestion, protected DNA fragments were electrophoresed on a 6% polyacrylamide-urea gel and visualized by autoradiography. Arrows denote the position of the full-length probe and correctly initiated transcripts The dotted arrow shows the position of a second V,21C initiation site used in MPCll cells. To monitor the relative transfection efficiencies, Hirt supernatant DNA was prepared from each sample of cells, digested with EcoRl and Hindlll, and analyzed by Southern blotting with nick-translated plasmid pUC18. EcoRIHindlll digestion excises the plasmid vector sequences from all constructs used, so that a common hybridizing band of identical size is obtained with all transfection samples.

W.Sp)

Figure 5. Si Nuclease Analysis lated from Stable Transformants

VK21 Probe-

(+ 1 .

v,21-

6). In addition to the two C-containing Hindlll fragments corresponding to the endogenous K alleles of S194 cells, the cells transfected with linearized XV21V19 (clones V21/19-2, -3, -6, and -7) exhibited a common 5.6 kb fragment extending from the Hindlll site in the J-C intron to a site in the pUC18 vector. For these clones, the number of integrated V21V19 genes was estimated by comparing the intensity of the 5.6 kb band with the intensity of bands representing the endogenous K alleles. ln cells transfected with the purified V21V19 BamHl fragment (clones V21/19-1, -4 and -5), the integrated genes yield novel Hindlll fragments extending from the intronic Hindlll site to a 3’site that is unique for each integration event. For

Tandem

-V,i9

Probe

of RNA Iso-

Total cytoplasmic RNA was isolated from cells stably transformed either with a 9.6 kb DNA fragment containing tandem V, genes (V21/191) or with the plasmid XV21V19 linearized with Sal1 (V21/19-2, V2lNl9-3). Total cytoplasmic RNA (50 ug from transformants and 100 pg from untransfected S194 cells) was assayed by Sl nuclease analysis with probes specific for V,21 (A) and V,l9 (B), shown in Figure 2. The amount of MPCll RNA used in each positive control hybridization is indicated in pg above the lanes, After hybridization and Sl digestion, protected DNA fragments were electrophoresed on an 8% polyacrylamide-urea gel and bands were visualized by autoradiography. Arrows show the position of the undigested probe and the position of the correctly initiated transcripts. The dotted arrow marks the position of the upstream transcription start site used by the V,2lC gene in MPCll cells. This same start site is used to a much lesser extent by the V,21E gene in our transfected constructs.

these clones, the number of integrated V21V19 genes is accurately given by the number of novel fragments. From this analysis we deduced that the various transformants contain from one to five copies of the V21V19 gene (Table 1). For some of the clones, it was possible to verify that the V21V19 and SV2gpt genes were integrated at separate locations in the recipient genomes. For example, SVgpt sequences in clone V21/19-1 reside on two comigrating 4.6 kb Hindlll fragments that are distinct from the 7.4 and 5.7 kb fragments that respectively contain the 5’and 3’flanks of the single integrated V21V19 gene (Figure 6B). Similarly, in clone V21/19-2, the SV2gpt genes and 3’ flanks of

Cell 258

Table 1. Expression and Copy Number of Transfected Stably Transformed S194 Cells

K Genes in

Relative RNA Content Clone

Distal (V,21)a

V21/19-

Proximal (V,l9)

1 2 3 4 5 6 7

1.6 1.1 1.3 1.5 2.7 1.3 2.0

Transformantb

$e:Fi:e,,

K

MPCll

Constructs

per CellC

v,19

-\

3’ (0) Hind

IU

0

S’(b) QPt Hind III

5’(c)< gpt(PVC) Eco RI

v2i I.

V21/19(1.4.5)

0

L

0.19 0.18 0.21 0.06 0.07 0.15 0.07

Cells

1

2 &5 3 1 $4 1

a Ratio of the intensities of the 1.4 and 1 .l kb components in Northern blots probed with V,19 V region probe (see Figure 7C). b Determined from the ratio of intensities of the 1 .I kb bands in transformants and MPCll cells, corrected for amounts of RNA loaded on gel; same autoradiograms as used for (a). c Determined by Southern blot analysis. Clones V21/19-1, V21/19-4, and V21/19-5 were transfected with the BamHl fragment isolated from the tandem promoter construct XV2lVl9; the other clones were transfected with the Sal1 linearized plasmid XV2lVl9.

PUC

s*BB9 R V2l/lSC2,3,67)-t

”

Figure 6. Southern

v21 !m

VI9 5:..._..

