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 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 . 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 -containing woodchuck N-myc gene also exhibited promoter activity in transient transfection assays. The high evolutionary conservation of these sequences in mammalian N-myc 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 . 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 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 (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 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 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 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 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 (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' (positions 44 to 62 in the transfers on Hybond-N+ (Amersham). The probes M13.13 N-myc2 sequence) (15) and the plus-strand OLI5 5'- and 5'N2 have been described previously (15). The 165-bp CCTAGGGAAAGGAAGCACCC-3' (positions -83 to RsaI-Hinfl (RsH) fragment covering N-myc2 exon 1, the -64), OLI6 5'-CGGTATTAAAAAGAGCTGAGCG-3' (po- 360-bp RsaI-BglII (RsB) fragment covering N-myc2 exon 1 sitions -61 to -39), and OLI4 5'-AAAGAAGCTCTCT and the beginning of exon 2, and the 2.2-kb Bglll-HindIII CAGTC-3' (positions -37 to -20), respectively; the position (BH2.2) fragment covering most of the N-myc2 exons 2 and of the 5' end of OL13 allows in-frame fusion with the LUC 3 were subcloned into pBS+ and used as probes. Hybrid- gene. The N-myc2 fragment in construct 8 was also obtained izations were carried out as previously described (42). by polymerase chain reaction with HB1150, the minus- strand oligonucleotide OLI7 5'-TACCGGGGGGTGCTIl RESULTS CCT-3' (positions -57 to -74), and the plus-strand oligonu- cleotide OL18 5'-GACCCCAGCGCAGAATCG-3' (positions Initiation and termination sites of N-myc2 transcription are -166 to -147). internal to the retroposon. We have previously shown that The N-mycl sequences contained in constructs 9, 10, and the N-myc2 gene is expressed at various levels in most 11 consisted of the 2.0-kb BglIl-BamHI fragment (positions woodchuck liver tumors induced by chronic WHV infection -3250 to -1250, numbering from the first N-mycl initiation (15). In some tumors, N-myc2 transcripts ranging from 3 to codon), the 250-bp SmaI-BamHI fragment (positions -1500 5 kb in size have been identified as N-myc2/WHV cotrans- to -1250), and the 268-bp AccI-XhoI fragment (positions cripts resulting from WHV DNA insertion into the 3' un- -214 to +54), respectively. translated region of N-myc2. Tumors carrying viral inser- The TK-LUC plasmid, carrying the thymidine kinase (TK) tions upstream of N-myc2 (45) or harboring apparently only promoter in front of the LUC gene (11), was designated wild-type N-myc2 alleles express a 2.3-kb N-myc RNA, construct 12. Insertion of the N-myc2 sequences extending therefore assumed to be the normal N-myc2 transcript. The from position, -166 to -57 (a polymerase chain reaction reduced size of the N-myc2 mRNA compared with the product also used for construction of plasmid 8), upstream of 3.0-kb N-myc mRNA suggested that N-myc2 mRNA start the TK promoter and in the same orientation, yielded or/and termination sites resided within the retroposon. construct 13. To characterize the 5' end of N-myc2 mRNA, RNase Plasmids pN2L and pNlL used in RNase mapping were protection experiments were carried out with SN560, an obtained by cloning the 290-bp BamHI-XbaI N-myc2-LUC antisense RNA probe which covers the boundary between fragment of construct 4 and the homologous 300-bp N-mycl- exons 1 and 2 in the N-myc2 gene, as referred to in the exon LUC fragment of construct 11 in pBS+. organization in the classical N-myc gene (Fig. 1C). Figure LA Transient expression assays. N-myc2/LUC constructs (13 shows that from all the woodchuck tumors tested ,ug) and the ,B-galactosidase expression vector pCH110 (3 protected a major fragment of identical size (322 + 2 ,ug), used as an internal control for transfection efficiency, nucleotides [nt]) and a minor one about 5 nt longer. The 3' 5338 FOUREL ET AL. MLMOL. CELL.EL BIOL.IL

