Expression of the human PDGF-B gene is regulated by both positively ...

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Gary C.Franklin, Mark Donovan, ... gene coding for the B-chain of the PDGF ligand is the ..... activity within the 1st intron, a series of deletion constructs.
The EMBO Journal vol.10 no.6 pp.1365- 1373, 1991

Expression of the human PDGF-B gene is regulated by both positively and negatively acting cell type-specific regulatory elements located in the first intron

Gary C.Franklin, Mark Donovan, Gail I.R.Adam, Lars Holmgren, Susan Pfeifer-Ohisson and Rolf Ohisson Laboratory for Molecular Development and Tumour Biology, Institute for Drug Research, Karolinska Institute, Box 60500, S-10401, Stockholm, Sweden Communicated by C.-H.Heldin

Potential cis-acting regulatory elements of the human platelet derived growth factor-B (PDGF-B) gene were identified by DNase I hypersensitive site mapping. The transcription unit was examined for the presence of hypersensitive sites in chromatin DNA isolated from human term placental cytotrophoblasts, human placental fibroblasts, the JEG-3 choriocarcinoma cell line and the U2-OS osteosarcoma cell line. A number of cell typespecific hypersensitive sites were identified, all within the 1st intron. Transient transfection of JEG-3 cells with CAT constructs containing regions of the c-sis 1st intron linked to the basal c-sis promoter identified a cell type-specific positive regulatory activity within the intron, composed of at least two distinct elements. One element appeared to be specific for JEG-3 cells, while the other was also active in U2-OS cells. The overall positive regulatory activity of the 1st intron region was specific for JEG-3 cells, but did not function as a classically defined enhancer, as it was orientation-dependent (unless stably integrated into chromatin DNA). In addition, the activator appears to require interaction with the c-sis promoter, as little or no activation was seen when either the SV40 or human 3-globin promoters were substituted for the c-sis promoter. The 1st intron also contained a negative regulatory element, which was specific for U2-OS cells and silenced an abnormally high basal c-sis promoter activity in these cells. The complexity of the transcriptional control of the PDGF-B gene is discussed. Key words: sis protooncogene/transcriptional regulation/ DNase I hypersensitivity/cytotrophoblasts

Introduction Human platelet derived growth factor (PDGF) is a potent growth factor for a number of cell types of mesenchymal origin (Ross et al., 1986). The PDGF ligand exists as a homo- or heterodimer of two distinct, but related gene products (PDGF-A and PDGF-B) and elicits its actions via homo- or heterodimers of the PDGF a- and/or ,3-receptor subunits (for review see Heldin and Westermark, 1989). The gene coding for the B-chain of the PDGF ligand is the cellular equivalent of the transforming gene v-sis of the simian sarcoma virus (Doolittle et al., 1983; Waterfield et al., 1983). The c-sis gene has been implicated in human cancer (Nister et al., 1988) and in addition acts dominantly Oxford University Press

to induce neoplastic transformation of receptor-positive cells in vitro (Clarke et al., 1985). PDGF may also play roles in other, non-neoplastic pathological conditions, such as tissue fibrosis (Martinet et al., 1987) and atherosclerosis (Ross, 1986). PDGF has also been implicated in the processes of tissue repair during wound healing (Pierce et al., 1989) and angiogenesis (Bowen-Pope et al., 1984) (for a recent review see Heldin and Westermark, 1990). Although the precise in vivo roles of PDGF remain somewhat speculative, its expression in the embryonic tissue of humans (Goustin et al., 1985; Ohlsson et al., 1991), mouse (Rappolee et al., 1988; Mercola et al., 1990) and Xenopus (Mercola et al., 1988), suggests an involvement in development. Indeed, we have recently found that the PDGF-BB homodimer is a potent growth factor for a subset of cytotrophoblasts which have invaded the maternal lining (L.Holmgren, L.Claesson-Welsh, C.-H.Heldin and R.Ohlsson, in preparation). In addition, the PDGF-B gene is coexpressed with the ,3 receptor gene in the growing tip of blood vessels in early human term placenta, suggesting a role in placental angiogenesis (L.Holmgren, R.Glaser, S.Pfeifer-Ohlsson and R.Ohlsson, in preparation). The maintenance of a strict spatial and temporal pattern of c-sis expression is likely, therefore, to be very important for normal placental development (Goustin et al., 1985). Surprisingly little is known about the control mechanisms involved in the regulation of such an important gene as c-sis, even outside the placental context. Transforming growth factor (TGF)-3 has been shown to induce c-sis in endothelial cells (Daniel et al., 1987), but this effect has not been characterized at the molecular level. It has been shown, however, that the K562 human haematopoietic stem cell line produces a 200-fold increase in c-sis mRNA levels when induced to differentiate into megakaryoblasts by phorbol esters, and that a region of the c-sis promoter, upstream of the cap site, is a target for such regulation (Pech et al., 1989). We decided to search the transcriptional unit of the human c-sis gene for potential regulatory regions involved in tissue-specific expression of the gene, in both placental and non-placental cell types. As the presence of cell type-specific DNase I hypersensitive sites (DHS) has been shown to correlate strongly with gene regulatory elements such as enhancers and silencers (reviewed in Gross and Garrard, 1988), we used the DHS mapping procedure (Wu, 1980) to search the c-sis transcription unit and identified a number of cell type-specific DHS in the 1st intron. Here we show that the c-sis 1st intron contains multiple elements which confer cell type-specific regulation on the transcriptional activity of the basal c-sis promoter.

