MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3325-3333 0270-7306/90/073325-09$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 10, No. 7

Molecular Organization of the Human Raf-1 Promoter Region T. W. BECK,1 U. BRENNSCHEIDT,2 G. SITHANANDAM,2 J. CLEVELAND,2t AND U. R. RApp2* Program Resources, Inc., Biological Carcinogenesis Development Program, Frederick, Maryland 21701,1 and Laboratory of Viral Carcinogenesis, National Cancer Institute Frederick Cancer Research Facility, Frederick, Maryland 217012 Received 22 November 1989/Accepted 27 March 1990

A genomic DNA fragment containing the Raf-1 promoter region was isolated by using a cDNA extension clone. Nucleotide sequencing of genomic DNA clones, primer extension, and S1 nuclease assays have been used to identify the 5' ends of Raf-l RNAs. Consistent with its ubiquitous expression, the Raf-l promoter region had features of a housekeeping gene in that it was GC-rich (HTF-like), lacked TATA and CAAT boxes, and contained heterogeneous RNA start sites and four potential binding sites for the transcription factor SP1. In addition, an octamer motif (ATTTCAT), a potential binding site for the octamer family of transcription factors, was located at -734 base pairs. The Raf-) promoter region drove reporter gene expression 30-fold over the promoterless reporter in Cos 7 cells.

Three raf family proto-oncogenes, A- raf(3, 25, 26), B-raf (27, 49a), and Raf-l (7, 8) have been identified in mice and humans. These genes encode cytoplasmic serine/threoninespecific protein kinases which function in mitogen signal transduction from the cell membrane to the nucleus (22, 45, 46). Consistent with such a function, treatment of cells with growth factors or the tumor promoter phorbol myristic acid or transformation of cells with nonnuclear oncogenes (e.g., src) increased the apparent molecular weight, phosphorylation state, and serine/threonine kinase activity of the Raf-l protein in rodent fibroblasts in vivo (39). More recently, a physical association between the ligand-activated plateletderived growth factor receptor and Raf-l protein has been demonstrated in a baculovirus overexpression system with concurrent phosphorylation of Raf-J protein on tyrosine (40). Nuclear events triggered by oncogenic versions of raf proteins include induction of transforming growth factor a RNA and activation of AP-1/PEA1- and PEA3-dependent transcription in transient transfection assays (22, 53, 54). Whether these events are catalyzed by activated raf directly or involve intermediaries remains to be established. Although there is accumulating evidence for regulation of Raf-l kinase at the posttranslational level, little is known about its transcriptional regulation. As with certain other proto-oncogenes including the epidermal growth factor receptor and c-Ha-ras, Raf-l RNA is overexpressed in certain human lung tumors, chemically induced mouse lung tumors, and chemically induced preneoplastic nodules of rat liver (4, 44, 45). Normal expression of raf family proto-oncogenes has been examined in RNA preparations from 36 different mouse tissues (49a). Raf-l transcripts were found to be expressed in all tissues examined, with the highest levels in striated muscle, cerebellum, and fetal brain and the lowest levels in the skin, small intestine, thyroid, and pancreas. The transcriptional control elements responsible for normal and aberrant raf gene expression have not previously been examined. Bonner et al. (7, 8) characterized a series of genomic clones which were shown to encode exons 2 to 17 of the human Raf-l gene in 45 kilobases (kb) of DNA. However, none of the genomic clones contained sequences encoding the putative untranslated exon 1 sequences of the Corresponding author. t Present address: Department of Biochemistry, St. Jude's Children's Hospital, Memphis, TN 38101. *

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human Raf-1 cDNA. In this report, we describe the isolation and characterization of the human Raf-1 gene promoter region and more of exon 1. MATERIALS AND METHODS Isolation of cDNAs and genomic DNA clones. A human