ends of the protected fragments mapped to positions -22 A B and -27 from the first N-myc2 translation initiation codon, in the 5' noncoding region of the second exon. In contrast, C_ c- ' .cn Z -- ("- > L- C, no protected fragment was detected with normal liver RNA 7: ::- c- -. 2 3 4 5:5 6 7 8 9 110 i' (Fig. IA, lane 11) confirming the absence of N-myc2 tran- C; A - -m in this tissue !I scripts (15). T Protected fragments appearing as a doublet together with ci the absence of a consensus splice acceptor site (30) in the T, - - . r 30 to 20 region strongly suggested that true RNA 5' ends I T were detected in the RNase protection analysis. This was c confirmed by primer extension analyses with an oligonucle- 6 A . G otide complementary to positions 44 to 62 of the N-myc2 -rII gene; three specific extended fragmnents were detected from cI W98 T RNA lane three initiation sites G MO (Fig. iB, 6), mapping 11 to positions -24 (A), -23 (G), and -27 (C), by decreasing order of relative use. Therefore, transcription of the N-myc2 retroposon initiates at clustered sites in the region homolo- gous to N-myc exon 2, giving rise to an mRNA devoid of the retroposed exon 1 region. Furthermore, the location of the transcription start sites was not affected in the tumors carrying a viral insertion in the N-myc2 3' untranslated region (Fig. 1A, lanes 8 and 9). Notably, inspection of the upstream sequence revealed a TATA-like motif (TATTA) located 34 bp upstream of the major transcriptional start C and the sequences the cap site include f. Bg (Fig. 1D) surrounding H' Ba consensus N-myc2 ---m i iBx2 .:..1. ... .:: ex3 the CJ7YANTCYYY sequences of the initiator I L-...... --- .:.Z. r - element, which mediates precise initiation by the transcrip- tional machinery (35). We then characterized the 3' end of N-myc2 RNA by RNase mapping, by using an antisense RNA probe comple- Il9DP pr me- mentary to the 3' noncoding region of N-myc2 and its 3' - 8- 8 flanking sequences (Fig. 2B). Protection of a 450 (±'- 20)-bp fragmnent (Fig. 2A) localized the RNA termination site around position 2045 in the published N-myc2 sequence (15). This result was in agreement with the finding of a poly(A) tail starting at position 2040 in an N-myc2 cDNA clone (unpub- lished results). Inspection of this region revealed a TATAAA motif located 23 nt upstream of the RNA 3' end and followed by a U-rich sequence, which might serve as a polyadenyla- tion signal. Interestingly, this N-myc2 motif appeared as a consequence of a G-->A transition in the homologous region of N-mycl (14). These results show that the transcription of the N-myc2 gene initiates and terminates at internal sites within the retroposed sequence. FIG. 1. Identification of the N-myc2 mRNA start sites. (A) Total N-myc2 uses cryptic promoter elements present in the RNA (20 ~i.g) from different woodchuck liver tumors and a normal parental N-mc gene. Initiation of N-myc2 transcription in woodchuck liver (lanes 6 to 11) was analyzed by RNase protection the region homologous to the N-myc exon 2, about 280 bp by using a 32P-labeled riboprobe spanning the 5' end of N-myc2 downstream of the 5' border of the retroposon, suggested coding sequences and the upstream region (SN560, as illustrated in that promoter elements might be confined to the retroposed panel C). Lane 5, control hybridization with 20 pg of yeast tRNA. region. To identify these elements, we inserted different End-labeled HpaII fragments of pBR322 (lane 1) and uniformly of the 5' of and labeled RNA derived from 2 and were used as size portions region N-myc2 upstream sequences pSN56O (lanes 4) a and markers. N-myc2-specific fragments are indicated by arrows. (B) in front of promoterless LUC reporter gene assayed Primer extension analysis was performed by using a labeled these hybrid constructs by transient transfection in HepG2, N-myc2-specific, 19-bp oligonucleotide and 5 pg of poly(A)' RNA a human hepatoma cell line. from the N-myc2-positive tumor W98T and the N-myc2-negative As shown in Fig. 3, the 1.1-kb region upstream of the first tumor W64T. Lanes G, A, T, and C, sequencing ladders obtained with the same primer from pSN560. The nucleotide sequence of the sense strand at the region of interest is indicated on the left, and the transcription start sites are noted as dots. (C) Schematic localization the first encountered AUG initiation codon, shown in boldface of the RNA probe and primer and of the protected and extended letters. The locations of the major and minor start sites are indicated fragments on the N-myc2 map. N-myc2 sequences are represented as by heavy and thin arrows. The TATA motif is boxed. Pu box, purine a box with the coding region shaded, and the flanking region is shown box, DR, short sequence directly repeated at each side of the as a thick line. The RNA probe, shown as a thick arrow, includes N-myc2 retroposon. The sequence complementary to the oligonu- plasmid sequences indicated by a hatched box. Circle, oligonucleo- cleotide used for primer extension analysis is underlined with an tide used for primer extension analysis. H, Hindlll; Bg, Bglll. (D) arrow. The 5' ends of the deletion mutants of the N-myc2 promoter Nucleotide sequence of the N-myc2 promoter. Numbering starts at are indicated by vertical, empty arrowheads. VOL. 12, 1992 EXPRESSION OF THE WOODCHUCK N-myc2 RETROPOSON 5339