Results Expression of the c-sis gene in a variety of placental and non-placental cell types Northern blot analysis (Figure 1), using a cDNA probe, showed that the steady state level of the principal 4.2 kb 1365

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Fig. 1. Northern blot analysis of c-sis expression in a variety of cell types. 10 itg total RNA from 1st trimester human placental fibroblasts (FB), term human placental cytotrophoblasts (TB), the JEG-3 choriocarcinoma cell line and the U2-OS osteosarcoma cell line was run on a 0.9% denaturing agarose gel and blotted. The filter was hybridized with a c-sis cDNA probe, PM-1 (sis cDNA), an oligonucleotide probe spanning the cap site of the c-sis 1st exon (sis oligo) and a fl-actin probe (PA-1). The sizes of the c-sis transcripts, in kb, are indicated at the side.

c-sis mRNA in purified human term placental cytotrophoblasts was comparable to that observed in the high c-sisexpressing osteosarcoma cell line, U2-OS. Both cell types also expressed the smaller, as yet uncharacterized c-sis transcript of 3.7 kb. This was in sharp contrast to in vitro passaged human 1st trimester placental fibroblasts, in which no c-sis mRNA could be detected. The 4.2 kb transcript was also undetectable in the JEG-3 choriocarcinoma cell line, although the 3.7 kb species was expressed. The use of an antisense oligonucleotide probe to the 1st exon (which covered the putative cap site of the c-sis gene) in place of the cDNA probe produced essentially the same expression pattern for the 4.2 kb transcript, but did not detect the 3.7 kb transcript in any cell type. In order to examine the promoter usage for c-sis in cytotrophoblast and JEG-3 cells, the position of the transcriptional initiation site was determined by SI mapping, using the oligonucleotide described above. Figure 2A shows that two protected fragments were observed using cytotrophoblast RNA, indicating the location of the major cap site to be 26 bp downstream of the 3' end of the consensus TATA box. This is consistent with the previous c-sis cap site determinations in K562 cells (Pech et al., 1989), total human placenta and EJ cells (Durga Rao et al., 1986). A second, minor cap site was located a further 7 bp downstream of the principal cap site. The localization of the cap sites within the promoter sequence is indicated in Figure 2B. No protected fragments were observed using RNA isolated from JEG-3 cells which, in combination with the lack of hybridization to the 1 st exon oligonucleotide probe, allows us to conclude that the frequently observed smaller transcript of c-sis does not originate from the same promoter as the principal 4.2 kb transcript. In fact, preliminary results show that the small transcript is antisense to the major c-sis transcript (unpublished observation). -

The c-sis 1st intron contains cell type-specific DNase I hypersensitive sites In order to identify potential cell type-specific regulatory elements within the c-sis transcription unit, DHS mapping 1 366

Fig. 2. Transcriptional initiation (cap) site determination for the c-sis gene in trophoblastic cell types. (A) SI mapping of the c-sis transcript in term cytotrophoblasts and JEG-3 cells. Total cytoplasmic cytotrophoblast RNA and total JEG-3 RNA were hybridized to a 32P-labelled, 72 bp oligomer (spanning the 5' boundary of the c-sis 1st exon) and digested with SI nuclease as described in Materials and methods. The digested RNA-DNA hybrids were then electrophoresed on an acrylamide gel, along with a sequence ladder (psisCAT plasmid, sequenced using the Sequenase system) and 32P end-labelled HaellIdigested PhiX 174 mol. wt markers. The lane details are indicated at the top of the figure; GATC, four sequence lanes for the psisCAT plasmid sequencing reaction; M, mol. wt markers; 1, JEG-3 RNA; 2, yeast tRNA; 3, cytotrophoblast RNA. The sizes of the protected fragments are indicated on the right hand side in bp. The 72 bp band represents residual, undigested oligonucleotide probe. (B) Location of cap sites within the c-sis promoter sequence. The major and minor transcriptional initiation sites, as determined by S1 mapping, are indicated by arrowed lines (marked 1 and 2, respectively) above the nucleotide sequence of the c-sis promoter. The consensus TATAAA motif is indicated by a box at the 5' end of the sequence, and the extent of the 72 bp oligomer used as the probe for the S1 mapping is indicated below the sequence.

of the gene was carried out using chromatin DNA purified from 1st trimester human placental fibroblasts, JEG-3 cells, term cytotrophoblasts and U2-OS cells. DHS mapping from the 5' end of a 23 kb region of genomic DNA (an EcoRI fragment), encompassing the promoter and all downstream exon/intron sequences, is shown in Figure 3. Term cytotrophoblast DNA exhibited two strong, as well

1st intron regulatory elements in the human c-sis gene P.

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Fig. 4. CAT activity generated by the psisCAT and E+psisCAT constructs in JEG-3 and U2-OS cells. Equimolar amounts of the constructs were co-transfected with an SV40 LacZ plasmid into the JEG-3 and U2-OS cell lines. Cytosolic protein extracts from the two cell types were used to make CAT assays. The amount of protein used for each CAT assay was standarized with regard to transfection efficiency by means of ,B-galactosidase assay. The relative CAT activities of the constructs in the two cell lines are shown in the form of autoradiographs exposed from the TLC separation of the 14C-labelled acetylated chloramphenicol products. The pSVCAT construct was employed as a positive control for the CAT assay. The constructs used are indicated below each lane, while the cell type is indicated above.