T-cell library in AgtlO (obtained from Tak Mak, Ontario Cancer Institute) was plated at 2 x 105 cells per plate (22 by 22 cm; Nunc, Roskilde, Denmark) and screened for Raf-1 cDNA extensions with 32P-labeled fragments of the Raf-1 cDNA from human liver described by Bonner et al. (8). A human placenta genomic DNA library in pWE15 (Stratagene, La Jolla, Calif.) was plated at 2 x 104 onto 150-mm plates, and the colonies were transferred to Whatman 541 paper. Hybridizations with 32P-labeled Raf-l cDNA fragments were performed in 50% formamide at 42°C. Phage and bacteria were isolated by three or four sequential rounds of screening. Restriction mapping was performed by standard methods, including partial digestion and transcription from the T7 and T3 promoters of pWE15 (15, 36). Subcloning and DNA sequencing. cDNA and genomic DNA fragments were subcloned into a pUC19 or pKS vector by established protocols (36). Templates for sequencing were generated by forced cloning into M13 vectors and sequenced by the dideoxynucleotide chain termination method (47) with Sequenase (U.S. Biochemicals, Cleveland, Ohio) or by the chemical method (37). Nucleotide sequence alignments and analyses were performed with the software package of the University of Wisconsin Genetics Computer Group at our Super Computer Facility (12). Northern (RNA) and Southern blot analyses. For Northern blots, total cellular polyadenylated [poly(A)+] RNA, prepared as described by Storm et al. (49a), was denatured with 50% formamide-6% formaldehyde (vol/vol), separated electrophoretically on 0.7% agarose-6% formaldehyde gels, transferred to nitrocellulose (BA85; Schleicher & Schuell, Inc., Keene, N.H.), and hybridized with 32P-labeled DNA probes. For Southern blots, 7 ,ug of genomic DNA was digested with appropriate restriction enzymes, fractionated by electrophoresis through 1% agarose gels, transferred to nylon membranes, and hybridized as described previously (49). S1 nuclease mapping. The 520-base-pair (bp) StuI-SacII fragment was subcloned into the Smal-SacII sites of pKS+ and designated pBBCO.52. This was linearized with Bsu36 I,

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dephosphorylated with calf intestinal phosphatase, and labeled with polynucleotide kinase and [_y-32P]ATP (37). The labeled DNA was then digested with XhoI, and the 420-bp fragment was isolated from a DNA sequencing gel. Hybridizations were carried out in 80% formamide as described previously (14) with 10 ,ug of poly(A)+ RNA and 100,000 to 250,000 cpm of labeled probe at 55°C overnight and treated with 200 U of Si nuclease (Boehringer Mannheim, Indianapolis, Ind.) per ml at 37°C for 1 h. Primer extension analysis. pBBCO.52, 5'-end labeled at the Bsu36 I site, was digested with ApaI, and the 40-nucleotide fragment was isolated as above. Hybridizations were carried out as described above except that the buffer was 10 mM Tris hydrochloride (Tris-HCl, pH 8)-i mM EDTA-100 mM NaCl (32) and the temperature was 65°C. Primer extensions were performed with murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Md.) as described by the manufacturer. Transient transfection assays. Cos 7 cells were allowed to grow to 60 to 70% confluence on 100-mm dishes in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated fetal calf serum. Cells were transfected with 10 ,ug of test plasmid DNA by the calcium phosphate method. After 48 h, cells were washed with ice-cold phosphate-buffered saline, lysed in 100 RI of 100 mM Tris-HCl (pH 7.8) by three cycles of freeze-thawing, and assayed for chloramphenicol acetyltransferase (CAT) activity by the liquid method of Neumann et al. (43). RESULTS Isolation of Raf-l cDNA extensions. Figure 1A shows a partial restriction map of the 600-bp portion of the human liver Raf-1 cDNA encoding exons 1 to 4 (8). This cDNA contained 100 bp of an untranslated exon 1 sequence, determined by comparison with genomic DNA sequences encoding exon 2 from the genomic clones X40 and A70, which, however, do not contain exon 1 sequences. In Southern blots of human DNA, we were unable to detect discrete restriction fragments by using the terminal 100-bp OxaNI fragment of the human liver cDNA as a probe. Therefore, a human T-cell cDNA library was screened for Raf-J cDNA extensions with exon 2-4 and exon 5-17 probes from the human-liver-derived Raf-J cDNA. Two clones with inserts of 1.8 and 1.3 kb, designated XCR7 and XCR8, respectively, were selected for further analysis, and the 5'-terminal HindIII fragments were subcloned into pUC18; the resulting constructs were designated pTUC7 and pTUC8, respectively. Figure 1A shows the restriction map of the longer Raf-l extension, pTUC8, relative to human liver cDNA. pTUC8 contained an additional 100 nucleotides at the 5' end as determined by DNA sequence analysis. pTUC7 contained exon 1 sequences found in pTUC8 and the liver cDNA, but was shorter at the 5' end than the others. Most importantly, we found that all three cDNAs contained the same exon 1-exon 2 splice junction originally described by Bonner et al. (8). To verify that the terminal sequences unique to pTUC8 were specific for Raf-J, we compared the hybridization pattern of the terminal 100-bp OxaNI fragment from pTUC8 with that of a fragment encoding exons 2 to 17 from the liver Raf-l cDNA. Northern blots with poly(A)+ RNA from 14 different human tumor cell lines were examined. Both cDNA probes detected only Raf-l-sized RNA (3.4 kb) in CEM, A3.01, Molt 3, and Colo 320 cells (Fig. 1B). Identical results were obtained with the 10 other cell lines examined (data not