and a CAAT box in inverted orientation, two purine (Pu) A boxes, known to be the core binding motif of Ets family proteins, and a TATA-like sequence (TATTA) (see Fig. 1D).

.- - 3: Further deletion of a 56-bp fragment containing the CAAT .1 box and the Spl binding site led to a twofold decrease in 32341 2 3 4 5 promoter activity (Fig. 3, construct 5), and extending the deletion to -61, which removed the Pu box 1 (construct 6), resulted in an overall three- to fourfold decrease in promoter activity. A deletion to -37 led to a complete loss of LUC activity, suggesting that the TATA box might be a critical element of the N-myc2 promoter (compare constructs 6 and 7). Finally, sequences between -166 and -57, spanning the inverted Spl binding site and CAAT box and the Pu box, while devoid of promoter activity (construct 8), led to a two- to threefold increase of TK promoter activity (con- struct 13), showing a behavior typical of proximal promoter elements. Transcription of the human and murine N-myc genes has B been shown to initiate at multiple sites at the 5' boundary of exon 1 (22, 24, 37, 38), and to date, no N-myc RNA initiating in exon 2 sequences has been characterized. However, the homology between N-myc2 promoter sequences and the 5' noncoding region of N-mycl exon 2 (see Fig. 5) suggested ex3 that the latter might also be endowed with promoter activity. To investigate this possibility, different N-mycl-LUC con- structs were designed and assayed by transient transfection in HepG2 cells (Fig. 3). Transfection of constructs 9 and 10 showed that the classical N-mycl promoter is contained in a 250-bp fragment spanning the 5' border of exon 1 and that addition of 1,750 bp of 5' flanking sequences does not ~polyA significantly increase promoter activity. Remarkably, ATCA-C ,,C;AACTAA-AAAT .ATAAAGAAA7 CAAGAA-GC-TT-CTTAGTACG-T-7G CATTTTA.-G N-mycl exon 2 sequences homologous to the N-myc2 pro- TTCTAGT'TzI,--TGCATAGAA.7-OGG7TA;ATGCAAAGTC rTGTGT,rTAATTCTTCAAkAACACAGTATATVTAGAA moter (construct 11) exhibited promoter activity at a level comparable to that achieved by the classical N-mycl pro- C iTIGAAATAcCTCATGTTATGAAATAAATAGCAG-TAAGTGACTrAAAA3-7,C'-TTIkAAAGTATAAAGAAA3 DR moter, and by so doing identified a new potential promoter. AAAAAA7ATAAA-TGTAGGAAATnAATCTAAATTAAGATGTC-TAAAAA Similar results were obtained in transient transfection assays FIG. 2. Identification of the N-myc2 mRNA termination site. (A) of constructs 1 to 13 in two other cell lines, SKNBe2, a Total W98T RNA was analyzed by RNase protection with a human neuroblastoma cell line producing high levels of 32P-labeled probe spanning the 3' end of N-myc2, as illustrated in mRNA as a consequence of , and panel B. The same reaction was carried out by using yeast tRNA N-nyc and normal liver RNA as negative controls, and the products were Ltk-, a mouse fibroblast cell line (data not shown). run on a sequencing gel. Molecular weight markers (pBR322 DNA The sites of RNA initiation were determined by RNase digested with HpaII) and the input probe are shown in lanes 1 and 2. mapping with RNA from HepG2 cells