Fig. 3. DNase I hypersensitive site (DHS) mapping of the c-sis transcription unit. (A) Southern blot analysis. Nuclei were prepared from term human cytotrophoblast, 1st trimester human placental fibroblasts, JEG-3 and U2-OS cells and digested with DNase I. DNA samples were digested with EcoRI, electrophoresed on 0.8% agarose gels and blotted. Filters were hybridized with a 32P-labelled probe which derived from the 5' end of a 23 kb EcoRI fragment of genomic DNA, encompassing the c-sis transcription unit. The cell type used is indicated above each panel. The four lanes shown in each panel correspond to increasing amounts of DNase I used, reading from left to right as indicated below each panel. The four lanes shown correspond to (from left to right) 0, 10, 100 and 750 units, respectively, of DNase I. The sizes (in kb) of the DNase I concentration-dependent bands detected are indicated by arrows. (B) Schematic representation of the locations of DHS within the c-sis transcription unit. The diagram represents a total of 27 kb of genomic DNA encompassing the c-sis transcription unit. The seven exons of the gene are indicated by black boxes.The four 1st intron DHS identified amongst the cell types investigated are indicated below the diagram by arrows. The presence (+) or absence (-) of each site is indicated directly beneath the corresponding arrow for each of the four cell types investigated. EcoRI (E) and BamHI (B) restriction sites are indicated on top of the diagram. The two BamHI sites shown here define the 1st intron-containing fragment used to make the E+psisCAT and E-psisCAT constructs.

as one much weaker, DNAse I concentration-dependent DHS (Figure 3A). JEG-3 choriocarcinoma cells also exhibited two of the DHS seen in cytotrophoblasts, although they lacked the strongest, most downstream DHS of the latter cell type. In sharp contrast to the trophoblast-derived DNA samples, no DHS were detected in DNA from 1st trimester placental fibroblasts. Although the U2-OS cell line expresses the c-sis gene at a level comparable to that of cytotrophoblasts, the DHS patterns of the two cell types were remarkably different. The U2-OS DNA did not exhibit any of the three DHS seen in cytotrophoblasts, but instead exhibited a single, strong site located -2 kb downstream of the most 3' cytotrophoblast site. Localization of these DHS on a map of the c-sis transcriptional unit (Figure 3B) indicated that they all lay within the 8 kb 1st intron of the gene. The weak and strong DHS, potentially common to both cytotrophoblasts and JEG-3 cells, were located -2 kb and 4.2 kb respectively 3' of the EcoRI site which defined the 5' end of the examined 23 kb restriction fragment (and which is located 375 bp upstream of the TATA box). Using the same reference point for site mapping, the unique strong DHS observed in cytotrophoblasts was located at 4.7 kb, while the unique U2-OS site was located at 6.7 kb. DHS mapping of a 4 kb region upstream of the c-sis promoter revealed no detectable sites (data not shown). -

Cell type-specific regulatory activity of the DHS-containing region of the c-sis 1st intron In order to investigate the significance of the hypersensitive regions present in the c-sis 1st intron reporter gene, constructs were made containing either the basal c-sis promoter alone, or a combination of the promoter and regions of the 1 st intron. The basal c-sis promoter was cloned as a 413 bp PstI fragment (included 13 bases of the 1st exon) into a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene to produce a basal c-sis promoter construct termed psisCAT. The DHS mapping suggested the possible presence of a number of control 1367

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Fig. 5. CAT activities generated by deletion constructs derived from the E+psisCAT plasmid. (A) Schematic representation of the deletion constructs. The constructs are identified at the right hand side of each diagram. The relative locations of the basal c-sis promoter (413 bp PstI fragment) and CAT coding region are indicated by hatched boxes and open boxes respectively (not to scale). The regions of the c-sis 1st intron present in each construct are indicated, to scale, in front of the promoter/CAT gene boxes. Restriction sites indicated are B, BamHI; H, HindIII; K, KpnI and X, XnaI. Note: the 8.3 kb BamHI fragment contains additional, unmapped XmaI sites which lie outside the XmaI fragment indicated. The dashed line in the E+psisCATAKK construct represents a segment of DNA which has been deleted, relative to the E+psisCAT plasmid. (B) Relative CAT activities generated by the deletion constructs in JEG-3 cells. The relative CAT activity is calculated as the ratio of deletion construct:psisCAT CAT conversion, as determined by scintillation counting. The values shown represent the mean value from at least three independent, ,B-galactosidasestandardized transfection experiments and error bars represent the standard deviations of each set of data. The identities of the constructs are detailed in the key at the right hand side of the graph: E+, E+psisCAT; XX+, XX+psisCAT; -KK, E+psisCATAKK; BK+, BK+psisCAT; KB+, KB +psisCAT. (C) Relative CAT activities generated by the deletion constructs in U2-OS cells. The data are presented as in (B). Note that the relative CAT activity is calculated using the psisCAT activity in the cell type in question. The activity of psisCAT was -4-fold higher in U2-OS relative to that found in JEG-3.

elements which encompassed a significant portion of the intron. An 8.3 kb BamHI fragment (indicated in Figure 3B) containing the entire 1st intron was subsequently cloned into a BamHI site located just upstream of the c-sis promoter in psisCAT. The 8.3 kb BamHI fragment was subcloned from the human c-sis-containing cosmid clone ALLW-1283, a kind gift of P.Bloemers. Resulting constructs were termed E+psisCAT and E-psisCAT, denoting the positive and negative orientations of the intron respectively (the schematic organization of the E+psisCAT construct is included within Figure 5A). Although the obvious choice of cells for transfection