MOL. CELL. BIOL.

shown). This strongly suggests that the terminal sequences unique to pTUC8 are present in mature Raf-l RNA. To establish the appropriateness of this probe for genomic library screening, we used this probe in Southern blot hybridizations to restriction digests of total human DNA. The pTUC8 terminal probe detected only a single-sized restriction fragment of 14, 12, and 9 kb in XhoI, HindIII, and EcoRI digests of human genomic DNA, respectively. Therefore, the pTUC8 probe detects a single-copy sequence. Isolation and characterization of genomic DNA sequences corresponding to exon 1 of Raf-1. A human placental cosmid library (200,000 colonies) was screened with the terminal 100-bp pTUC8 probe. A single positive colony designated cBBC1 was identified and isolated. Figure 2 shows the restriction map of the resulting ca. 30-kbp cosmid insert. The pTUC8 cDNA sequences localized to the 14-, 12-, and 8.5-kb fragments in XhoI, HindIII, and EcoRI digests, respectively, closely corresponding to restriction fragments detected in Southern blots of total human DNA (Fig. 1C). Therefore, there are no major structural alterations in the wider vicinity of Raf-J exon 1 in cBBC1. Additional Southern blot mapping experiments and comparison of the cosmid restriction map with that of the cDNAs localized the pTUC8 probe to sequences flanking the SacIl site within the 1.2-kb SallEcoRI fragment. Since the direction of transcription is from left to right (see below), cBBC1 contains 25 kbp of 5'flanking sequence and 5 kbp of intron 1. The exon 2-17 probe of Raf-J did not hybridize to cBBC1, nor did the restriction map of the cosmid insert overlap with previous Raf-l genomic clones encoding exons 2 to 17. Therefore, we believe that intron 1 must be significantly longer than 25 kbp (see discussion). Fine mapping of the Raf-) transcription initiation sites. Having localized pTUC8 terminal sequences to the SallSacII fragment of cBBC1, we used two independent techniques, Si nuclease mapping and primer extension assays, to fine-map the 5' end of the Raf-J gene. Probes for both experiments were labeled at the same site to facilitate direct comparison of the products obtained by the two techniques (Fig. 3A). The products were analyzed in parallel with a DNA sequence ladder generated from the Si probe. For Si nuclease mapping, the 420-bp StuI-OxaNI fragment was 5'-end labeled with 32P at the OxaNI site, hybridized with H-9 cell poly(A)+ RNA, and treated with Si nuclease. For primer extension assays, the 40-nucleotide fragment ApaIOxaNI was 5'-end labeled with 32P at the OxaNI site, hybridized with H-9 cell poly(A)+ RNA, and extended with reverse transcriptase. The sizes of the Si nuclease-protected products and the primer extension products were determined by denaturing polyacrylamide gel electrophoresis. With both techniques, we detected a similar pattern of two major clusters of products with an estimated size range of between 106 and 133 nucleotides (Fig. 3B). No products were detected with either technique in control reactions containing Escherichia coli tRNA in place of H-9 RNA. Additional control experiments established a hybridization temperature optimum of 55°C for Si nuclease-protected products and 65°C for primer extension products with these probes. The intensity of the products of both reactions was dependent on the level of input RNA in the range of 2 to 20 ILg; however, the overall pattern was essentially unaffected. Comparison of the reaction products to the DNA sequence ladder generated from the Si probe allowed us to assign directly the sites of transcriptional initiation to specific nucleotides in the DNA sequence. The results demonstrate that there are multiple sites of Raf-J transcriptional