transfected with the (B) Schematic localization of the RNA probe and protected frag- N-myc2-LUC construct 4 or with the N-mycl-LUC con- ment on the map of N-myc2 exon 3. (C) Nucleotide sequence at the struct 11 (see Fig. 3) and homologous anti-sense riboprobes 3' end of the N-myc2 retroposon. Poly(A) tailing proceeds from the spanning 80 bp of the LUC coding region and upstream position indicated by an arrow. The deduced N-Mc2 polyadenyla- N-myc sequences (Fig. 4B). Protection of a 167-nt fragment tion signal and the AATAAA motif of the parental N-myc gene are mapped the transcription start sites of N-myc2-LUC RNA to boxed. The diverged 3' poly(A) sequence and the flanking direct positions -23 to -27 on the sequence (Fig. 4A), as repeat (DR), which are marks of N-myc2 retroposon genesis, are N-myc2 indicated by a continuous line and by a broken line, respectively. in the case of N-myc2 RNA produced in vivo. These results, showing that transcription of hybrid N-myc2-LUC con- structs in transfected cells started at authentic sites in the eukaryotic sequences, validate the preceding data based on AUG of the N-myc2 open reading frame (construct 1) luciferase expression. Similarly, N-mcl-LUC RNA pro- exhibits promoter activity in the same range as the TK tected a 160-nt fragment of the homologous probe (Fig. 4A), promoter (construct 13). Removal of the 200 bp immediately demonstrating that transcription from the N-myc2 promoter flanking the initiation site abolished promoter activity (con- and the N-mycl exon 2 promoter initiates at homologous struct 2). In contrast, promoter activity was not significantly positions in the second N-myc exon, approximately 35 bp reduced by 5' deletions of the initial fragment to position downstream of the conserved variant TATA box. -139 (constructs 3 and 4). These results map cis-acting In conclusion, N-myc2 transcription initiation is governed elements responsible for efficient transcription of N-myc2 by a promoter located in exon 2 sequences, which remains within -a 115-bp region upstream of the transcription initia- cryptic in the parental N-mc gene. Alignment of the homol- tion site and indicate that no distal transcription stimulating ogous sequences of the woodchuck N-mc2 retroposon, the element is located in N-myc exon 1 and in the 800-bp region woodchuck N-mycl gene, the human, mouse, and rat N-myc flanking the retroposon (Fig. 3). Different potential binding genes and the rat s-mc gene in this region (Fig. 5) showed a sites for known transcription factors can be recognized in remarkable degree of conservation for untranslated se- this short promoter sequence: a consensus Spl binding site quences, particularly between the woodchuck and human 5340 FOUREL ET AL. MOL. CELL. BIOL.

H Bg Cla H N-myc2 1 F Bg

- / Relative luciferase activity (Arbitrary Units) 0.1 0.5 1 2 l.Afl Il _____j 1 -1100 / i ex I leX41LU .. .. 2

3 -308 ex le

-139 4 U I ...... >

5 re ...... i4~ 6 -61 ex LUC| -37exjLUCj 7 -57 -1661 2LUC 8

BgRH SmBa X Ba BaCia H N-mycl II / /T 1' I_3 1.1250 1 \ 9 -3250/ lA \ I -1250 1 10 -1500 LUCI I CA -214 1* 1< XLC