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experiments were human term placental cytotrophoblasts, we were unable to transfect these cells at anything approaching a useful degree of efficiency, despite using a variety of established techniques. In view of these difficulties, the psisCAT, E+psisCAT and E-psisCAT constructs were transfected into JEG-3 cells. While JEG-3 cells do not express the principal c-sis mRNA, we nevertheless decided to test their acceptance of the 1st intron as a transcriptional regulator of the c-sis promoter for a number of reasons: the JEG-3 cell line was originally derived (via the malignant choriocarcinoma tumour) from cytotrophoblasts; the cells exhibit two out of the three DHS observed in primary

1st intron regulatory elements in the human c-sis gene

Fig. 6. The influence of proper chromatin structure on the orientation effect of the transcriptional activation properties of the c-sis 1st intron region. The relative CAT activities of the E+psisCAT and E-psisCAT constructs were compared in both transient and stable transfections of JEG-3 cells. The constructs are indicated below each lane and the type of transfections is indicated above. The pSVneo construct (SV40-driven neomycin construct) was included as a negative CAT control for neomycin-resistant stable JEG-3 cells, while pSVCAT was included as a positive control. (A) Results of stable JEG-3 transfections. (B) Results of transient JEG-3 transfections.

cytotrophoblasts and, in addition, it is likely that the lack of the principal c-sis transcript in JEG-3 cells results from hypermethylation of certain key CpG dinucleotides within the promoter region in this cell line (G.Adam et al., in preparation). This may be an in vitro culturing effect, as primary choriocarcinoma cells do express significant levels of c-sis mRNA (unpublished data). Figure 4 shows the primary CAT assay data obtained after transient transfection of the psisCAT and E+psisCAT plasmids into JEG-3 and U2-OS cells. The CAT activity of cytosolic extracts prepared from E+psisCAT transfected cells was of the order of 26-fold greater than that observed for cells transfected with the psisCAT plasmid. A dramatically different situation was observed when these constructs were transfected into U2-OS cells. The activity of the basal c-sis promoter in the psisCAT construct was higher in U2-OS cells than that observed in JEG-3 cells ( 4-fold). Conversely, the CAT activity of the E+psisCAT construct was 3-fold lower than that of the psisCAT in U2-OS, indicating a negative regulation of the basal c-sis promoter activity in the U2-OS cell line by the 1st intron (Figure 4). The positive regulatory activity of the 1st intron is composed of at least two distinct elements In order to define more closely the location of the regulatory activity within the 1st intron, a series of deletion constructs was made, containing a number of subfragments of the 8.3 kb BamHI fragment linked to the basal c-sis promoter. These constructs were transfected in both JEG-3 and U2-OS cells in order to assess their relative activities. Figure 5 shows both the plasmid construction details and the results of the CAT assays. In JEG-3 cells, the relative CAT activity generated by the E+psisCAT construct was 26-fold higher, compared to that of the basal c-sis promoter construct. The XX+psisCAT construct, containing the central 1.95 kb XmaI intron fragment, produced a relative CAT activity increase of 8-fold. As this region, which contains a strong DHS in JEG-3 cells, obviously did not account for all the positive regulation exhibited by the E+psisCAT construct,

a new construct was made in which the central 3.5 kb KpnI fragment of the intron (containing the 1.95 kb XmaI fragment) was deleted. The resulting E+psisCATAKK construct showed an increased relative CAT activity of 10-fold, indicating that the positive regulatory activity of the 1st intron was made up of at least two distinct elements. In order to locate the other element(s), the 1.7 kb 5' BamHI-KpnI and the 3.3 kb 3' KpnI-BamHI fragments, which make up the E +psisCATAKK construct, were cloned separately into the psisCAT vector, to generate constructs termed BK +psisCAT and KB +psisCAT respectively. The BK +psisCAT construct showed a relative CAT activity of 4-fold higher, whereas the KB +psisCAT was inactive in promoting any activation of the basal c-sis promoter. These data indicate that a second, positive regulatory region exists within the 5' BamHI -KpnI segment of the intron, which may correlate with the weak DHS mapped within the same restriction fragment in JEG-3 cells and cytotrophoblasts. The 1st intron contains both negative and positive regulatory elements for the c-sis promoter in U2-OS cells The same deletion constructs were also transfected into U2-OS cells, with somewhat surprising results. The relative CAT activities provided by the deletion constructs in U2-OS cells are shown in Figure SC. Relative to the CAT activity produced by psisCAT (which was of the order of 4-fold higher than for the same construct in JEG-3 cells), that of E+psisCAT was 3-fold lower. The E+psisCATAKK, BK +psisCAT and KB +psisCAT constructs also generated CATactivities lower than did psisCAT (3-fold, 2.5-fold and 2-fold respectively). In contrast, however, the XX +psisCAT construct showed a 4.5-fold increase in CAT activity relative to psisCAT. Thus it appears that in U2-OS cells the c-sis 1st intron contains elements which can, in isolation, confer activation (XmaI fragment) or repression (5' BamHI -KpnI and 3' KpnI -BamHI fragments) on the c-sis promoter. The overall effect of the intact BamHI fragment is, however, to negatively regulate the c-sis promoter in U2-OS cells.