Blot Analysis of Integrated

DNA Sequences

DNA from various stable transformants was digested with either Hindlll (A and B) or EcoRl (C), blotted onto nitrocellulose, and hybridized with the probes listed below each panel. Arrows denote the positions of the newly integrated DNA sequences recognized by each probe; in (C) all of the bands hybridizing with the gpt (pUC) probe represent exogenous sequences. Fragment sizes in kb are indicated. The organization of the integrated genes and probe segments are diagrammed below. Wavy lines represent mouse genomic sequences flanking the integration sites, thin lines represent pUC18 sequences, and open and closed boxes represent K locus DNA as shown in Figure 1. The gpt probe is plasmid SV2gpt, which recognizes SV2gpt sequences in addition to the pUC18 sequences that constitute the 3’ends of the integrated DNA in clones V21/19-2, 3, 6, and 7.

the two integrated V21V19 genes are on EcoRl fragments that are distinct from those containing the 5’ flanks of the V21V19 genes (Figure 6C). Thus, the two V21V19 genes in this clone are neither contiguous nor located downstream of an SV2gpt sequence. From these and similar analyses of other clones we conclude that the observed activity of the tandem promoters is not attributable to contiguous integration of multiple K genes or of K genes and SV40 enhancer elements. Splicing of Tandem K Transcripts Since the tandem V, promoters were both clearly being expressed, it was of interest to determine the splicing pattern of their resultant transcripts. RNA from the stable transformants was analyzed on Northern blots that were hybridized with an appropriate set of probes (Figure 7). A 1.1 kb component that comigrated with authentic mRNA from MPCll cells was revealed with probes for the V,19 leader exon and V,19 variable region sequences (Fig-

ures 7A and 7C), indicating that the transcripts from the proximal promoter are spliced in the conventional manner. The V,19 variable region probe revealed an additional component of approximately 1.4 kb, which was also observed with the V,21 leader exon probe (Figures 76 and 7C). This result indicates that the transcripts initiated from the distal promoter are spliced so as to produce a novel RNA species containing both V,19 and V,21 sequences (Figure 7F). The same splicing pattern was observed for all seven transformants. Relative Level of Expression of Distal and Proximal Promoters The fortuitous occurrence of V,19 sequences in the products of both promoters enabled us to quantitate accurately the relative level of each RNA product by a comparison of band intensities. From densiometric scans of the 1.4 and 1.1 kb bands in blots hybridized with V,19 probe, we ascertained that the relative amount of RNA initiating from the distal (V,21) and proximal (V,19) promoters ranges from approximately 1 :l to nearly 3:l (Table 1). Thus, in all of the transformants, the quantity of transcripts from the distal promoter is greater than or equal to that from the proximal promoter. In agreement with the Sl nuclease protection assays, this analysis also indicated reasonably high levels of expression for some of the newly integrated genes, the content of V,19 mRNA per gene in one of the single copy transformants being about 20% that of the endogenous V,19 gene in MPCll cells. The same analysis was used to quantitate the relative amounts of mRNA produced from distal and proximal promoters in a typical set of transient expression experiments. S194 cells were transfected with plasmids XV21, XV21V19, or XV19V21, and 40 hr later cytoplasmic poly(A)+

Interaction 259

between

K Enhancer

and Promoter

S(L21)

A (Li9)

c (Vi91

D&l

E(Vi9)

-0.8

LV,Z~E

F”

Figure 7. Northern

L V,l9A

CK

!IiMMs#cDNA (H.Sp)

Blot Analysis of RNA Isolated from Ceils either Stably or Transiently

Expressing

Transfected

Genes

(A-C) Triplicate 10 fig samples of total cytoplasmic RNA from V21/19-1, V21/19-2, and V21/19-3 cells and 10 trg (A and B) or 1 ug (C) of RNA from MPCll cells were electrophoresed on a 1.5% agarose-formaldehyde gel and transferred to nitrocellulose. The blots were hybridized with the following nick-translated probes: (A) a 320 bp DNA fragment (L19) containing the V,19A leader exon, 198 bp of 5’-flanking, and 46 bp of intron DNA sequence; (8) a 470 bp fragment (L21) containing the V,21E leader exon, 230 bp of 5’-flanking, and 170 bp of intron DNA sequence; and (C) a 165 bp (Hincll-Hpall fragment (V19) containing sequences derived from the variable region of the V,19A gene. (D) A similar blot of a 5 ug sample of MPCll RNA was hybridized with a 1.7 kb Hindlll-Bglll fragment containing the C, region. The sizes of the MPCll V,21C (0.6 kb) and V,19A (1.1 kb) mRNAs and that of the novel 1.4 kb transcript found in the transfected cells are indicated in kilobases. The relative abundance of 0.8 and 1.1 kb mRNAs in MPCll cells, determined from the corresponding band intensities in (D), is 0.6:1. (E) S194 cells were transfected with construct XV21, XV21V19, or XV19V21 in transient expression experiments and harvested 40 hr later. Two ug of cyioplasmic poly(A)+ RNA from each transfection experiment and 5 ttg of MPCll total cytoplasmic RNA were analyzed on a Northern blot with the V19 fragment. Southern blots of DNA in the Hirt supernatant fraction were used to monitor transfection efficiencies as in Figure 4. (F) Illustrates the probable splicing of transcripts initiating at the distal (V,21E) promoter, which yields a 1.4 kb RNA component, and transcripts initiating at the proximal (V,19A) promoter, which yields a 1.1 kb component.

