.=) 1992 Oxford University Press
Nucleic Acids Research, 1992, Vol. 20, No. 23 6355-6361
Organization of the unriN-ras locus: characterization of the promoter region of the human unr gene Helene Jacquemin-Sablon and Francois Dautry Laboratoire d'Oncologie Moleculaire, CNRS UA 1158, Institut Gustave Roussy, 94805 Villejuif Cedex, France Received August 7, 1992; Revised and Accepted November 5, 1992
ABSTRACT Investigations of the structure and expression of the N-ras gene in mammals has led to the identification of another gene designated unr , which is located immediately upstream of N-ras. These two genes are transcribed in the same orientation and the intergenic distance is of the order of 150 nucleotides. This genetic organization has been observed in the genome of guinea pig, rat, mouse and man with a very high level of sequence conservation in the intergenic region. This unusual gene clustering suggests that the transcriptional regulation of this locus could involve common regulatory sequences as well as transcriptional interference between the two genes. In this study, we have isolated and characterized the human unr promoter. A cluster of transcription initiation sites was mapped by primer extension and RNase protection and shown to be located in a CpG island devoid of TATA and CAAT boxes. Functional organization of the promoter was investigated by measuring the ability of a set of 5' deletions within al kb promoter region to drive the expression of the luciferase gene. These studies indicated a very strong promoter actvity in NIH 3T3 cells and the presence of positive and negative regulatory domains. Nevertheless, a 90 bp fragment showed the same level of promoter activity as the 1 kb fragment. We also showed that ras genes can transactivate the unr promoter activity and that the 90 bp fragment responded to this transactivation. INTRODUCTION Harvey, Kirsten and N-ras are members of the ras gene family which have been implicated in the pathogenesis of human cancer. This is based on the observation that a large proportion of tumors contain a mutated ras gene which can transform cells in vitro (1), and predispose transgenic mice to cancer (2). While oncogenic activation of the ras genes by point mutations has been well documented, many experiments have also shown that the level of expression is critical for the biological activity of both normal and mutated ras proteins. Although most of these studies deal with the Harvey-ras gene, analysis of the revertants of the fibrosarcoma cell line HT 1080 clearly indicates that this is also true for N-ras(3).
EMBL accession no. X68286
It has been shown that the N-ras gene is part of an unusual genetic structure which comprises a tandem of closely linked transcription units in the same orientation. This organization has been observed in the genome of guinea pig (4), rat (5), mouse (5,6) and man (6). N-ras corresponds to the downstream transcription unit, while the upstream one does not match with any previously identified gene and has been designated unr (upstream of N-ras) (4, 5) or NRU (N-ras upstream) in our previous study (6). Sequence analysis of the predicted unr protein product has revealed the presence of five internal repeats which are distantly related to the E.coli cold shock protein (7). Since it has been proposed that these motifs are the hallmark of a new class of DNA binding proteins, this raises the possibility that unr could be a DNA binding protein. However, at this stage, the function of unr and what relation, if any, it may have with the function of N-ras is unknown. The unusual nature of the unrlNras locus comes from the small intergenic distance (of the order of 150 nucleotides) between two ubiquitously expressed genes. Moreover, there are three unr messages which have the same coding capacity and differ only by their polyadenylation site (6) (O.Boussadia, H.J.-S. and F.D., submitted for publication). While the first polyadenylation signal is located 1.2 kb upstream of the N-ras gene, at a standard intergenic distance, it is the less frequently used of the three signals. This observation, and the extremely high level of sequence conservation of the unrlN-ras junction suggest that there is a selective pressure to maintain this compact organization. Genomic transfection experiments have established that the Nras gene was functional in the absence of upstream sequences and both the murine and the human N-ras promoter regions have been partially characterized(8 9). Nevertheless, the presence of the unr transcription unit suggests that other levels of regulation could contribute to the control of N-ras expression. On the one hand, usage of the last unr polyadenylation signal implies that the two transcription units overlap, raising the possibility of transcriptional interferences (10). On the other hand, it is likely that unr and N-ras are part of the same transcriptional domain and therefore are subjected to common transcriptional regulations. As a part of our efforts to evaluate the relative contributions of these different levels of regulation within the unr/N-ras locus, we present in this paper a characterization of the human unr promoter. We have cloned and sequenced the 5' end of the unr gene and localized the transcription initiation sites. We have used a transient expression assay in NIH 3T3 cells to analyze the
6356 Nucleic Acids Research, 1992, Vol. 20, No. 23 functional organization of this promoter and established that a 90 pb fragment in which the only known binding site is an SPI site, has a very high promoter activity. Finally, the unr promoter activity is increased by cotransfection with an activated ras gene, indicating the possibility of interactions in trans between N-ras and unr.
RESULTS Cloning and sequence analysis of the human unr promoter region We have previously reported the isolation and nucleotide sequence of cDNA clones of the human unr gene (6). In most cell types, three unr mRNA can be detected with apparent mobilities of approximately 4.4, 4 and 3.4 kb. We have established that these mRNA are generated by the use of ftree different polyadenylation sites located in the last unr exon (6,7) (O.Boussadia, H.J.-S. and F.D. unpublished results). To isolate genomic clones corresponding to the 5' end of the unr gene, we used the first 800 nucleotides of our longest cDNA clone to screen a human leucocyte genomic DNA library. Overlapping positives clones A
x P7 Sal
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were isolated and rescreened with a 20mer oligonucleotide corresponding to the 5' end of the reported cDNA sequence (6). One phage (X P7) which contained a 13 kb insert was selected for further study and its restriction map is presented in Fig. lA. This clone contained two fragments which hybridized to the 5' untranslated region of the unr cDNA. Sequence analysis of these fragments indicated the presence of two exons, tentatively numbered exons 1 and 2. Exon 2 was colinear with the cDNA sequence from position + 26 to + 341 and was surrounded by potential splice acceptor and donor sites. The 1.2 kb SalI-SacI fragment containing the putative exon 1 along with 1 kb of upstream sequences was then sequenced (Fig. IB).
Mapping of the transcriptional start sites The transcription initiation sites were determined by a combination of RNase mapping and primer extension. We first used a genomic probe to analyse by RNase mapping the size of exon 1. A 190 nucleotides long 32P-labelled RNA probe spanning the presumptive 5' end of the unr gene was hybridized with total mRNA isolated from different cell lines. Fig.2A shows the presence of a major protected fragment of 60 nucleotides in all the cell lines that we have analyzed (MCF 7, K 562, RD). On longer exposure a cluster of fragments with sizes ranging from 55 to 65 nucleotides can also be observed. Since RNase protection does not discriminate between the end of the mRNA and the 5' end of an exon, we performed a primer extension analysis to confirm that these protected fragments corresponded to the transcription start sites. The primer used is located in exon
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Organization and nucleotide sequence of the 5' end of the human unr Map of the 7 kb Sail-Afll fragment which contains exons 1 and 2. The sites for the restriction enzymes SacI (Sac), Bglll (Bgl) and AflII (Afl) are indicated. The Sail site was introduced by the bacteriophage vector. (B) Nuckeotide sequence of the 1.2 kb Sail-SacI frgment. Nucleotides are numbered with respect to the major transcription initiation site (+ 1) and exon 1 is boxed. The SPI and Mt4 consensus sequences are boxed and a 12 bp palindrome (PAL) is underlined. Figure 1. gene. (A)
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Figure 2. Determination of the transcription initiation sites in the unr gene. (A) RNase mapping analysis of the size of exon 1. As indicated at the bottom of the figure the probe was obtained by in vitro transctiption of the 190 bp SaclNruI genomic fragment. Lanes: 1, undigested probe; 2, DNA molecular weight markers (MspI-digested pBR322 DNA); 3, RNA molecular weight markers (65 and 54 nucleotides); 4, cytoplasmic MCF 7 RNA; 5, MCF 7 polyA+ RNA; 6, K 562 total RNA; 7, K 562 polyA+ RNA; 8, cytoplasmic RD RNA; 9, tRNA. (B) Primer extension analysis of the unr messages. The primer used is a 20mer oligonucleotide located 46 nucleotides downstream of the exon 2 as indicated at the bottom of the figure. The sequence ladder on the left was used to determine the size of the extended products. Lanes: 1, cytoplasmic MCF 7 RNA; 2, MCF 7 polyA+ RNA; 3, K 562 total RNA; 4, RD cytoplasmic RNA; 5, Jurkat total RNA; 6, tRNA. An arrow indicates the major extension product.