Orientation/distance dependence and specific promoter requirements of the 1st intron regulatory activity The question of whether or not the regulatory activity identified within the c-sis 1st intron qualifies as a classically defined enhancer was addressed by assessing the orientation/ distance dependence and promoter requirements of the region. The E -psisCAT (negative orientation of the 8.3 kb BamHI fragment) construct showed little or no increased CAT activity, relative to psisCAT, in transiently transfected JEG-3 cells (Figure 6). Previous work has suggested that the dependence upon the physical relationship between enhancer and promoter, which has been observed for some promoter elements in transient transfection assay systems (Oshima et al., 1990), may derive from the absence of normal chromatin structure (Muller and Schaffner, 1990). With this in mind, the psisCAT and E+/E-psisCAT constructs were used to make stable transfections of JEG-3 cells. The relative CAT activity of E-psisCAT was markedly higher in the stable transfections than that observed in the transient transfections (Figure 6). The 8.3 kb BamHI fragment was also cloned, both upstream and downstream, of the basal SV40 viral promoter

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Fig. 7. The effect of the 8.3 kb BamHI c-sis 1st intron fragment on a heterologous promoter. The intron-containing region from E+psisCAT was cloned upstream of the SV40 basal promoter, in positive and negative orientations (E+pSVCAT and E+pSVCAT, respectively), as well as downstream in the positive orientation (pSVCATE+) in the pCATPromoter CAT vector (Promega). These constructs were transfected into JEG-3 cells. The relative CAT activity data are calculated and presented in Figure 5B and C.

linked to a CAT reporter gene in the pCATPromoter vector (Promega). These constructs were transfected into JEG-3 cells to test for the activity of the 1st intron region on the heterologous SV40 promoter. Figure 7 shows that the c-sis 1 st intron region had little or no regulatory effect upon the SV40 promoter. The E+psisCAT construct (containing the BamHI fragment cloned in the positive orientation and upstream of the SV40 promoter) showed a very slight (2-fold) increase relative to the basal SV40 promoter construct, but this compared to a 26-fold activation of the c-sis promoter by the same fragment of the c-sis 1st intron (Figure 5B). The XX+psisCAT construct exhibited a positive regulatory activity on the basal c-sis promoter, but despite this the same 1.96 kb XmaI intron fragment had no effect when cloned in front of the human f-globin promoter, as assayed by RNase protection of the f-globin reporter gene construct (data not shown). It appears, therefore, that the regulatory elements within the 1st intron of the c-sis gene require specific interaction with their own promoter. The regulatory element described here fails, therefore, to meet the criteria sometimes applied to qualify the term 'enhancer' (ability to drive a heterologous promoter, orientation/distance independence of activity), but appears to be a strong, cell type-specific, positive regulator of c-sis transcription in placental cells. The same region also confers a net negative regulatory effect on the c-sis promoter in the U2-OS osteosarcoma cell line.

Discussion The c-sis 1st intron contains both positive and negative regulatory elements, some of which show cell type specificity Here we report the presence of cell type-specific transcriptional regulatory elements located in the 1st intron of the c-sis gene. Despite the long-recognized biological importance of PDGF, this is the first description of the complexity of the transcriptional control elements for the human PDGF-B gene. The finding of cell type-specific regulatory elements for the c-sis was predictable, given the distinct spatial and temporal expression patterns observed

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Fig. 8. Schematic overview of the contribution of the elements of the c-sis 1st intron to the overall regulation of c-sis in JEG-3 and U2-OS cells. The E+psisCAT construct is shown at the top of the diagram, as in Figure 5A. The regulatory effect on c-sis promoter activity shown by various segments of the 1st intron is indicated within open boxes aligned beneath the E+psisCAT construct: + + +, strong activation; +, moderate activation; N, no net effect and -, repression. The c-sis promoter is represented by a hatched box, with the relative activity indicated beneath it, for the two cell types.

during development (Ohlsson et al., 1981; L.Holmgren, R.Glaser, S.Pfeifer-Ohlsson and R.Ohlsson, in preparation). However, in comparison to many genes, which are regulated

in a strictly limited cell type-specific manner via enhancer elements, the c-sis gene is expressed by a wide range of cell lineages, although this occurs in very specific spatial and temporal 'windows' for these cell types (Ross et al., 1986; Smeland et al., 1988; Ohlsson et al., 1991). For this reason, it is perhaps not surprising that the control mechanisms involved in the regulation of c-sis expression appear more complex and diverse than those associated with genes which are more strictly and simply limited to a specific cell type (or types). It is clear that the c-sis 1st intron is multifunctional with regard to transcriptional regulation, containing both positive and negative regulatory elements which show cell type specificity (JEG-3 and U2-OS cells respectively). The CAT activities of the E +psisCAT, E +psisCATAKK and BK + psisCAT constructs were considerably greater than the low basal promoter activity of psisCAT when assayed in JEG-3 cells. In general, the reverse situation was observed in U2-OS cells, where a higher basal promoter activity was repressed by the presence of 1st intron sequences in the E+psisCAT, E+psisCATAKK, BK+psisCAT and KB+psisCAT constructs. Comparison of the activities of the various c-sis promoter/intron-CAT constructs in JEG-3 and U2-OS cells implies that a very complex set of distinct transcriptional control mechanisms do indeed exist for this gene. The presence of a transcriptionally enhancing activity which is active in trophoblast-derived JEG-3 cells could explain the relatively high level of c-sis expression seen in placental cytotrophoblasts. In U2-OS cells, however, it would appear that the enhancing activity of the 1st intron does not function. The higher basal activity of the c-sis promoter in U2-OS cells may compensate for the absence of the activator activity to some degree, but cannot itself