RNA was isolated and submitted to Northern blot analysis with the V,19 variable region probe (Figure 7E). Nearly identical levels of transfected DNA were observed in the Hirt supernatant fractions, indicating equivalent transfection efficiencies in the various experiments. As observed with the stable transformants, there are nearly equal amounts of 1.4 and 1.1 kb component in the cells transfected with XVZlV19, indicating roughly equivalent accumulation of product from distal and proximal promoters. Since this probe does not recognize V,21 sequences, it does not detect any product in the cells transfected with XV21, and recognizes only the product of the distal promoter in cells transfected with XV19V21. The level of 1.4 kb transcript in cells transfected with XV19V21 is comparable to that of the 1.1 kb transcript in cells transfected with XV21V19, indicating that the V,19 promoter produces roughly the same amount of transcript whether it is located distal or proximal to the enhancer. These results are entirely consistent with those obtained by Sl nuclease protection assays in the experiments of Figure 2 and Figure 4. Discussion The foregoing results definitively establish that the immunoglobulin K enhancer can activate tandem V, promoters irrespective of whether they are located 1.7 or

7.7 kb away. This conclusion is based on results obtained with both transient and stable expression assays of constructs in which transcription from the tandem promoters occurs at the authentic initiation sites and is totally dependent upon the presence of the K enhancer. A quantitative evaluation of the relative activity of the promoters associated with the V,19A and V,21E genes indicated that these promoters function equivalently when positioned singly, in tandem, or at a variety of distances from the K enhancer. In none of our experiments was there any indication of promoter dampening, i.e., the modulation of the activity of a distal promoter by the presence of an intervening functional promoter. Our results also rule out another, more complicated interpretation for the equal activity of the tandem promoters, namely, that it is due to a balance between promoter dampening and promoter occlusion (Adhya and Gottesman, 1982). According to this model, the distal promoter would be weakly activated by the enhancer, but its expression would be sufficient to occlude the high level of expression from the proximal promoter, resulting in roughly similar activities of both promoters. If this explanation were correct, one would expect the level of expression in the tandem promoter constructs, e.g., XV21V19, XV19V21, and XL21L19, to be much lower than that observed with the corresponding single promoter constructs, e.g., XV21 and XL19. On the contrary, we observe the same promoter

Cell 260

activity whether the construct contains a single or tandem promoters. The assessment of the relative expression of tandem and single promoters was based on measurements of accumulated amounts of RNA, either by Sl nuclease protection assays or by Northern blot analysis. In using such measurements for this purpose, we have assumed that the relative RNA levels are a reflection only of transcriptional activity and that they are not influenced by differences in RNA stability or processing efficiency. Several of our findings confirm the validity of this assumption. First, the results based on the 36-40 hr accumulation of transcripts in transient expression assays were in complete agreement with those based on measurements of steady state levels of RNA in stable transformants. Since the turnover of mRNA in plasmacytoma cells is normally very slow (tl,, > 20 hr [Marcu et al., 1978]), we would not expect agreement between these two types of measurement if RNA stability differences made a significant contribution to the results. Second, among the various constructs used to demonstrate roughly equal amounts of RNA from proximal and distal promoters are those that produce transcripts which are processed into identical molecules with presumably identical stabilities, e.g., products of the V,21 promoter in V21 or V19V21 vs. V21L19 (Figure 2A, lane 3 or 5 vs. lane 6) and in XL21L19.0- vs. XL21L19 (Figure 4A, lane 8 vs. lane 7). Third, the amount of RNA derived from a particular promoter (distal or proximal) was the same regardless of the splicing pattern of the transcript. For example, transcripts from the distal V,21 promoter in XV21V19 or V21V19, V21L19, and XL21L19 are processed respectively into equally abundant 1.4,l .l, and 0.8 kb components (Figure 2A, lane 4 vs. lane 6; Figure 4A, lane 4 vs. lane 7) and transcripts from the proximal V,19 promoter in V21V19 and V21L19 into equally abundant 1.1 and 0.8 kb components (Figure 28, lane 3 vs. lane 5). Since transcription rates are presumably equivalent for such comparisons, the equal abundance of these different components indicates that they have similar stabilities and processing efficiencies. Finally, we obtained the same results in transient expression experiments, irrespective of whether total cellular RNA or cytoplasmic RNA was used for the assay (data not shown), again indicating that the various transcripts are processed with similar efficiencies. An important feature of our experiments is that the equivalent activity of tandem promoters was established with stable transformants as well as with transient expression assays. Transient expression experiments are very effective in definitively demonstrating enhancer dependence of transcription and lack of promoter occlusion, as well as in discriminating between effects on transcription and RNA stability. However, since enhancers act over large distances to stimulate transcription from promoters located in either direction, the use of circular plasmids in transient expression experiments may introduce a certain ambiguity into the interpretation of results. This ambiguity is eliminated in stable transformation experiments in which linear DNA is integrated into the genome and isolated copies of the tandem promoter constructs are ex-