Nucleic Acids Research, 1992, Vol. 20, No. 23 6357
2 (nucleotides 71 to 52 of the published cDNA sequence). Similar results were obtained with either total RNA from different cell lines, or poly (A)+ RNA from MCF-7 cells. The abundance of the extension products matched the accumulation of unr mRNA observed by Northern blot analysis (data not shown). By comparaison to a DNA sequence ladder, the major extension products ranged in size between 90 and 110 nucleotides, with a prominent band of 106 nucleotides (Fig.2B). Since 46 nucleotides should be derived from exon 2, this indicated that the size of exon 1 was 60 nucleotides, in good agreement with the RNase mapping results. Additional start positions, upstream and downstream of the major site were also detected, but with lower relative abundance. Taken together, these results indicated that the unr initiation sites were located in the SailI-SacI fragment of the clone X P7, and we designated + 1 the major initiation site in the sequence of Fig. 1.
Sequence of the unr promoter region Inspection of the unr promoter sequence revealed that it lacked TATA or CCAAT box sequences upstream of the transcription initiation sites. The unr first exon and the surrounding sequences (from -84 to +87) contained 64% G+C and in this region the ratio of CpG to GpC is 1.1, indicating the absence of CpG suppression. These properties established that the 5' end of the unr gene was located within a CpG island (ll). The sequence of the 1 kb region upstream of exon 1 appeared remarkably devoid of known binding sites for transcriptional regulators. Indeed, in spite of the high G/C content only one potential SPI site (matching the minimal consensus sequence GGCGGG) could be found at position -60. Further upstream, the sequence Mt4 (CTCACCA) which is common to several nuclear genes encoding mitochondrial proteins (12) was found at position -784. A 12 bp palindromic sequence (5'-CTCTCGCGAGAG-3', underlined in Fig.lB) was observed at position -22. Functional analysis of the unr promoter The promoter activity of the cloned 5' end of the unr gene was studied by transient expression assays with the luciferase gene as a reporter. Initially, the 1076 nucleotides Sal1-HaeII fragment from X P7, which contains 1044 nucleotides of upstream sequences and 32 nucleotides of exon 1 was cloned in front of the luciferase gene in the plasmid pcluc (see Material and Methods). This fragment was cloned in either the natural or the reverse orientation, giving the constructs unrluc and unrlucAS, respectively. These constructs were introduced by calcium phosphate-mediated transfection into different cell lines and the luciferase activities in the cell extracts were analyzed. The Table 1. Activities of the unr-luciferase and CMV-luciferase constructs in NIH 3T3 cells. Plasmids CMVluc unrluc unrlucAS p90
p90A pcluc
Luciferase activity (RLU)
SEE
160000 40000 1500 45000 51000 1100
7 10 12 7 10 9
linearity of the assay was checked by measuring the luciferase activity for two extract dilutions. Two different plasmid preparations were used for each construct to check for anomalies due to a particular plasmid preparation. A construct (CMVluc) containing the human cytomegalovirus immediate early promoter was included as a positive control. As shown in table 1, unrluc yielded high level of luciferase activity in NIH3T3 cells (25% of that of CMVluc) while unrlucAS did not behave differently from the promoterless pcluc. The activity levels in other cell lines, in particular in human cell lines, were much lower (see below) and therefore we first carried out an analysis of the unr promoter in NIH 3T3 cells. A series of 5' deletions of the upstream region was constructed to search for regulatory elements and to determine a minimum sequence needed for promoter activity. Bal 31 exonuclease was used to generate progressive 5' deletions within this promoter (Fig.3A). We observed no significant difference in luciferase activities among the constructs containing more than 600bp of flanking sequences (Fig.3B). In contrast, a sharp reduction of luciferase activity was observed when sequences between -600 and -370 were deleted, suggesting the presence of positive regulatory sequences within this region. Further deletions progressively restored a high level of luciferase activity, indicating the presence of a negative regulatory domain between -370 and -90. In particular, plasmid p90 which contains only 90 nucleotides of the unr promoter was as active as the full length promoter construct (unrluc). Finally, a deletion up to-22 (plasmid p22), which removed 6bp of the 12 bp palindrome, reduced unr promoter activity to about 10% of its maximum level. These results established that, in NIH 3T3 cells, 90 nucleotides of upstream sequences and 32 nucleotides of exon 1 constituted a highly efficient promoter. Because of the very small amount of unr sequences present in p90, it became important to assess the possible contributions of the plasmid sequences to the promoter activity. It should be pointed out that, in the absence of unr promoter sequences (i. e. in the vector pcluc), the level of luciferase activity is fifty fold lower than in p90, indicating a very low level of promoter activity within the plasmid B
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Figure 3. Functional analysis of the unr promoter by 5' deletions. (A) Schematic representation of the unr-luciferase constructs. Progressive 5' deletion fragments of the unr promoter were linked at position +32 of exon 1 to the luciferase gene as indicated in Material and Methods. The amount of sequences present upstream of the transcription initiation sites is indicated on the left. (B) Luciferase activities observed after transfection in NIH 3T3 cells. Each construct was assayed by transient expression in NIH 3T3 cells. The activities are expressed as a percentage of that of the 'full length' unr promoter construct p1O44 (unrluc). The data represent the mean from three experiments, each performed in triplicate. Standard errors were less than 15%.
6358 Nucleic Acids Research, 1992, Vol. 20, No. 23 Hela %
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Figure 4. Functional organization of the unr promoter in human cell lines. Three unr-luciferase constructs, unrluc, p370 and p90 containing respectively 1 kb, 370 bp and 90 bp of sequences upstream of the transcription initiation sites were transfected in the indicated cell lines. The promoterless constuct pcluc was included as a negative control. The results are expressed as a percentage of the activity of the unrluc construct in the corresponding cell line (the absolute measurements were: Hela, 60; RD, 158; SW 613, 240; MCF 7, 80 relative light units). sequences. Furthermore, RNase mapping experiments indicated that the transcription initiation sites were the same in unrluc and in p90, excluding the activation of a cryptic promoter within the plasmid (data not shown). Finally, we deleted 188 bp of plasmid sequences upstream of the unr sequences in p90, yielding p90A (see Material and Methods). This deletion removed, among other things, all of the polylinker sequences present in the unrluc constructs. As shown in table 1, the luciferase activity of p90A did not differ from that of p90. From all these data, we therefore concluded that the plasmid sequences did not significantly contribute to the promoter activity of p90.
Activity of the unr promoter in human cell lines We then investigated whether the same functional organization of the unr promoter (i. e. positive and negative upstream domains and a small full strength promoter) could be observed in human cell lines. We transfected unrluc, p370 and p90 as representing the 'full length', the 'repressed' and the minimal unr promoter, respectively. Four cell lines were used, Hela (derived from a cervical carcinoma), RD (derived from a rhabdomyosarcoma), SW613 (derived from a colon carcinoma) and MCF 7 (derived from a breast carcinoma). The luciferase vector pcluc was included as a negative control The results of these experiments are presented Fig. 4 and clearly established that the unr promoter had the same functional organization in human cells as in NIH 3T3 cells. As previously mentioned, the levels of luciferase activity in the human cell lines were about 200 fold lower than in NIH 3T3 cells. This probably reflects the difference in transfection efficiency between these human cell lines and the NIH 3T3 cells, since the activity of the CMVluc construct was similarly reduced a hundred fold (data not shown). .
