MOLECULAR AND CELLULAR BIOLOGY, Dec. 1992, p. 5336-5344 Vol. 12, No. 12 0270-7306/92/125336-09$02.00/0 Copyright © 1992, American Society for Microbiology Expression of the Woodchuck N-myc2 Retroposon in Brain and in Liver Tumors Is Driven by a Cryptic N-myc Promoter GENEVIEVE FOUREL,1 CATHERINE TRANSY,' BUD C. TENNANT,2 ANDMARIE ANNICK BUENDIAL* Unitede Recombinaison et Expression Genetique (Inserm U163), Institut Pasteur, 28 rue du Dr. Roux, Paris, France,1 and College of Veterinary Medicine, Cornell University, Ithaca, New York 148532 Received 21 April 1992/Returned for modification 10 June 1992/Accepted 31 August 1992 The woodchuck intronless proto-oncogene N-myc2 was initially discovered as a frequent target site for hepadnavirus integration in hepatoceliular carcinoma. N-myc2 possesses characteristics of a functional retroposon derived from the woodchuck N-myc gene. We have investigated the regulatory signals governing N-myc2 expression and found that a short promoter, including a variant TATA box and potential binding sites for several transcription factors, is localized in the N-myc2 sequences homologous to the 5' untranslated region of the second N-myc exon. The corresponding region in the intron-containing woodchuck N-myc gene also exhibited promoter activity in transient transfection assays. The high evolutionary conservation of these sequences in mammalian N-myc genes suggests that they contain a cryptic N-myc promoter which may be unmasked in the particular context provided by the N-myc2 retroposon. Although N-myc2, like the woodchuck N-myc gene, contributes to an extended CpG island and was found constitutively hypomethylated, it presents a highly restricted expression pattern in adult animals. Whereas the intron-containing N-myc gene is expressed at low levels in different tissues, N-myc2 mRNA was detected only in brain tissue, raising questions about the functional significance of the maintenance of a second N-myc gene in the woodchuck genome. The family of myc genes contains three well-characterized have been found to be activated by retroviral insertion in proto-oncogenes, c-myc, N-myc, and L-myc, present in all T-cell lymphoma and by hepadnavirus insertion in wood- vertebrate species analyzed so far (for a review, see reference chuck hepatocellular carcinoma (7, 15, 17, 44). 10). These genes share a three-exon structure and encode Other myc-related, species-specific genes, including the structurally related proteins of similar sizes and short half- human L-myc2 (8), the rat B-myc (18) and s-myc (40), and the lives which exhibit transforming potential. Among different woodchuck N-myc2 (15) genes, have recently been described. functions consistent with their nuclear localization and DNA- s-myc and N-myc2 are two intronless genes encoding N-myc- binding capacity, a role of myc genes in the regulation of gene related proteins. Deletion of an acidic domain highly con- expression has been supported by several recent data (1, 13, served among myc genes might be responsible for the tumor- 19, 25, 31). The carboxyl terminus of Myc proteins contains suppressor activity described for s-myc. In contrast, the DNA binding and dimerization motifs previously identified in N-myc2 retroposon, first identified as a common target for distinct families of transcription factors: a leucine zipper (Zip) hepadnavirus insertion in woodchuck liver tumors, shows and a helix-loop-helix domain preceded by a basic domain extensive coding and transforming homology with the paren- (bHLH). The amino-terminal domain of Myc proteins func- tal woodchuck N-myc gene. Activation of N-myc2 in liver tions as a transcriptional activation domain (21). In addition, tumors induced by chronic infection with woodchuck hepati- this region is involved in the binding of Myc proteins to the tis virus (WHV) results in most cases from cis activation by retinoblastoma gene product, pRb (34), and such an interac- inserted WHV DNA, although unknown trans-acting mecha- tion might couple myc activity to the cell cycle. The transcrip- nisms may also operate in some cases (15). The maintenance tional activation and DNA binding domains of Myc are both of a second functional N-myc gene in woodchucks, whereas necessary for transformation, suggesting that this process most processed pseudogenes show untranscribed and altered might be mediated by direct regulation of a set of genes sequences (43), suggests that N-myc2 could be expressed in implicated in cellular growth and differentiation. physiological situations in a way compatible with the expres- Differential expression of myc genes during normal devel- sion pattern of other myc family genes. opment has suggested distinct physiological functions. To address this question and get insight into a possible Whereas