Relative luciterase activity (Arbitrary Units) 3 6 12 -57 ,* ...... -166 ...... 13 ------...... I. N-myc2 FIG. 3. Identification of N-myc2 promoter sequences. The physical maps of the N-myc2 and N-mycl loci, and the different N-myc- luciferase (LUC) constructs used in transient assays are shown on the left part. Symbols are as for Fig. 1C. H, HindIII; Bg, BgIII; Cla, CMaI, R, EcoRI; Sm, SmaI; Ba, BamHI; X, XhoI. Numbering of the N-mycl and N-myc2 sequences starts at the first AUG initiation codon. The length of the N-mycl first intron is estimated. TK, thymidine kinase promoter. Transcription start sites are indicated by arrows. The results diagrammed on the right are the averages of at least three independent experiments. genes (>80% homology). The TATA-like box is preserved W134TB, a liver tumor producing high levels of both N-mycl and its surrounding sequences including the purine box are and N-myc2 RNAs, protected a 140-nt fragment in addition well conserved, but the inverted CAAT box is specific to to the N-myc2-specific 322-nt fragment. The shortest pro- N-myc2. Short deletions are observed in the mouse and rat tected fragment likely results from partial protection of the sequences, and the s-myc gene is clearly more divergent. N-myc2 probe by the N-mycl RNA species. Therefore, our Expression of the N-myc2 retroposon in woodchuck tissues. assay allowed simultaneous detection of N-mycl and We have previously reported that N-myc2 is expressed in a N-myc2 mRNAs. Of the various tissues tested (Fig. 6, lanes majority of woodchuck hepatomas but remains silent in 3 to 10), only the brain expressed detectable levels of normal liver (15). To investigate N-myc2 expression in N-myc2 mRNA (lane 10). In addition, the N-myc2 RNA healthy animals compared with the parental N-mycl gene level was more than 100-fold lower in the brain than in the expression, we performed RNase protection experiments control hepatoma W98T (compare lanes 2 and 10). No using the probe SN560 (Fig. 1) and total RNA from various evidence of a regionalized pattern of expression of N-myc2 adult tissues. As shown in Fig. 6, lane 14, RNA from in the brain was obtained, since N-myc2 mRNA was barely VOL. 12, 1992 EXPRESSION OF THE WOODCHUCK N-myc2 RETROPOSON 5341

I1M 2 3 4 5 6 7 8 9 10 11 M 12 examined (Fig. 6, lanes 4 to 10) except liver (lane 3), which A is consistent with results obtained by Northern (RNA) 309 analysis (data not shown). In agreement with previous investigations performed with mice and rats (41, 48), N-mycl 309 expression was significantly higher in brain and testis (lanes S~~~ 9 and 10) than in spleen, kidney, heart, lung, and intestine tissues (lanes 4 to 8). -242 Because overexpression of normal N-myc2 mRNA in - 242 238 some of the woodchuck liver tumors might correspond to the -21 7 resurgence of a fetal expression program, we also investi- 238- in near term *_ gated N-myc2 expression fetal livers and in 217_ _20 1 livers from newborn or 4-day-old animals. No detectable expression of N-myc2 in RNase protection assays was found 4004 90 20 1_ 1l at these stages of liver development (data not shown). -180 However, the possibility remains that a small percentage of 1 90- cells like periportal liver stem cells might express the 180_ N-myc2 gene. Further analyses using RNA in situ hybrid- so~~~~~~4 -1 = ization of newborn liver should allow clarification of this point. 160- _147 N-myc2 is a nonmethylated CpG-rich locus. DNA methyl- ation is thought to play a key role in the regulation of tissue-specific genes in animal. We therefore investigated 147 - N-myc2 methylation patterns in genomic DNA from adult 410~~~~~~5 *~~~~~~~~~d brain and various liver samples, including normal adult or neonatal liver, and tumorous and nontumorous liver from -122 WHV-infected animals, by using Southern analysis. As shown in Fig. 7A (lanes 1, 4, 7, 10, and 13), the probe 1l5 0 RsB360, spanning the 5' region and the promoter of N-myc2, 122- detected a 3,450-bp HindIII fragment covering the entire gene (see Fig. 1C). Subsequent digestions of the HindIII 110- fragment by HpaII orMspl, two restriction enzymes that are 0o respectively methylation sensitive and insensitive, gave identical patterns in all samples analyzed (Fig. 7), showing 90 that the N-myc2 promoter region is unmethylated in normal liver as in liver tumors. Similar results were obtained upon hybridization of the same Southern blot with various N-myc2 probes, indicating the absence of methylation 90 - 76 throughout the whole locus, including the AT-rich regions flanking the retroposon (data not shown). Inspection of N-myc2 sequences revealed the presence of a CpG island M 2 3 4 5 6 7 8 9 10 11 M 12 covering the entire gene, with a peak of CpG dinucleotide residues located 300 bp downstream of the 5' end of the coding region (Fig. 7B). Therefore, N-myc2 appears to be Ba,H,N-myc2 X: 52-, N-mycl X:a! - e 2~~-''LCx ccr5'snc!4 ex2 L- UC~ ccrs;--.V2 constitutively unmethylated in this island, as are all known - t0-:tNAe ^e 3 :, A D;=e CpG island-containing genes (5). -7-t :-olteztez'2- e 4ocmt _* r- ~ec -a-ne,! Owing to cross-hybridization with homologous N-mycl sequences, the probe RsB360 also detected a 7.2-kb HindIII