Transactivation of the unr promoter by activated ras genes Although the levels of unr and N-ras mRNA accumulation in vivo differ widely between tissues (5, 6), we have repetitively observed that cell lines in vitro express an average level of each gene, irrespectively of their proliferation or differentiation status (H.J.-S., G.Triqueneaux and F.D. unpublished results). To extend our functional analysis of the unr promoter, we searched for stimuli that could alter the expression of unr in cells in culture. We had observed that in NIH 3T3 cells expressing a Kirsten
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Figure 5. Transactivation of the unr promoter by mutated ras genes. (A) Transactivation of the 'full length' unr promoter. The unrluc construct was cotransfected in NIH 3T3 cells with either a normal (gly 12, light bar) or an activated (val 12, dark bar) ras expression vector. For each ras gene, the luciferase activity observed in the presence of the normal form of p2lras was normalized to 1. (B) Transactivation of the 'minimal' unr promoter. The same experiments as in (A) were carried out with the p9O construct.
cDNA with a valine 12 mutation there was a two to three fold increase in the level of unr mRNAs. This suggested that the unr promoter could be transactivated by ras oncogenes. To explore this possiblity, we cotransfected unr-luciferase constructs with expression vectors containing either normal (gly 12) or activated (val 12) ras coding sequences. Ki-ras and N-ras cDNAs were expressed from the Moloney LTR of the pZipneo vector (13), while Ha-ras was expressed from its own promoter in the genomic clones EC and EJ. We first cotransfected the three couples of ras expression vectors with unrluc (Fig. 5A). When compared with the corresponding normal sequence, the presence of a valine 12 activating mutation led to a 2.5 to 7 fold increase in the luciferase activity. It is interesting to note that despite the very close similarity of ZipKi-ras and ZipN-ras, the latter is less potent both as a transactivator of the unr promoter and as an inducer of the transformed phenotype in NIH 3T3 cells (unpublished observations). We then tested the short promoter fragment p90, which contains 90 bp upstream of the start site, for its ability to respond to ras oncogenes (Fig. SB). The pattern of p90 transactivation by the different ras genes was almost identical with that observed with unrluc, indicating that this basal promoter structure was transactivated as efficiently by ras oncogenes as the 'full length' promoter.
DISCUSSION As a part of our investigation of the unr/N-ras locus, we have begun a characterization of the human unr promoter. To tis end, we have isolated and characterized genomic clones corresponding to the 5' end of the unr gene.We have mapped the transcription
Nucleic Acids Research, 1992, Vol. 20, No. 23 6359 initiation sites by a combination of RNase mapping and primer extension experiments. Sequence analysis of a genomic clone encompassing the transcription initiation sites indicates that they are located within a CpG island since the sequence has a high G/C content and an equal frequency of CpG and GpC dinucleotides (O1). Moreover, as it is often the case for promoters located in CpG islands, the unr promoter lacks typical TATA or CAAT box. Inspection of 1 kb of sequence upstream of the transcription initiation sites as well as of the first exon reveals the presence a remarkably small number of potential binding sites for known transcription factors. In the proximal promoter region there is only one consensus sequence, a potential Spl binding site (GGCGGG), located at-60. This site is likely to be important since it has been proposed that, in G/C rich promoters, Spl sites substitute for TATAA sequences in nucleating assembly of the transcription machinery (14). Further upstream, a potential Mt4 binding site (12) is located at -784. Besides these two sites for known factors, one remarkable feature of the unr promoter is the presence at -20 of a 12 bp palindrome. The only other occurence of this sequence in GENBANK (release 71) is in the promoter of the mitochondrial muscle adenine nucleotide translocator at position -42(15). This observation and the fact that many transcription factors bind to DNA as dimers and recognize palindromic sequences are in favor of a functional significance of this sequence. Moreover, it is worth noting that the highest level of NRU expression has been observed in the skeletal muscle (6), indicating the presence of some muscle specific regulation. We have used a set of 5' deletions to investigate the functional organization of the unr promoter. In NIH 3T3 cells, sequences extending from -1044 to +32 confered a high level of expression to a luciferase reporter gene. Indeed, the level of expression reached 25 % of that of the cytomegalovirus immediate early promoter, one of the strongest mammalian promoters known. Deletion analysis indicated that sequences from -90 to + 32 were sufficient to confer this high level of expression. Several studies of G/C rich promoters have reported that a basal promoter structure, including less than one hundred nucleotides upstream of the transcription initiation sites, was sufficient for maximal promoter activity in transient expression assays (16-18). However, the case of unr is remarkable by the level of this basal promoter activity. Moreover, the nucleotide sequence provides little clue as to the mechanism which leads to this efficient transcription initiation, since the only known binding site in this region is an Spl site located at -58. Although this short fragment is sufficient for maximal promoter activity, analysis of the other deletions indicates that sequences located upstream are also important for the regulation of transcription. The simplest interpretation of our results is that there is a negative regulatory domain located between -190 and -370, the effect of which is neutralized by a positive regulatory element located between 440 and -600. The same functional organization was observed by transfection of human cell lines of different origins. Since, in vivo there are large variations in the level of unr mRNA accumulation between tissues (5, 6), these upstream sequences are likely to play a role in the tissue specific expression. Thus, for instance, the expression of unr is particularly high in skeletal muscles, where it closely follows the expression of cytochrome oxydase (G.Triqueneaux and F.D. unpublished observations), suggesting that the Mt4 sequence located at -784 could be involved. However, we have so far failed to identify a cellular system that would reproduce this muscle specific expression in vitro, possibly because none of the myogenic cell lines available
has an adult phenotype. Thus, at this time, analysis of the role of the upstream sequences would have to be carried out in vivo. Despite the lack of an in vitro system that would reproduce the tissue-specific expression of unr, some variations in the level of unr mRNA accumulation can be observed in cell lines(6). Our observation that NIH 3T3 cells transformed by a Ki-ras gene expressed a two to three fold higher level of unr mRNA, as well as the results of Doniger (4) showing that tumorigenic cells harboring a mutated N-ras gene had a higher level of unr mRNA, suggested the possibility of a coupling between the ras transduction pathway and the expression of unr. Cotransfection experiments with either Harvey, Kirsten or N-ras expression vectors showed that the presence of an activating mutation within the ras gene led to a 3 to 7 fold stimulation of the unr promoter activity. This effect was also observed with the p90 'basal' promoter construct, indicating that the factors mediating the transactivation should bind within this short DNA fragment. This is of particular interest since, although several transcription factors like AP1('9), NFkB(20), PEA3 (21) have been shown to be regulated by ras, none of these has a canonical binding site in p90, suggesting that other transcription factors are involved in the ras transduction pathway. The close proximity of the unr and N-ras transcription units suggests that multiple levels of regulation could operate within this locus. First, the previous characterization of the N-ras promoter and the present study establish that the two genes have their own promoter regions. Second, since the 3' end of unr coincides with the 5' end of N-ras, it is likely that the two genes belong to the same domain of active chromatin (22). As a consequence, they should not be insulated from the transcriptional regulation of each other or from the influence of transcriptional regulators acting on the whole locus (23-25). Third, RNA polymerases transcribing the unr gene could limit the access to the N-ras promoter (10). However, since this phenomenon of promoter occlusion can be relieved by polyadenylation/termination signals (10, 26), the potential transcriptional interference between unr and N-ras should be considered in relation with the usage of the three polyadenylation signals of the unr gene. Fourth, we have shown in this study that the activation of the ras signaling pathway leads to the transactivation of the unr promoter, indicating a possible interaction in trans within the locus. Analysis of the pattern of unr and N-ras mRNA accumulation in murine tissues (5, 6) reveals no obvious signature of one of these modes of regulation. Indeed, the global results are most easily interpreted if one assumes that several of these regulations can act simultaneously on the locus. Nevertheless, the examples of co-regulation or opposite regulation that can be observed in some tissues indicate that the relative importance of these regulations varies with the cellular context. This is illustrated by the co-regulation of unr and N-ras during lymphocyte activation (6), which excludes that at these levels of expression there is a significant promoter occlusion. On the other hand, the extremely high level of unr expression in skeletal muscles (6) is associated with a very low level of N-ras expression which is in agreement with either a promoter occlusion or the extinction of the N-ras promoter in a tissue where the unr promoter is particularly active (hence an efficient insulation from the regulatory elements of unr). A priori, a detailed knowledge of both unr and N-ras promoters should make it possible to evaluate the relative importance of the individual promoters and of the interactions within the locus. However, the available structural informations ((89 9) and this study) only indicate that these promoters share no obvious regulatory element beyond the fact that they are G/C rich and
6360 Nucleic Acids Research, 1992, Vol. 20, No. 23 contain positive and negative regulatory domains. Moreover, no data on their strength in vivo are available. As an alternative strategy, we have chosen to use the gene targeting technique ('homologous recombination') to investigate in vivo the interactions within the locus. As a first step, we have created a null allele of N-ras in embryonic stem cells (27). The characterization of the unr promoter will now enable us to delete this promoter in ES cells and to generate unr- mice. Finally, crosses between the unr- and N-ras- heterozygotes will allow us to analyze the interactions in cis within the locus by following the expression of N-ras.