1 st intron regulatory elements in the human c-sis gene

account for the high level of transcription of c-sis in this cell line.The appearance of a silencing activity within the intron in U2-OS cells (presumably due to the production of factors specific for the elements involved) may reflect a counter-balancing mechanism, which responds to a higher promoter activity. It would obviously be of interest to see if this silencing activity is present in other situations in which the basal c-sis promoter activity is elevated above normal levels, in TPA-induced K562 cells (Pech et al., 1989) for example. Indeed, in the studies on TPA induction of the c-sis promoter, Pech and co-workers suggested that negative control elements must exist outside of the basal promoter region (Pech et al., 1989). The observation that JEG-3 cells accept the 1st intron region as an activator of the c-sis promoter, despite failing to express the principal c-sis transcript, may seem surprising at first. There is, however, a conceptually simple explanation of this observation, namely that JEG-3 cells produce the necessary positive trans-acting factors required by the cis-acting regulatory sequences, but that an epigenetic, dominant negative regulatory mechanism exists in these cells. A similar phenomenon has been observed for the mouse keratin 18 gene enhancer, which can function in transient transfection assays in mouse fibroblasts, despite the fact that these cells do not express the endogenous gene (Kulesh and Oshima, 1988). Interestingly, the endogenous gene was found to be hypermethylated in these fibroblasts and, furthermore, the epigenetic repression appeared to dominate over the positive trans-acting factors, as the keratin 18 gene of fibroblasts could not be induced by somatic cell hybridization (Oshima et al., 1988). Similarly, here it appears that the putatitive negative epigenetic control of c-sis in JEG-3 cells occurs via specific CpG methylation within the c-sis promoter and, furthermore, that this regulation operates specifically in conjunction with the 1st intron activater elements (G.Adam, G.Franklin and R.OhIsson, in preparation). The positive regulatory elements of the c-sis 1st intron do not function as a classically defined enhancer While a large number of genes have now been shown to possess cis-acting regulatory elements which can be classified as 'enhancers', the positive regulatory elements which we describe here for the c-sis gene do not appear to operate via a classically defined enhancer mechanism. The failure of the E-psisCAT construct to show any real transcriptional activation of the c-sis promoter in transient assays indicates a marked orientation/distance dependence on activation. This is not in keeping with the originally defined enhancer concept, but it should be mentioned that the term 'enhancer' has been used more recently to describe regulatory sequences which show orientation/distance dependence (Oshima et al., 1990). In any case, as the activator elements exist in the positive orientation in vivo, the orientation effect could be considered a purely academic point. However, the observation that different transcriptional regulatory elements

show differences in their positional dependences with respect

to promoters could signify basic differences in their mechanisms of action. Whatever mechanism of action the c-sis activator elements may have, the observation that the negative orientation of the BamHI fragment (E-psisCAT) appeared to function more efficiently in stable transfections

suggests that chromatin structure may have an influence on activity, as has been observed previously (Muller and Schaffner, 1990). Perhaps more interesting than the orientation/distance dependence exhibited by the 1st intron activator was the apparent specificity which it showed for the c-sis promoter. While the possibility obviously remains that the c-sis activator could exert its effect on other, as yet untested, promoters the failure of either the XmaI intron fragment to drive the human 3-globin promoter, or the BamHI fragment to efficiently drive the SV40 promoter strongly suggests that the activator requires some specific interaction with sequence elements within the basal c-sis promoter in order to function as an efficient activator of transcription. In this regard in particular the activator element we describe here differs fundamentally from previously characterized 'enhancer' elements. One enhancer-like element which does show specificity towards a limited set of promoters has been described previously, namely the locus activation region (LAR) of the human f-globin cluster (Tuan et al., 1985; Forrester et al., 1986). The c-sis 1st intron activator and negative regulatory regions are composed of multiple elements The positive regulatory region consists of at least two distinct elements, one of which is active in both JEG-3 and U2-OS cells (the 1.95 kb XmaI fragment) and another which is active only in JEG-3 cells (the 5' 1.7 kb BamHI-KpnI fragment). A similar situation has been observed for the enhancer of the az-crystallin gene, where a complex synergism operates between non-specific and highly cell type-specific enhancer components in order to drive the correct expression pattern of the gene (Goto et al., 1990). As the transcriptional activation of the c-sis promoter by the 8.3 kb BamHI fragment was significantly greater than the combined activation by the Xmal- and KpnI-deleted fragments (as assayed by CAT activity in JEG-3 transfections), it appears that the two components of the activator sequence operate together in a synergistic, rather than an additive manner. Furthermore, as the relative CAT activity of the BK+psisCAT construct did not account for all the activity shown by the E+psisCATAKK construct, it is possible that the 5' BamHI-KpnI region requires a synergistic interaction with some element within the 3' KpnI -BamHI region, which does not itself show any activity when tested in isolation. The observation that the XmaI fragment can activate the c-sis promoter in U2-OS cells while the intact intron fragment acts as a transcriptional repressor merely indicates that the factor(s) required for interaction with the regulatory sequences within the Xnal fragment are present in U2-OS cells. Suppression of the positive regulatory region in the XmaI fragment could be due either to an active down-regulation (perhaps via chromatin constraints on the access of trans-acting factors), or at the level of interaction with the promoter, where the activating and (stronger) suppressing elements compete for control of the transcriptional machinery. The absence of a DNase I hypersensitive site which maps within the XmaI fragment in U2-OS cells, despite the presence of factors which can activate the c-sis promoter via sequences within this fragment, would seem to support the latter possibility. Whatever the function of the silencing activity of the c-sis 1st intron may be in U2-OS cells, the fact that they express