Raf-J PROMOTER REGION

VOL. 10, 1990

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FIG. 1. Characterization of Raf-J cDNAs. (A) Partial restriction maps of the 5' region of the human liver Raf-1 cDNA (7) and the human T-cell cDNA extension pTUC8. The location of the exon 1-exon 2 splice junction as determined by nucleotide sequencing is indicated by an arrow, and the Raf-1 exon 1 probe from pTUC8 is shown. (B) Comparison of the hybridization pattern of the human liver Raf-1 cDNA encoding exons 2 to 17 and the Raf-J exon 1 probe from pTUC8 to Northern blots of poly(A)+ RNA from human cell lines CEM, A3.01, Molt 3, and Colo 320. (C) Hybridization of the Raf-J exon 1 probe to human genomic DNA. Hut 9 cell DNA (5 ,ug) was digested with XhoI (lane 1), HindIll (lane 2), or EcoRI (lane 3), fractionated through 1% agarose, and transferred to a nylon membrane. The sizes of the fragments detected (in kilobase pairs) are indicated.

initiation located between 276 and 303 nucleotides upstream of the exon 1-exon 2 splice junction. To determine whether Raf-i start sites were independent of cell type, we performed primer extension assays with poly(A)+ RNA from eight additional cell lines. As shown in Fig. 3C, a pattern of cDNA extensions similar to H-9 was observed for all four RNA samples shown. However, an additional cDNA extension product of ca. 230 nucleotides was clearly detected with MCF-7 cell RNA. Assuming that the additional 100 nucleotides are contiguous with exon 1, this would place the additional start site 403 nucleotides

upstream of the exon 1-exon 2 splice junction. However, Si nuclease assays with the same MCF-7 RNA preparation failed to yield a similarly sized nuclease-resistant product (data not shown), and it was not examined further. We conclude that the Raf-1 promoter described here is functional in a variety of cell types and that the RNA start sites

heterogeneous. Nucleotide sequence of the 5'-flanking region of the human Raf-1 gene. The Raf-J gene transcriptional start sites, exon 1, and the 5'- and 3'-flanking regions are shown in relation to the pertinent nucleotide sequences of cBBC1 in Fig. 4. A are

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FIG. 3. 5'-End mapping of the human Raf-1 gene. (A) Partial restriction map of exon 1 and the 5'-flanking region of the Raf-1 gene. The Si nuclease probe (the StuI-OxaNI fragment) and primer extension (PE) probe (ApaI-OQaNI), both labeled at the OxaNI site, are indicated, with a summary of the resulting products of the assays. (B) The Si and primer extension (PE) probes were hybridized to 10 pLg of poly(A)+ RNA from H-9 cells (+) or 10 ,ug of E. coli tRNA (-), treated with Si nuclease or reverse transcriptase, and the products were analyzed on a 6% polyacrylamide-8 M urea gel as described in Materials and Methods. The Raf-1-specific primer extension and Si nuclease-resistant products are indicated by dots. Lane P shows the 417-nucleotide Si probe; lanes C, CQT, A/G, and G show the nucleotide sequence of the Si probe generated by chemical sequencing; and lanes Ml and M2 are HpaII and HaeIII digests, respectively, of pBR322 for size markers. (C) Comparison of the Raf-1 RNA start sites from different human cell lines by primer extension analysis. Primer extensions were carried out as described above with 10 jig of RNA from the four human tumor cell lines indicated or 10 gLg of E. coli tRNA (lane c). The primer extension product detected with MCF-7 RNA is indicated with an arrow.

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Molecular organization of the human Raf-1 promoter region.

A genomic DNA fragment containing the Raf-1 promoter region was isolated by using a cDNA extension clone. Nucleotide sequencing of genomic DNA clones,...
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