FIG. 4. (A) RNase protection analysis of the N-nyc transcripts fragment covering the entire N-mycl gene (Fig. 7A, lanes 1, produced in HepG2 cells transformed with the N-rn 'cl- and 4, 7, 10, and 13). This fragment disappeared upon digestion N-nyc2-LUC plasmids. Total RNA (25 ,ug) isolated fronn HepG2 with HpaII (Fig. 7A, lanes 2, 5, 8, 11, and 14), suggesting cells transformed with construct 4 (lanes 5 and 10) or 12 (laI nes 6 and absence of methylation in the N-mycl locus as expected 11), or the vector pSVOALA5' (lanes 4 and 8), and 5 Fg of W98T from the similar CpG profiles of N-mycl and N-nyc2. total RNA (lanes 2 and 7) or 20 ,±g of yeast tRNA (lanes 3 and 8), was annealed either with the N-nyc2-LUC probe deri%ved from pN2L (lanes 2 to 6, input probe in lane 1) or with the N-rn 'cl-LUC DISCUSSION probe derived from pNlL (lanes 7 to 11, input probe in lane 12) as a a illustrated in panel B. Protected fragments revealing specific tran- Previous analysis of hepadnavirus insertion site in scription initiation sites are indicated. Smaller fragments result from woodchuck liver tumor has led to the identification of partial protection due to regions of homology between the different N-myc2, a novel intronless, functional N-myc gene that has RNA species.~'; retained transforming potential and is overexpressed in a majority of woodchuck liver tumors (15). Thus far, N-ryc2 sequences have been found only in rodent species of the Sciuridae family (Marmota monax, Marmota mannota, and detectable in isolated cerebellum, cerebrum, and batsal gan- Spermophilus beechyi) (42; our unpublished results), that glia tissues (Fig. 6, lanes 11 to 13). evolved about 15 million years ago from a common ancestor. In contrast, low levels of the N-mycl mRNAm vere de- Although many processed genes have been described in tected by partial protection of the probe in most tissues mammals (for a review, see reference 43), only a few of them 5342 FOUREL ET AL. MOL. CELL. BIOL.

W N-myc2 -119 GTTGGAGATTGGCTATTGCCCCCGCCCTCCGCACACCCTAGGG AAAGGAAGCP CCCCCCGGTATTAAAAAGAGCTGAGCG

W N-myc 1 ...... GCGG ...... T.- ..C.C .....A...... -G ......

H N-myc ...... G.C GCCG ...... T G.C C.CG.C...... C A.G.G...