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the c-sis gene at a relatively high level implies that other positive regulatory mechanisms exist in these cells. A schematic overview of how the various fragments of the c-sis st intron contribute to the regulation of the c-sis promoter in JEG-3 and U2-OS cells, both individually and in concert, is presented in Figure 8. The c-sis gene encodes a potent growth factor which has been implicated to have pivotal roles in development and neoplasia. The control of PDGF-B gene transcription should, therefore, be of fundamental importance in both neoplasia and development (Donovan et al., 1991). Our observation here, that complex arrangements of cell type-specific positive and negative elements control the c-sis protooncogene, are in keeping with this notion. We are currently attempting to establish which cis-acting elements are active in other cell types important for development and neoplasia (such as endothelial cells), by transgenic mouse approaches. Other genetic and epigenetic control mechanisms for this gene are also currently under investigation.

Materials and methods Cell purification and culture Human term placentae were obtained from routine Caesarian sections performed at Huddinge University Hospital. Cytotrophoblasts were purified by selective trypsinization of the placental -decidual interface, followed by separation on Percoll (Pharmacia) density gradients, following established methodology (Kliman et ol., 1986; Ohlsson et al., 1989). Purified cytotrophoblasts (routinely >95% pure as assayed by immunostaining with the cytokeratin antibody PKK1) were used directly to prepare nuclei and were not, therefore, exposed to serum or other potential culturing artefacts prior to RNA extraction (see below). Primary placental fibroblasts, U2-OS and JEG-3 cells were maintained as described previously (Goustin et al., 1985; Ohlsson et al., 1989). RNA preparation and Northern analysis Total RNA was prepared from fibroblasts and cancer cell lines using the LiCl-urea method (Auffrey and Rougeon, 1980). Total cytoplasmic RNA was prepared directly from purified cytotrophoblasts, followed by proteinase K digestion and phenol-chloroform extraction (Sambrook et al., 1989). Electrophoresis and transfer of total RNA, as well as subsequent hybridization were carried out as described previously (Pfeifer-Ohlsson et ol., 1984). The blots were probed with a c-sis cDNA probe (PM-1), an oligonucleotide probe covering the cap site of the 1st exon (see below) and to normalize RNA input, a /3-actin probe (PA-1). S mapping of c-sis transcript in cytotrophoblasts and JEG-3 cells Total cytoplasmic RNA was prepared from cytotrophoblasts and total RNA from JEG-3 cells, as described above. An oligonucleotide was synthesized which spanned 72 bp of the c-sis promoter, starting within the consensus TATA box and ending downstream of this region into the 1st exon (see Figure 2B). SI nuclease protection analysis was performed with the 72 bp oligomer, 32P-labelled at its 5' end, using standard techniques (Sambrook et al., 1989). 1

Dnase I hypersensitive site mapping Cells were washed extensively in PBS, pelleted at low speed (600 g) and suspended in ice-cold lysis buffer (500 mM sucrose, 0.5% Triton X-100, 0.15 mM spermine, 0.5 mM spermidine, 15 mM Tris, 60 mM KCI, 15 mM NaCl, 2 mM EDTA, 0.2 mM EGTA and 1 mM PMSF, adjusted to pH 7.4) at 2 x 107 cells/ml. Nuclei were prepared by careful use of a Dounce homogenizer, checked microscopically and spun down at 600 g, 4°C. Pelleted nulcei were resuspended in wash buffer (made up as lysis buffer, but with 350 mM sucrose and without Triton X-100), counted in a haemocytometer and spun down a second time at 600 g, 4°C. Nuclei were then resuspended in digestion buffer (equivalent to wash buffer without sucrose) at 1.2 x 108 cells/ml. Aliquots of 2.3 x 107 nuclei were then digested with DNase I (ranging from 0 to 750 units), reactions were activated by adding MgCl2 to a final concentration of 5 mM and incubated at 40C for 30 min. DNase I digestion was terminated by the addition of EDTA to 10 mM. Nuclei were pelleted at 600 g and incubated overnight at 37°C with 0.4 mg/ml proteinase K in a buffer consisting of 50 mM Tris (pH 1372

8), 2 mM EDTA and 0.1 I% SDS. DNA samples were then purified by multiple ethanol precipitation/resuspension cycles and RNase A digestion (50 itg/ml). After a final ethanol precipitation. 35yg aliquots of DNA were digested with EcoRI, Southern blotted and hybridized with 32P-labelled, 1 kb EcoRI-XoI probe (Multiprime kit, Amersham), using standard techniques (Sambrook et al., 1989). The 5' end of the probe corresponded exactly with the 5' end of the 23 kb genomic c-sis EcoRI fragment examined