MATERIALS AND METHODS Cloning and sequencing A human genomic library generated from human leucocytes DNA cloned into the lambda phage EMBL3 (Clontech) was screened according to Sambrook et al. (28). Hybridization studies indicated that sequences complementary to the 5' end of unr cDNAs were present in a lambda clone (1P7), as a 1.3 kb SalI-SacI and a 3.5 kb HindIl-HindIII Fragment. These fragments were subcloned into the plasmid Bluescript (Stratagene) and sequence analysis was performed on both strands, using the sequenase sequencing kit (United States Biochemicals). Primer extension A 20 base oligonucleotide (5'-TGAAGCAGCAGTTTCAGGTGG-3') complementary to bases +52 to +71 in the 5' untranslated region of the human unr message(6) was endlabelled with ATP (.y-32p) to a specific activity of 8 x 108 cpm/1g. 10 pg of primer were hybridized with either 2 Ag of poly (A)+ or 10 jig of total RNA for two hours at 50°C in 4 tu of 0.25 M KCI, 10 mM Tris-HCl pH8. Reverse transcription was performed with 200 units of Moloney virus reverse transcriptase (BRL) for 30 minutes at 37°C in a final volume of 20 j1A according to Ausubel et al. (29).
RNase mapping The RNA probe was transcribed in vitro to a specific activity of 6 x 107 cpm/4g, from the plasmid containing the SailI-Sacd fragment linearized at the NruI site. Samples containing 0.5 ng of probe and 10 Ag of total RNA were hybrized at 42°C for 16 h and then processed according to a standard protocol (28). RNA isolation RNA was prepared by the guanidinium-thiocyanate method (30). Poly (A)+ RNA was selected by oligo (dT) chromatography according to Sambrook et al. (28). Plasmids The pZIP-Kiras (gly12), pZIP-Kiras* (val12), pZIP-Nras (gly12), and pZIPNras* (val12) plasmids contain the corresponding human ras cDNAs (with either the normal glycine or the activating valine at codon 12) cloned into the BamHI site of pZIP-Neo (13). The EC and EJ plasmids contain the BamHI genomic fragment encompassing the c-Ha-ras gene isolated from either normal human DNA or the EJ bladder carcinoma cell line (31)
Luciferase expression vectors Plasmids unrluc and unrlucAS contain the 1 kb SalI-HaeHI genomic fragment corresponding to nucleotides -1044 to + 32 of the human unr gene, cloned in either orientation in front of
the luciferase gene into the HindIl site of the pcluc plasmid (32). A series of 5' unidirectional deletions was created by Bal3l digestion of the plasmid unrluc linearized at the SaUl site (position -1044 of the NRU promoter). The Bal3l-XbaI fragments were then inserted in the KpnI-XbaI digested pcluc. Plasmid p22 was obtained by restriction of NRUluc at the unique NruI site located 22 bp upstream of the unr transcription initiation site. As a negative control, pcluc vector was also digested by KpnI and HindM, so that all constructs analyzed in transfections, including the promoterless pcluc, have identical plasmid sequences. Plasmid p9OD was generated by cloning the SacI-XbaI fragment of the Bal3l generated -90 deletion into the pcluc vector digested by PvuIl and XbaI. As a positive control we used the CMVluc construct, which contains the immediate early promoter of the human cytomegalovirus cloned upstream of the luciferase gene in the pcluc vector.
Cell culture and transfections experiments NIH3T3, Hela, SW613, RD and MCF 7 cells were grown in Dulecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. 8 hours before transfection, cells were seeded a density of 4 x 105 cells per 6 cm dish. For each dish, 8Isg of plasmid were transfected by the CaPO4 technique. For cotransfection experiments, 4tsg of the reporter construct were mixed with 4 isg of the effector plasmid. All transfections were carried out in triplicate, and each plasmid was tested in three independent experiments. 48h after transfection, the cells were lysed with 0.4 ml of 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, 15% glycerol, 0.4 mM PMSF, and luciferase activity was measured in a luminometer (Lumac) as described (33). Enzymatic reactions were initiated by injecting 100 1l of 0.25 mM luciferin and performed at 25° for 60 sec. The results were expressed as relative light units (RLU), for 11AI of extract (33).
ACKNOWLEDGEMENTS We are grateful to H.Neel for providing the pZipKi-ras and pZipN-ras vectors and to U.Hazan for the pcluc and CMVluc plasmids. We are indebted to C.Lavialle, G.Triqueneaux and D.Weil for helpful discussions. This work was supported by the CNRS and the Association pour la Recherche sur le Cancer (grant
6307).
REFERENCES 1. Barbacid, M. (1987) Ann. Rev. Biochem. 56, 779-827. 2. Quaife, C.J., Pinkert, C.A., Ornitz, D.M., Palmiter, R.D. & Brinster, R.L. (1987) Cell 48, 1023-1034. 3. Paterson, H., Reeves, B., Brown, R., Hall, A., Furth, M., Bos, J., Jones, P. & Marshall, C. (1987) Cell 51, 803-812. 4. Doniger, J. & DiPaolo, J.A. (1988) Nucleic Acids Res. 16, 969-980. 5. Jeffers, M., Paciucci, R. & Pellicer, A. (1990) Nucleic Acids Research 18, 4891 -4899. 6. Nicolaiew, N., Triqueneaux, G. & Dautiy, F. (1991) Oncogene 6, 721-730. 7. Doniger, J., Landsman, D., Gonda, M.A. & Wistow, G. (1992) The New Biologist 4, 389-395. 8. Paciucci, P. & Pellicer, A. (1991) Mol. Cell. Bio. 11, 1334-1343. 9. Thorn, J.T., Yodd, A.V., Warrilow, D., Watt, F., Molloy, P.L. & Iland, H.J. (1991) Oncogene 6, 1843-1850. 10. Proudfoot, N.J. (1986) Nature 322, 562-565. 11. Bird, A.P. (1986) Nature 321, 209-213. 12. Suzuki, H., Hosokawa, Y., Tosa, H., Nishikimi, M. & Ozawa, T. (1990) J. Biol. Chem. 265, 8159-8163. 13. Cepko, C.L., Roberts, B.E. & Mulligan, R.C. (1984) Cell 37, 1053-1052. 14. Pugh, B.F. & Tjian, R. (1991) Genes and Development 5, 1935 - 1345.