pressed at levels approaching those of endogenous K genes. The fact that the activities of the tandem V, promoters are nearly identical when located at a variety of distances from the K enhancer, ranging from 1.7 to 7.7 kb, means that the enhancer effect is distributed evenly over a broad domain of influence and that promoters falling within this domain are activated independently. This property is contrary to the idea that the immunoglobulin K enhancer functions as a bidirectional entry site for RNA polymerase or other factors associated with the transcription complex. More likely, the enhancer may function either by initiating a general domain opening of the K locus, perhaps by altering the torsional stress in DNA (Ryoji and Worcel, 1984), or by directing the K locus to transcriptionally active nuclear structures. Another possibility is that transcription is stimulated by interactions between proteins separately bound at the enhancer and promoter sequences. For this possibility to be consistent with our results, one would have to assume that such interactions are not sterically hindered, even when concurrently activated promoters are separated by as little as 440 bp. Experimental Plasmid

Procedures

Constructions

A 3.9 kb Xbal-BamHI DNA fragment containing the C, region was inserted at the BamHl and Xbal sites of plasmid pUC18 to produce plasmid pXBC,. Plasmid pXBC, was linearized with Xbal and ligated to a 4.1 kb Xbal fragment that contains the V,21E gene expressed in plasmacytoma PC6684 and 1.8 kb of 5’-flanking sequence and extends 3 to the intronic Xbal site. This clone, XV21, represents the intact, expressed K gene from plasmacytoma PC6664, including 1.8 kb of 5’flanking sequence. The XV21 plasmid was partially digested with Xbal, and the single-cut linear molecules were isolated from an agarose gel and ligated to a 2.3 kb Xbal fragment that contains the V,19A gene expressed in plasmacytoma MPCll and 400 bp of 5’-flanking sequence and extends 3’to the intronic Xbal site. Plasmid XV21V19 was produced by insertion of the 2.3 kb Xbal fragment at the Xbal site in the J-C, intron, while plasmid XV19V21 was made by insertion of the 2.3 kb Xbal fragment at the Xbal site that lies 1.8 kb upstream of the V,21E gene. Plasmids with both V genes in the correct orientation were selected by restriction endonuclease mapping. Plasmids XV21, XV21V19,and XV19V21 were linearized at the vector Smal site just 3 of the BamHl site, and a 3.6 kb BamHl fragment containing the polyoma early region was inserted by blunt-end ligation. Plasmids that contained the K gene and polyoma early region in opposite orientation were selected and designated V21, V21V19, and V19V21. To produce plasmid XL19, a 320 bp Avalldhalll DNA segment containing the V,19A leader exon, 196 bp of 5’-flanking, and 46 bp of intron DNA sequence was inserted by blunt-end ligation at the Xbal site of plasmid pXBC,. This plasmid was linearized at the vector Sall site just 5’of the Xbal site, and a 4.1 kb Xbal fragment containing the V,21E gene (see above) was inserted by blunt-end ligation to yield plasmid XV21L19. The polyoma early region was then inserted at the vector Smal site to produce V21Ll9. To produce plasmid XL21119, a 470 bp Ahalll DNA fragment containing the V,21E leader exon, 230 bp of 5’ flanking, and 170 bp of intron DNAsequence was inserted at thevector Sall site of plasmid XLIS. In plasmid XLl9.0-, a 169 bp Pvull-Ahalll DNA fragment containing the V,19A leader exon, 42 bp of 5’-flanking, and 48 bp of intron DNA sequence was inserted by blunt-end ligation at the Xbal site of plasmid pXBC,. Plasmid XL19.0- was linearized at the vector Sall site just upstream of the Xbal site, and a 470 bp Ahalll fragment containing the V,21E leader exon and promoter (see above) was inserted by blunt-end ligation to yield XL21Ll9.0-. To produce constructs lacking the K enhancer, plasmids XV21 and XV21V19were digested with Ncol, resulting in the excision of a 2.6 kb DNA segment encompassing the K enhancer and C, (see Figure 1).