(Figure 3). Plasmid constructions The psisCAT, E+psisCAT and E-psisCAT constructs were made as described in Results. XX +psisCAT was constructed by cloning the 1.95 kd XinaHI fragment from E + psisCAT into the unique XmnaI site just upstream of the promoter in psisCAT. E+psisCATAKK was made by deleting the central 3.5 kb KpnI fragment from E+psisCAT and religating the resulting plasmid. The BK+psisCAT and KB+psisCAT plasmids were made by double-digesting E+psisCATAKK with BamHI and KpnI, blunt ending the resulting fragments with T4 DNA polymerase and ligating the mixture of blunt ended fragments. E+pSVCAT and pSVCATE+ were constructed by cloning the 8.3 kb BaniHI fragment from E+psisCAT into the BglII and BamznHI sites, respectively, of the pCATProt"oter vector (Promega). Large-scale DNA preparations were made using the alkaline lysis and polyethylene glycol precipitation method of R.Treisman (Sambrook et ol., 1989). This method was found to produce DNA of sufficient quality for transfection, equal to that of CsCl gradient purified DNA. Transfections and CAT assays JEG-3 and U2-OS cells were trypsinized and plated out at a density of 1.0 x 106 cells per 100 mm culture dish and allowed to attach overnight, prior to transfection. The overnight culture medium was aspirated and replaced and the cells were incubated for a further 3 h in fresh medium. Equimolar amounts of construct DNAs were added to cells as CaPO4 precipitates (Graham and Van Der Eb, 1973). After 4 h incubation with the DNA precipitates, the cells were washed with PBS to remove serum and shocked (Parker and Start, 1979) by incubating with a 15% glycerol solution (in 25 mM HEPES, 140 mM NaCl and 0.75 mM Na2HPO4) for 140 s. Fresh culture medium was then added and replaced the following day. All CATconstructs were co-transfected with an SV40 promoter/enhancer 3-galactosidase reporter gene construct (pSVLacZ) in order to normalize experiments for variations in transfection efficiency. Cells were harvested -48 h post-transfection. Cell monolayers were washed twice in cold PBS, scraped off the dish and pelleted at 1200 g at 4°C. Cell pellets were resuspended in 0.25 M Tris (pH 7.4) and protein extracts were obtained by three freeze-thaw cycles (dry ice-ethanol bath and 37°C heat block). Protein yields were determined spectroscopically (Bio-Rad Protein Assay) and 200 ,ug aliquots taken for 3-galactosidase (LacZ) assay. LacZ assays were carried out using o-nitrophenyl-o-D-galcatosidase (ONPG) as substrate (0.8 mg/ml) in an assay buffer consisting of 60 mM Na,HPO4, 30 mM NaH,PO4, 10 mM KCI, 1 mM MgCl, and 50 mM 0-mercaptoethanol. Reactions were incubated at 37°C for 2-3 h and OD415 nm readings were measured using a Bio-Rad model 3550 microplate reader. Protein aliquots were taken for CATassay with the amounts adjusted with respect to the relative transfection efficiencies of the different constructs (as determined by relative LacZ activities). Protein extracts were diluted with 0.25 M Tris (pH 7.4), adjusted to 5 mM EDTA and heated at 60°C for 10 min to selectively deactivate contaminating de-acetylases (Sambrook et al., 1989). Acetyl coenzyme A was added (to 2.9 mM) and 3 t l [14C]chloramphenicol (Amersham, 0.025 mCi/ml, 57 mCi/mmol) in a total reaction volume of 150 pi. Reactions were incubated at 370C for 2 h, extracted with ethyl acetate and dried down in a vacuum centrifuge. Samples were then resuspended in a small volume of ethyl acetate and analyzed on 0.2 mm silica gel TLC plates (Merck), using a 95:5 chloroform-methanol mix as the running solvent. After obtaining suitable autoradiographs of the TLC runs, CATconversions were calculated by cutting out the radioactive spots, followed by scintillation counting. Stable transfections of JEG-3 cells with the c-sis promoter/intron - CAT constructs were made by co-transfecting with pSVLacZ and an SV40 promoter/enhancer-driven neomycin resistance gene construct (pSV,Neo). Transfection was carried out as detailed above, after which selection was applied by growing the cells in normal medium supplemented with 700 ,ug/ml Geneticin G418 sulphate (Gibco) for 10 days. Surviving neomycin-resistant clones were pooled and expanded in normal medium, after which protein extraction, 3-galactosidase assay and CATassay were carried out as described above. The f-galactosidase activities of the various cell extracts were consistent and, in a control transfection, all cells transfected with a promoterless neomycin resistance vector (pUCNeo) were dead before day 10 of the G-418 treatment.

1st intron regulatory elements in the human c-sis gene

Acknowledgements We would like to thank P.Bloemers for providing the superCAT vector and human c-sis cosmid clone, ALLW-1283. We are also grateful to S.Petterson for providing advice and reagents for the RNase protection experiment and to C.Walsh for helpful comments on the manuscript preparation. This work was supported by grants from the Swedish Cancer Research Foundation (RMC): 2777-B91-024, 1955-B91-05PBC and from the Swedish Natural Science Research Foundation (NFR): B-BU 4494-301, B-BU 4494-302 and B-BU 4494-303.

Waterfield,M.D., Scrace,G.T., Whittle,N., Stroobant,P., Johnsson,A., Wasteson,A., Westermark,B., Heldin,C-H., Huang,J.S. and Deuel,T. (1983) Nature, 304, 35-39. Wu,C. (1980) Nature, 286, 854-860. Received on January 29, 1991 Note added in proof

This work was carried out at the Centre for Biotechnology, Karolinska Institute, Novum S-141 57, Huddinge, Sweden.

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