Nucleic Acids Research, 1992, Vol. 20, No. 23 6361 15. Li, K., Warner, C.K., Hodge, J.A., Minoshima, S., Kudoh, J., Fukuyama, R., Maekawa, M., Shimizu, Y., Shimizu, N. & Wallace, D.C. (1989) J. Biol. Chem. 264, 13998-14004. 16. Widen, S.G., Kedar, P. & Wilson, S.H. (1988) J. Biol. Chem. 263, 16992-16998. 17. Somma, M.P., Pisano, C. & Lavia, P. (1991) Nuc. Acids Res. 19, 2817-2824. 18. Raymond, M. & Gros, P. (1990) Mol. Cell. Biol. 10, 6036-6040. 19. Binetruy, B., Smeal, T. & Karin, M. (1991) Nature 351, 122-127. 20. Arenzana-Seisdedos, F., Israel, N., Bachelerie, F., Hazan, U., Acalmi, J., Dautry, F. & Virelizier, J.-L. (1989) Oncogene 4, 1359-1362. 21. Wasylyk, C., Flores, P., Gutman, A. & Wasylyk, B. (1989) 7he EMBO Journal 8, 3371-3378. 22. Wolffe, A. Chronatin, structure and function (Academic Press, London, 1992). 23. Stief, A., Winter, D.M., Stratling, W.H. & Sippel, A.E. (1989) Nature 341, 343-345. 24. Kellum, R. & Schedl, P. (1991) Cell 64, 941-950. 25. McKnight, R.A., Shamay, A., Sankaran, L., Wall, R.J. & Hennighausen, L. (1992) Proc. Natl. Acad. Sci. USA 89, 6943-6947. 26. Irniger, S., Egli, C.M., Kuenzler, M. & Braus, G.H. (1992) Nuc. Acids Res. 20, 4733-4739. 27. Cases, S. & Dautry, F. (1992) Oncogene, in press. 28. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). 29. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Stnrhl, K. Current protocols in nolecular biology (John Wiley and sons, New-York, 1990). 30. Berger, S.L. & Kimmel, A.L. Guide to molecular cloning techniques (Academic Press, 1987). 31. Shih, C., Padhy, L.C., Murray, M. & Weinberg, R.A. (1981) Nature 290, 261-264. 32. Schwartz, O., Virelizier, J.L., Montagnier, L. & Hazan, U. (1990) Gene 88, 197-205. 33. De Wet, J.R., Wood, K., DeLuca, M., Helinski, D.R. & Subramani, S. (1987) Mol. Cell. Biol. 7, 725-737.
Nucleic Acids Research, 1992, Vol. 20, No. 23 6355-6361
Organization of the unriN-ras locus: characterization of the promoter region of the human unr gene Helene Jacquemin-Sablon and Francois Dautry Laboratoire d'Oncologie Moleculaire, CNRS UA 1158, Institut Gustave Roussy, 94805 Villejuif Cedex, France Received August 7, 1992; Revised and Accepted November 5, 1992
ABSTRACT Investigations of the structure and expression of the N-ras gene in mammals has led to the identification of another gene designated unr , which is located immediately upstream of N-ras. These two genes are transcribed in the same orientation and the intergenic distance is of the order of 150 nucleotides. This genetic organization has been observed in the genome of guinea pig, rat, mouse and man with a very high level of sequence conservation in the intergenic region. This unusual gene clustering suggests that the transcriptional regulation of this locus could involve common regulatory sequences as well as transcriptional interference between the two genes. In this study, we have isolated and characterized the human unr promoter. A cluster of transcription initiation sites was mapped by primer extension and RNase protection and shown to be located in a CpG island devoid of TATA and CAAT boxes. Functional organization of the promoter was investigated by measuring the ability of a set of 5' deletions within al kb promoter region to drive the expression of the luciferase gene. These studies indicated a very strong promoter actvity in NIH 3T3 cells and the presence of positive and negative regulatory domains. Nevertheless, a 90 bp fragment showed the same level of promoter activity as the 1 kb fragment. We also showed that ras genes can transactivate the unr promoter activity and that the 90 bp fragment responded to this transactivation. INTRODUCTION Harvey, Kirsten and N-ras are members of the ras gene family which have been implicated in the pathogenesis of human cancer. This is based on the observation that a large proportion of tumors contain a mutated ras gene which can transform cells in vitro (1), and predispose transgenic mice to cancer (2). While oncogenic activation of the ras genes by point mutations has been well documented, many experiments have also shown that the level of expression is critical for the biological activity of both normal and mutated ras proteins. Although most of these studies deal with the Harvey-ras gene, analysis of the revertants of the fibrosarcoma cell line HT 1080 clearly indicates that this is also true for N-ras(3).
EMBL accession no. X68286
It has been shown that the N-ras gene is part of an unusual genetic structure which comprises a tandem of closely linked transcription units in the same orientation. This organization has been observed in the genome of guinea pig (4), rat (5), mouse (5,6) and man (6). N-ras corresponds to the downstream transcription unit, while the upstream one does not match with any previously identified gene and has been designated unr (upstream of N-ras) (4, 5) or NRU (N-ras upstream) in our previous study (6). Sequence analysis of the predicted unr protein product has revealed the presence of five internal repeats which are distantly related to the E.coli cold shock protein (7). Since it has been proposed that these motifs are the hallmark of a new class of DNA binding proteins, this raises the possibility that unr could be a DNA binding protein. However, at this stage, the function of unr and what relation, if any, it may have with the function of N-ras is unknown. The unusual nature of the unrlNras locus comes from the small intergenic distance (of the order of 150 nucleotides) between two ubiquitously expressed genes. Moreover, there are three unr messages which have the same coding capacity and differ only by their polyadenylation site (6) (O.Boussadia, H.J.-S. and F.D., submitted for publication). While the first polyadenylation signal is located 1.2 kb upstream of the N-ras gene, at a standard intergenic distance, it is the less frequently used of the three signals. This observation, and the extremely high level of sequence conservation of the unrlN-ras junction suggest that there is a selective pressure to maintain this compact organization. Genomic transfection experiments have established that the Nras gene was functional in the absence of upstream sequences and both the murine and the human N-ras promoter regions have been partially characterized(8 9). Nevertheless, the presence of the unr transcription unit suggests that other levels of regulation could contribute to the control of N-ras expression. On the one hand, usage of the last unr polyadenylation signal implies that the two transcription units overlap, raising the possibility of transcriptional interferences (10). On the other hand, it is likely that unr and N-ras are part of the same transcriptional domain and therefore are subjected to common transcriptional regulations. As a part of our efforts to evaluate the relative contributions of these different levels of regulation within the unr/N-ras locus, we present in this paper a characterization of the human unr promoter. We have cloned and sequenced the 5' end of the unr gene and localized the transcription initiation sites. We have used a transient expression assay in NIH 3T3 cells to analyze the
6356 Nucleic Acids Research, 1992, Vol. 20, No. 23 functional organization of this promoter and established that a 90 pb fragment in which the only known binding site is an SPI site, has a very high promoter activity. Finally, the unr promoter activity is increased by cotransfection with an activated ras gene, indicating the possibility of interactions in trans between N-ras and unr.
RESULTS Cloning and sequence analysis of the human unr promoter region We have previously reported the isolation and nucleotide sequence of cDNA clones of the human unr gene (6). In most cell types, three unr mRNA can be detected with apparent mobilities of approximately 4.4, 4 and 3.4 kb. We have established that these mRNA are generated by the use of ftree different polyadenylation sites located in the last unr exon (6,7) (O.Boussadia, H.J.-S. and F.D. unpublished results). To isolate genomic clones corresponding to the 5' end of the unr gene, we used the first 800 nucleotides of our longest cDNA clone to screen a human leucocyte genomic DNA library. Overlapping positives clones A
x P7 Sal
Sac
I
AfI
Bgl
AfI
I
I
AfI
I
l
were isolated and rescreened with a 20mer oligonucleotide corresponding to the 5' end of the reported cDNA sequence (6). One phage (X P7) which contained a 13 kb insert was selected for further study and its restriction map is presented in Fig. lA. This clone contained two fragments which hybridized to the 5' untranslated region of the unr cDNA. Sequence analysis of these fragments indicated the presence of two exons, tentatively numbered exons 1 and 2. Exon 2 was colinear with the cDNA sequence from position + 26 to + 341 and was surrounded by potential splice acceptor and donor sites. The 1.2 kb SalI-SacI fragment containing the putative exon 1 along with 1 kb of upstream sequences was then sequenced (Fig. IB).