Interaction 261

between K Enhancer

and Promoter

The larger of the two DNA segments in each digest, which contained the V, region and vector sequences, was purified on an agarose gel, then blunt-end ligated to a 788 bp Banll-Ncol fragment containing the C, region. The resulting plasmids (XE-V21 and XEV21V19) were identical to the original plasmids except for a 1.8 kb deletion encompassing the enhancer region. Cell Culture and DNA Transfection Plasmacytoma S194 cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% horse serum. For transient expression assays, cells were transfected by the DEAE-dextran (Pharmacia) procedure followed by treatment with chloroquine diphosphate (Sigma) as described by Grosschedl and Baltimore (1985). Stable transformants were prepared by the electroporation method according to Potter et al. (1984) and isolated by mycophenolic acid selection beginning 2 days after transfection, essentially as described by Mulligan and Berg (1981). RNA Praparation and Analysis For transient expression experiments, cells were harvested 36-40 hr after transfection and total cytoplasmic or poly(A)+ cyloplasmic RNA was isolated (Schibler et al., 1978). For Sl nuclease analysis, l-100 pg of total cytoplasmic RNA was hybridized with 2-4 ng of strandseparated, 5’-end-labeled probes in a 50 ~1 volume containing 50% formamide, 0.6 M NaCI, 10 m M PIPES, 1 m M EDTA, pH 6.5, at 45OC for 3-6 hr. Total cytoplasmic RNA from untransfected S194 cells was used to adjust the amount of RNA in each hybridization to 100 kg. After hybridization, samples were diluted with 0.45 ml of Sl buffer (3 m M zinc acetate; 30 m M sodium acetate, pH 4.5; 250 m M NaCI) and digested with 2000 U of Sl nuclease (Miles) at 20°C for 30 min. Protected DNA fragments were then analyzed on polyacrylamide-urea gels. Restriction endonuclease fragments to be labeled for use as Sl probes were dephosphorylated with bacterial alkaline phosphatase (Millipore) and then 5’-end-labeled with T4 polynucleotide kinase (Pharmacia) to spacific activities of approximately 1 x lo6 to 1.5 x 10” cpmlpmole (V,19A probe) or 0.3 x lo6 to 0.5 x lo6 cpmlpmole (V,21E probe). DNA strands were separated on strand-separating gels according to Maniatis et al. (1982). Northern blot analysis of RNA was performed on 1.50/o agarose-formaldehyde gels followed by transfer to nitrocellulose filters (Schleicher and Schuell, Inc.) as described by Thomas (1980). Blots were hybridized with nick-translated probes (Rigbyet al., 1977) in 50% formamide, 5x SSC (lx SSC: 0.15 M NaCI, 0.015 M sodium citrate), 0.1% sodium dodecylsulfate, 100 pglml denatured, sonicated salmon sperm DNA, and 5x Denhardt’s solution (0.1% each of bovine serum albumin, polyvinylpyrolidone 360, and ficol 400).

The authors are indebted to Dawn E. Kelley for providing us with the cloned V,21E gene from PC6684 and to Dr. Cary Queen for helpful advice in the initial phase of this work. The research was supported by a research grant from the National Institutes of Health and an appropriation from the Commonwealth of Pennsylvania. M. L. A. was supported by an NIH postdoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. February

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DNA from infected

Kadesch, T R., and Berg, P (1983). Effects of the position of the 72 bp enhancer segment on transcription from the SV40 early region promoter. In Current Communications in Molecular Biology, Y. Gluzman and T. Shenk, eds. (Cold Spring Harbor, New York: Cold Spring Harbor Press), pp. 21-27. Kelley, D. E., Coleclough, C., and Perry, R. I? (1982). Functional significance and evolutionary development of the 5’.terminal regions of immunoglobulin variable-region genes. Cell 29, 681-689. Khoury, G., and Gruss, l? (1983). Enhancer elements. Cell 33,313-314.

Acknowledgments

Received

Choi, E., Kuehl, M., and Wall, R. (1980). RNA splicing generates a variant light chain from an aberrantly rearranged K gene. Nature 286. 776-779.

3, 1986; revised April 24, 1986

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cell to