c-myc is expressed at many developmental stages specific function of the woodchuck N-myc2 retroposon, the and in a wide variety of adult tissues, the expression of regulatory signals governing N-myc2 expression and its N-myc or L-myc is more regionalized in developing embryos normal tissue distribution were investigated. In this report, and is restricted to a limited set of tissues in adults (41, 48). we identify the N-myc2 promoter and regulatory elements Different transcriptional and posttranscriptional mechanisms within the retroposed region and show that detectable may account for these specific expression patterns (10). expression of the gene in healthy adult woodchucks is Overexpression of c-myc has been associated with a broad restricted to brain tissues. range of human neoplasms. In contrast, N-myc has been implicated in a narrow spectrum of tumors, mainly of neuroectodermal origin. However, both c-myc and N-myc MATERIALS AND METHODS Animals and cell lines. Woodchucks (Marmota monax) were born in captivity in the woodchuck breeding colony at * Corresponding author. the College of Veterinary Medicine, Cornell University 5336 VOL. 12, 1992 EXPRESSION OF THE WOODCHUCK N-myc2 RETROPOSON 5337 (Ithaca, N.Y.). Various tissues were resected from newborn were transfected into subconfluent HepG2 cells (1.5 x 106 and adult animals, quickly frozen in liquid nitrogen, and per 25 cm2) or semiconfluent SKNBe2 and Ltk- cells by the stored at -70°C. calcium phosphate precipitation method. LUC activity was Liver tumors and adjacent liver tissues were obtained determined from cells extracts prepared 30 to 48 h after from other animals chronically infected with WHV and kept transfection as described previously (12). For RNA analysis, in the laboratory animal facility at the Unite de Recherches the transfections were scaled up to 107 cells. sur les Hepatites (Lyon, France). RNA analysis. Total RNA was extracted from woodchuck The human hepatoma HepG2 cells, the human neuroblas- samples and cultured cells by the hot phenol procedure as toma SKNBe2 cells, and the murine Ltk- cells were grown previously described (29) and then by two rounds of acidic in Dulbecco modified Eagle medium with 10% fetal calf extraction (23) to remove contaminating DNA. serum and antibiotics in 5% Co2. RNase protection assays were performed essentially as Plasmid constructions. All recombinant DNA manipula- described by Melton et al. (28). 32P-labeled antisense RNA tions described in this work involved standard techniques probes were synthesized by using the plasmids pSN560, (26). The 534-bp NAeI-SmaI fragment (positions -238 to pBH810, pN2L, and pNlL as templates and hybridized with +296, numbering started at the first N-myc2 initiation codon) total RNA (20 p.g, unless otherwise specified) for 16 h at and the 715-bp BglI-HindIII fragment (positions 1592 to 56°C. Samples were digested with 2 ,g of RNase Ti per ml 2317) of N-myc2 (15) were subcloned in pBS+ vector, and and 20 Fg of RNase A per ml for 1 h at 37C. The digestion the resulting plasmids (pSN560 and pHB810) were used for products were run on denaturing 6% polyacrylamide gels mapping the N-myc2 transcription initiation and termination along with size markers. sites. The N-myc-luciferase (LUC) chimeric constructs For primer extension analysis, the oligonucleotide OLI3 were obtained by inserting different N-myc2 and N-mycl was end labeled by using T4 polynucleotide kinase, and 0.8 fragments at the HindIII site of pSVOALA5' (12), a plasmid pmol of labeled primer (specific activity, 5 x 10' cpm/jLg) carrying the promoterless LUC and simian virus 40 polyad- was annealed with 5 p.g of poly(A)+ RNA. Elongation was enylation signal. Construct 1 contains the 1,150-bp HindIII- carried out at 42°C for 1 h in the presence of the four BglII fragment (HB1150) of N-myc2 (positions -1100 to unlabeled deoxynucleotides and RNasin by using 40 U of +50). In constructs 2, 3, and 4, the 948-bp HindIII-Hinfl Rous-associated virus type 2 reverse transcriptase (Amer- fragment of N-myc2 (positions -1100 to -152), the 358-bp sham, Amersham, United Kingdom). The extension prod- RsaI-BglII fragment (positions -308 to +50), and the 189-bp ucts were electrophoresed on a denaturing 6% polyacryl- Hinfl-BglIH fragment (positions -139 to +50), respectively, amide gel. were placed upstream of the LUC gene in pSVOALA5'. The DNA analysis. DNA was extracted from frozen biopsy inserted fragments in constructs 5, 6, and 7 were obtained by samples as previously described (29). A total of 20 ,ug of polymerase chain reaction with HB1150 as a template and DNA was sequentially digested by HindIII and MspI or the following oligonucleotides: the minus-strand OL13 5'- HpaII and analyzed by Southern blotting with alkaline AACTCGAGATCTGCGTTCC-3'