Mapping of the transcriptional start sites The transcription initiation sites were determined by a combination of RNase mapping and primer extension. We first used a genomic probe to analyse by RNase mapping the size of exon 1. A 190 nucleotides long 32P-labelled RNA probe spanning the presumptive 5' end of the unr gene was hybridized with total mRNA isolated from different cell lines. Fig.2A shows the presence of a major protected fragment of 60 nucleotides in all the cell lines that we have analyzed (MCF 7, K 562, RD). On longer exposure a cluster of fragments with sizes ranging from 55 to 65 nucleotides can also be observed. Since RNase protection does not discriminate between the end of the mRNA and the 5' end of an exon, we performed a primer extension analysis to confirm that these protected fragments corresponded to the transcription start sites. The primer used is located in exon
I
Exonl
Exon2
L1Kb 1 Kb
-1044 -
9S4
3
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924
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-
864
TYYAYCCAAYACWDYCMAZ¶.65L0CCIUWGA&TCYGTG&AAA&C&G&AOC&9YGA&T
-
744
-
684
-
624
-
564
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II *'Us
-
504
-
444
-
384
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324
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-
264
-
204
-
144
CGTGYT0A4AGfOCYCGTCACOGTCACGGTAGCCCTOCTA&CCAAG
-
84
-
24
PAL
Sac GOCTCYGA&GCYAGGT0GGAG TCACCGATYCC CATACGTCCCTTCA=CTOCCTGGOCCTCA?TCAGTC^GAGCTGAACA
_
Organization and nucleotide sequence of the 5' end of the human unr Map of the 7 kb Sail-Afll fragment which contains exons 1 and 2. The sites for the restriction enzymes SacI (Sac), Bglll (Bgl) and AflII (Afl) are indicated. The Sail site was introduced by the bacteriophage vector. (B) Nuckeotide sequence of the 1.2 kb Sail-SacI frgment. Nucleotides are numbered with respect to the major transcription initiation site (+ 1) and exon 1 is boxed. The SPI and Mt4 consensus sequences are boxed and a 12 bp palindrome (PAL) is underlined. Figure 1. gene. (A)
t%
Hse2
* 37
* 156
_a
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ACOCTCG&&G&CYAGAG&COCCA&AGCC&CCCGCCCCCYAG?G?CAOG&GAG?GCAG ATATAA CCCA TTCTC CCAC TAAACCC
+ 216
a
,F,
-
* 96
*u
Figure 2. Determination of the transcription initiation sites in the unr gene. (A) RNase mapping analysis of the size of exon 1. As indicated at the bottom of the figure the probe was obtained by in vitro transctiption of the 190 bp SaclNruI genomic fragment. Lanes: 1, undigested probe; 2, DNA molecular weight markers (MspI-digested pBR322 DNA); 3, RNA molecular weight markers (65 and 54 nucleotides); 4, cytoplasmic MCF 7 RNA; 5, MCF 7 polyA+ RNA; 6, K 562 total RNA; 7, K 562 polyA+ RNA; 8, cytoplasmic RD RNA; 9, tRNA. (B) Primer extension analysis of the unr messages. The primer used is a 20mer oligonucleotide located 46 nucleotides downstream of the exon 2 as indicated at the bottom of the figure. The sequence ladder on the left was used to determine the size of the extended products. Lanes: 1, cytoplasmic MCF 7 RNA; 2, MCF 7 polyA+ RNA; 3, K 562 total RNA; 4, RD cytoplasmic RNA; 5, Jurkat total RNA; 6, tRNA. An arrow indicates the major extension product.
Nucleic Acids Research, 1992, Vol. 20, No. 23 6357
2 (nucleotides 71 to 52 of the published cDNA sequence). Similar results were obtained with either total RNA from different cell lines, or poly (A)+ RNA from MCF-7 cells. The abundance of the extension products matched the accumulation of unr mRNA observed by Northern blot analysis (data not shown). By comparaison to a DNA sequence ladder, the major extension products ranged in size between 90 and 110 nucleotides, with a prominent band of 106 nucleotides (Fig.2B). Since 46 nucleotides should be derived from exon 2, this indicated that the size of exon 1 was 60 nucleotides, in good agreement with the RNase mapping results. Additional start positions, upstream and downstream of the major site were also detected, but with lower relative abundance. Taken together, these results indicated that the unr initiation sites were located in the SailI-SacI fragment of the clone X P7, and we designated + 1 the major initiation site in the sequence of Fig. 1.
Sequence of the unr promoter region Inspection of the unr promoter sequence revealed that it lacked TATA or CCAAT box sequences upstream of the transcription initiation sites. The unr first exon and the surrounding sequences (from -84 to +87) contained 64% G+C and in this region the ratio of CpG to GpC is 1.1, indicating the absence of CpG suppression. These properties established that the 5' end of the unr gene was located within a CpG island (ll). The sequence of the 1 kb region upstream of exon 1 appeared remarkably devoid of known binding sites for transcriptional regulators. Indeed, in spite of the high G/C content only one potential SPI site (matching the minimal consensus sequence GGCGGG) could be found at position -60. Further upstream, the sequence Mt4 (CTCACCA) which is common to several nuclear genes encoding mitochondrial proteins (12) was found at position -784. A 12 bp palindromic sequence (5'-CTCTCGCGAGAG-3', underlined in Fig.lB) was observed at position -22. Functional analysis of the unr promoter The promoter activity of the cloned 5' end of the unr gene was studied by transient expression assays with the luciferase gene as a reporter. Initially, the 1076 nucleotides Sal1-HaeII fragment from X P7, which contains 1044 nucleotides of upstream sequences and 32 nucleotides of exon 1 was cloned in front of the luciferase gene in the plasmid pcluc (see Material and Methods). This fragment was cloned in either the natural or the reverse orientation, giving the constructs unrluc and unrlucAS, respectively. These constructs were introduced by calcium phosphate-mediated transfection into different cell lines and the luciferase activities in the cell extracts were analyzed. The Table 1. Activities of the unr-luciferase and CMV-luciferase constructs in NIH 3T3 cells. Plasmids CMVluc unrluc unrlucAS p90
p90A pcluc
Luciferase activity (RLU)
SEE
160000 40000 1500 45000 51000 1100
7 10 12 7 10 9
linearity of the assay was checked by measuring the luciferase activity for two extract dilutions. Two different plasmid preparations were used for each construct to check for anomalies due to a particular plasmid preparation. A construct (CMVluc) containing the human cytomegalovirus immediate early promoter was included as a positive control. As shown in table 1, unrluc yielded high level of luciferase activity in NIH3T3 cells (25% of that of CMVluc) while unrlucAS did not behave differently from the promoterless pcluc. The activity levels in other cell lines, in particular in human cell lines, were much lower (see below) and therefore we first carried out an analysis of the unr promoter in NIH 3T3 cells. A series of 5' deletions of the upstream region was constructed to search for regulatory elements and to determine a minimum sequence needed for promoter activity. Bal 31 exonuclease was used to generate progressive 5' deletions within this promoter (Fig.3A). We observed no significant difference in luciferase activities among the constructs containing more than 600bp of flanking sequences (Fig.3B). In contrast, a sharp reduction of luciferase activity was observed when sequences between -600 and -370 were deleted, suggesting the presence of positive regulatory sequences within this region. Further deletions progressively restored a high level of luciferase activity, indicating the presence of a negative regulatory domain between -370 and -90. In particular, plasmid p90 which contains only 90 nucleotides of the unr promoter was as active as the full length promoter construct (unrluc). Finally, a deletion up to-22 (plasmid p22), which removed 6bp of the 12 bp palindrome, reduced unr promoter activity to about 10% of its maximum level. These results established that, in NIH 3T3 cells, 90 nucleotides of upstream sequences and 32 nucleotides of exon 1 constituted a highly efficient promoter. Because of the very small amount of unr sequences present in p90, it became important to assess the possible contributions of the plasmid sequences to the promoter activity. It should be pointed out that, in the absence of unr promoter sequences (i. e. in the vector pcluc), the level of luciferase activity is fifty fold lower than in p90, indicating a very low level of promoter activity within the plasmid B
A unr
Luciferase activity
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The results are expressed in relative light units and represent the average of four independent experiments performed with two distinct plasmid preparations. SEE indicates the standard error of estimates.
Figure 3. Functional analysis of the unr promoter by 5' deletions. (A) Schematic representation of the unr-luciferase constructs. Progressive 5' deletion fragments of the unr promoter were linked at position +32 of exon 1 to the luciferase gene as indicated in Material and Methods. The amount of sequences present upstream of the transcription initiation sites is indicated on the left. (B) Luciferase activities observed after transfection in NIH 3T3 cells. Each construct was assayed by transient expression in NIH 3T3 cells. The activities are expressed as a percentage of that of the 'full length' unr promoter construct p1O44 (unrluc). The data represent the mean from three experiments, each performed in triplicate. Standard errors were less than 15%.
6358 Nucleic Acids Research, 1992, Vol. 20, No. 23 Hela %
MCF7
SW613
RD
B
0 40 80 120 0 40 80 120 0 40 80 120 0 40 80 120 L
a
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I
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.
-
-
unriuc p370
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Figure 4. Functional organization of the unr promoter in human cell lines. Three unr-luciferase constructs, unrluc, p370 and p90 containing respectively 1 kb, 370 bp and 90 bp of sequences upstream of the transcription initiation sites were transfected in the indicated cell lines. The promoterless constuct pcluc was included as a negative control. The results are expressed as a percentage of the activity of the unrluc construct in the corresponding cell line (the absolute measurements were: Hela, 60; RD, 158; SW 613, 240; MCF 7, 80 relative light units). sequences. Furthermore, RNase mapping experiments indicated that the transcription initiation sites were the same in unrluc and in p90, excluding the activation of a cryptic promoter within the plasmid (data not shown). Finally, we deleted 188 bp of plasmid sequences upstream of the unr sequences in p90, yielding p90A (see Material and Methods). This deletion removed, among other things, all of the polylinker sequences present in the unrluc constructs. As shown in table 1, the luciferase activity of p90A did not differ from that of p90. From all these data, we therefore concluded that the plasmid sequences did not significantly contribute to the promoter activity of p90.
Activity of the unr promoter in human cell lines We then investigated whether the same functional organization of the unr promoter (i. e. positive and negative upstream domains and a small full strength promoter) could be observed in human cell lines. We transfected unrluc, p370 and p90 as representing the 'full length', the 'repressed' and the minimal unr promoter, respectively. Four cell lines were used, Hela (derived from a cervical carcinoma), RD (derived from a rhabdomyosarcoma), SW613 (derived from a colon carcinoma) and MCF 7 (derived from a breast carcinoma). The luciferase vector pcluc was included as a negative control The results of these experiments are presented Fig. 4 and clearly established that the unr promoter had the same functional organization in human cells as in NIH 3T3 cells. As previously mentioned, the levels of luciferase activity in the human cell lines were about 200 fold lower than in NIH 3T3 cells. This probably reflects the difference in transfection efficiency between these human cell lines and the NIH 3T3 cells, since the activity of the CMVluc construct was similarly reduced a hundred fold (data not shown). .
Transactivation of the unr promoter by activated ras genes Although the levels of unr and N-ras mRNA accumulation in vivo differ widely between tissues (5, 6), we have repetitively observed that cell lines in vitro express an average level of each gene, irrespectively of their proliferation or differentiation status (H.J.-S., G.Triqueneaux and F.D. unpublished results). To extend our functional analysis of the unr promoter, we searched for stimuli that could alter the expression of unr in cells in culture. We had observed that in NIH 3T3 cells expressing a Kirsten
VI S
4
2
Ki-ras N-ras
Ha-ras
Ki-ras N-ras Ha-ras
Figure 5. Transactivation of the unr promoter by mutated ras genes. (A) Transactivation of the 'full length' unr promoter. The unrluc construct was cotransfected in NIH 3T3 cells with either a normal (gly 12, light bar) or an activated (val 12, dark bar) ras expression vector. For each ras gene, the luciferase activity observed in the presence of the normal form of p2lras was normalized to 1. (B) Transactivation of the 'minimal' unr promoter. The same experiments as in (A) were carried out with the p9O construct.
cDNA with a valine 12 mutation there was a two to three fold increase in the level of unr mRNAs. This suggested that the unr promoter could be transactivated by ras oncogenes. To explore this possiblity, we cotransfected unr-luciferase constructs with expression vectors containing either normal (gly 12) or activated (val 12) ras coding sequences. Ki-ras and N-ras cDNAs were expressed from the Moloney LTR of the pZipneo vector (13), while Ha-ras was expressed from its own promoter in the genomic clones EC and EJ. We first cotransfected the three couples of ras expression vectors with unrluc (Fig. 5A). When compared with the corresponding normal sequence, the presence of a valine 12 activating mutation led to a 2.5 to 7 fold increase in the luciferase activity. It is interesting to note that despite the very close similarity of ZipKi-ras and ZipN-ras, the latter is less potent both as a transactivator of the unr promoter and as an inducer of the transformed phenotype in NIH 3T3 cells (unpublished observations). We then tested the short promoter fragment p90, which contains 90 bp upstream of the start site, for its ability to respond to ras oncogenes (Fig. SB). The pattern of p90 transactivation by the different ras genes was almost identical with that observed with unrluc, indicating that this basal promoter structure was transactivated as efficiently by ras oncogenes as the 'full length' promoter.
DISCUSSION As a part of our investigation of the unr/N-ras locus, we have begun a characterization of the human unr promoter. To tis end, we have isolated and characterized genomic clones corresponding to the 5' end of the unr gene.We have mapped the transcription
Nucleic Acids Research, 1992, Vol. 20, No. 23 6359 initiation sites by a combination of RNase mapping and primer extension experiments. Sequence analysis of a genomic clone encompassing the transcription initiation sites indicates that they are located within a CpG island since the sequence has a high G/C content and an equal frequency of CpG and GpC dinucleotides (O1). Moreover, as it is often the case for promoters located in CpG islands, the unr promoter lacks typical TATA or CAAT box. Inspection of 1 kb of sequence upstream of the transcription initiation sites as well as of the first exon reveals the presence a remarkably small number of potential binding sites for known transcription factors. In the proximal promoter region there is only one consensus sequence, a potential Spl binding site (GGCGGG), located at-60. This site is likely to be important since it has been proposed that, in G/C rich promoters, Spl sites substitute for TATAA sequences in nucleating assembly of the transcription machinery (14). Further upstream, a potential Mt4 binding site (12) is located at -784. Besides these two sites for known factors, one remarkable feature of the unr promoter is the presence at -20 of a 12 bp palindrome. The only other occurence of this sequence in GENBANK (release 71) is in the promoter of the mitochondrial muscle adenine nucleotide translocator at position -42(15). This observation and the fact that many transcription factors bind to DNA as dimers and recognize palindromic sequences are in favor of a functional significance of this sequence. Moreover, it is worth noting that the highest level of NRU expression has been observed in the skeletal muscle (6), indicating the presence of some muscle specific regulation. We have used a set of 5' deletions to investigate the functional organization of the unr promoter. In NIH 3T3 cells, sequences extending from -1044 to +32 confered a high level of expression to a luciferase reporter gene. Indeed, the level of expression reached 25 % of that of the cytomegalovirus immediate early promoter, one of the strongest mammalian promoters known. Deletion analysis indicated that sequences from -90 to + 32 were sufficient to confer this high level of expression. Several studies of G/C rich promoters have reported that a basal promoter structure, including less than one hundred nucleotides upstream of the transcription initiation sites, was sufficient for maximal promoter activity in transient expression assays (16-18). However, the case of unr is remarkable by the level of this basal promoter activity. Moreover, the nucleotide sequence provides little clue as to the mechanism which leads to this efficient transcription initiation, since the only known binding site in this region is an Spl site located at -58. Although this short fragment is sufficient for maximal promoter activity, analysis of the other deletions indicates that sequences located upstream are also important for the regulation of transcription. The simplest interpretation of our results is that there is a negative regulatory domain located between -190 and -370, the effect of which is neutralized by a positive regulatory element located between 440 and -600. The same functional organization was observed by transfection of human cell lines of different origins. Since, in vivo there are large variations in the level of unr mRNA accumulation between tissues (5, 6), these upstream sequences are likely to play a role in the tissue specific expression. Thus, for instance, the expression of unr is particularly high in skeletal muscles, where it closely follows the expression of cytochrome oxydase (G.Triqueneaux and F.D. unpublished observations), suggesting that the Mt4 sequence located at -784 could be involved. However, we have so far failed to identify a cellular system that would reproduce this muscle specific expression in vitro, possibly because none of the myogenic cell lines available
has an adult phenotype. Thus, at this time, analysis of the role of the upstream sequences would have to be carried out in vivo. Despite the lack of an in vitro system that would reproduce the tissue-specific expression of unr, some variations in the level of unr mRNA accumulation can be observed in cell lines(6). Our observation that NIH 3T3 cells transformed by a Ki-ras gene expressed a two to three fold higher level of unr mRNA, as well as the results of Doniger (4) showing that tumorigenic cells harboring a mutated N-ras gene had a higher level of unr mRNA, suggested the possibility of a coupling between the ras transduction pathway and the expression of unr. Cotransfection experiments with either Harvey, Kirsten or N-ras expression vectors showed that the presence of an activating mutation within the ras gene led to a 3 to 7 fold stimulation of the unr promoter activity. This effect was also observed with the p90 'basal' promoter construct, indicating that the factors mediating the transactivation should bind within this short DNA fragment. This is of particular interest since, although several transcription factors like AP1('9), NFkB(20), PEA3 (21) have been shown to be regulated by ras, none of these has a canonical binding site in p90, suggesting that other transcription factors are involved in the ras transduction pathway. The close proximity of the unr and N-ras transcription units suggests that multiple levels of regulation could operate within this locus. First, the previous characterization of the N-ras promoter and the present study establish that the two genes have their own promoter regions. Second, since the 3' end of unr coincides with the 5' end of N-ras, it is likely that the two genes belong to the same domain of active chromatin (22). As a consequence, they should not be insulated from the transcriptional regulation of each other or from the influence of transcriptional regulators acting on the whole locus (23-25). Third, RNA polymerases transcribing the unr gene could limit the access to the N-ras promoter (10). However, since this phenomenon of promoter occlusion can be relieved by polyadenylation/termination signals (10, 26), the potential transcriptional interference between unr and N-ras should be considered in relation with the usage of the three polyadenylation signals of the unr gene. Fourth, we have shown in this study that the activation of the ras signaling pathway leads to the transactivation of the unr promoter, indicating a possible interaction in trans within the locus. Analysis of the pattern of unr and N-ras mRNA accumulation in murine tissues (5, 6) reveals no obvious signature of one of these modes of regulation. Indeed, the global results are most easily interpreted if one assumes that several of these regulations can act simultaneously on the locus. Nevertheless, the examples of co-regulation or opposite regulation that can be observed in some tissues indicate that the relative importance of these regulations varies with the cellular context. This is illustrated by the co-regulation of unr and N-ras during lymphocyte activation (6), which excludes that at these levels of expression there is a significant promoter occlusion. On the other hand, the extremely high level of unr expression in skeletal muscles (6) is associated with a very low level of N-ras expression which is in agreement with either a promoter occlusion or the extinction of the N-ras promoter in a tissue where the unr promoter is particularly active (hence an efficient insulation from the regulatory elements of unr). A priori, a detailed knowledge of both unr and N-ras promoters should make it possible to evaluate the relative importance of the individual promoters and of the interactions within the locus. However, the available structural informations ((89 9) and this study) only indicate that these promoters share no obvious regulatory element beyond the fact that they are G/C rich and
6360 Nucleic Acids Research, 1992, Vol. 20, No. 23 contain positive and negative regulatory domains. Moreover, no data on their strength in vivo are available. As an alternative strategy, we have chosen to use the gene targeting technique ('homologous recombination') to investigate in vivo the interactions within the locus. As a first step, we have created a null allele of N-ras in embryonic stem cells (27). The characterization of the unr promoter will now enable us to delete this promoter in ES cells and to generate unr- mice. Finally, crosses between the unr- and N-ras- heterozygotes will allow us to analyze the interactions in cis within the locus by following the expression of N-ras.
MATERIALS AND METHODS Cloning and sequencing A human genomic library generated from human leucocytes DNA cloned into the lambda phage EMBL3 (Clontech) was screened according to Sambrook et al. (28). Hybridization studies indicated that sequences complementary to the 5' end of unr cDNAs were present in a lambda clone (1P7), as a 1.3 kb SalI-SacI and a 3.5 kb HindIl-HindIII Fragment. These fragments were subcloned into the plasmid Bluescript (Stratagene) and sequence analysis was performed on both strands, using the sequenase sequencing kit (United States Biochemicals). Primer extension A 20 base oligonucleotide (5'-TGAAGCAGCAGTTTCAGGTGG-3') complementary to bases +52 to +71 in the 5' untranslated region of the human unr message(6) was endlabelled with ATP (.y-32p) to a specific activity of 8 x 108 cpm/1g. 10 pg of primer were hybridized with either 2 Ag of poly (A)+ or 10 jig of total RNA for two hours at 50°C in 4 tu of 0.25 M KCI, 10 mM Tris-HCl pH8. Reverse transcription was performed with 200 units of Moloney virus reverse transcriptase (BRL) for 30 minutes at 37°C in a final volume of 20 j1A according to Ausubel et al. (29).
RNase mapping The RNA probe was transcribed in vitro to a specific activity of 6 x 107 cpm/4g, from the plasmid containing the SailI-Sacd fragment linearized at the NruI site. Samples containing 0.5 ng of probe and 10 Ag of total RNA were hybrized at 42°C for 16 h and then processed according to a standard protocol (28). RNA isolation RNA was prepared by the guanidinium-thiocyanate method (30). Poly (A)+ RNA was selected by oligo (dT) chromatography according to Sambrook et al. (28). Plasmids The pZIP-Kiras (gly12), pZIP-Kiras* (val12), pZIP-Nras (gly12), and pZIPNras* (val12) plasmids contain the corresponding human ras cDNAs (with either the normal glycine or the activating valine at codon 12) cloned into the BamHI site of pZIP-Neo (13). The EC and EJ plasmids contain the BamHI genomic fragment encompassing the c-Ha-ras gene isolated from either normal human DNA or the EJ bladder carcinoma cell line (31)
Luciferase expression vectors Plasmids unrluc and unrlucAS contain the 1 kb SalI-HaeHI genomic fragment corresponding to nucleotides -1044 to + 32 of the human unr gene, cloned in either orientation in front of
the luciferase gene into the HindIl site of the pcluc plasmid (32). A series of 5' unidirectional deletions was created by Bal3l digestion of the plasmid unrluc linearized at the SaUl site (position -1044 of the NRU promoter). The Bal3l-XbaI fragments were then inserted in the KpnI-XbaI digested pcluc. Plasmid p22 was obtained by restriction of NRUluc at the unique NruI site located 22 bp upstream of the unr transcription initiation site. As a negative control, pcluc vector was also digested by KpnI and HindM, so that all constructs analyzed in transfections, including the promoterless pcluc, have identical plasmid sequences. Plasmid p9OD was generated by cloning the SacI-XbaI fragment of the Bal3l generated -90 deletion into the pcluc vector digested by PvuIl and XbaI. As a positive control we used the CMVluc construct, which contains the immediate early promoter of the human cytomegalovirus cloned upstream of the luciferase gene in the pcluc vector.
Cell culture and transfections experiments NIH3T3, Hela, SW613, RD and MCF 7 cells were grown in Dulecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. 8 hours before transfection, cells were seeded a density of 4 x 105 cells per 6 cm dish. For each dish, 8Isg of plasmid were transfected by the CaPO4 technique. For cotransfection experiments, 4tsg of the reporter construct were mixed with 4 isg of the effector plasmid. All transfections were carried out in triplicate, and each plasmid was tested in three independent experiments. 48h after transfection, the cells were lysed with 0.4 ml of 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, 15% glycerol, 0.4 mM PMSF, and luciferase activity was measured in a luminometer (Lumac) as described (33). Enzymatic reactions were initiated by injecting 100 1l of 0.25 mM luciferin and performed at 25° for 60 sec. The results were expressed as relative light units (RLU), for 11AI of extract (33).
ACKNOWLEDGEMENTS We are grateful to H.Neel for providing the pZipKi-ras and pZipN-ras vectors and to U.Hazan for the pcluc and CMVluc plasmids. We are indebted to C.Lavialle, G.Triqueneaux and D.Weil for helpful discussions. This work was supported by the CNRS and the Association pour la Recherche sur le Cancer (grant
6307).
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