M N-myc ...... GCGACT. TG.TG. TCT------C.C.. GG .T...... C.G AT R N-myc ...... AGCGACT. G...... ------G.C.G... . CA ... G. ..AT R s-myc ...... C.T.GCGT. .A.A.... .ATATGAC ..ACCAC.G.C ... AT.--... A T A . GA

W N-myc2 -39 GAAAGAAGCTCTCTCAGTCGCTGGCGGGCAGGCGAGCTGATGCGGAGCTGCACCGTGTCCACCATGCCGAGGATGATTT W N-mycl --G...... C... .A.G . C.C. C. G . C.

H N-myc . --C. C....C..G ...... C ....C . T..AC .G.C . C. M N-myc ATT.A. .A C------C...... C CC . C. G . C. R N-myc ATT.AG ---.CC.... CC-...------..CC.C.. G . C. R s-myc A.. A.G.-.G.T.ACT.GT.A ..T--.G . A.G . TC . TACA .CG.A . C. FIG. 5. Conservation of N-myc exon 2 noncoding sequences in mammals. The nucleotide sequence of the woodchuck (W) N-myc2 promoter was aligned with the homologous region of WN-mycl (14); human (H) (24), mouse (M) (9), and rat (R) (41) N-myc; and rat s-myc (40). Only the nucleotides differing from the woodchuck N-myc2 sequence are represented. N-myc2 transcriptional start sites are marked by arrows, putative transcription factor binding sites are boxed, and potential translation initiation codons are shown in boldface letters. appear to be functional. This likely reflects the constraints parental gene have been retroposed, presumably because of for emergence and maintenance of a functional retroposon the reverse transcription of an abnormal transcript initiated that require both capacity of autonomous expression and upstream of the usual start site. In others, such as the positive selection operating on the gene product. glutamine synthetase retroposon (GSr) (4), the cDNA copy Regarding the constraints for transcriptional capacity, two may have accidentally inserted downstream of a functional situations have been documented in known functional retro- promoter provided by the new locus. Our study of N-myc2 posons. In some cases, such as the human testis-specific promoter elements shows that autonomous expression can phosphoglycerate kinase gene (Pgk-2) (27, 33) and the rat be achieved in a third way, i.e., by unmasking cryptic preproinsulin I gene (36), the promoter sequences of the regulatory signals present in the 5' untranslated region of the intron-containing gene. The woodchuck N-myc2 retroposon arose from reverse transcription of an N-myc transcript similar to the major murine N-myc transcript (22), initiated 36 bp upstream of a TATA-like box conserved in mammalian N-myc genes, and spliced about 200 bp 3' of the initiation site (14). In the 3 _!7 3 4 5 6 7 8 g0 M1 1112 13 14

Neonatal Adult Infected Liver Brain Liver Liver Liver Tumour 30 g N-myc2 A 1 2 3 4 5 6 7 8 9 ID 11 12 13 14 15 7.2 kb- - __ -a-_ -_ - 3.5 kb- _ 0 242 4 238 - 27- 1 kb_ - _ 2C0 _ 360 bp_ 4w 40"m ~ 1 9I, 200 bp_ _^_ ^_ ^ 1 81- -_ 75 bp- go 2 3 4 5 6 7 8 9K) 11 12 13 14 15 B N-myc2 ex. ex2 i-N-myc CoG

Hipa! /isp I ,. .I i: r- 7 6 _:Z . 6 RsB360 FIG. 7. Methylation pattern of the N-myc2 gene. (A) Genomic DNA from woodchuck neonatal adult 3 4 5 9 (10 ,ug) brain, liver, liver, 2 6 7 8 ^' 2 '3 *4 chronically WHV-infected liver and an N-myc2-positive liver tumor FIG. 6. N-myc2 expression in adult tissues. Total RNA (20 pLg) was digested with HindIII (lanes 1, 4, 7, 10, and 13), HindIII and from various woodchuck adult tissues, indicated above the lanes, HpaII (lanes 2, 5, 8, 11, and 14), or Hindlll and MspI (lanes 3, 6, 9, was analyzed by RNase protection with the SN560 riboprobe (see 12, and 15). Samples were electrophoresed in 1.2% agarose-0.5% Fig. 1D). RNAs from W98T, a tumor producing abundant N-myc2 NuSieve gels, blotted, and hybridized with the probe RsB360 (B). transcripts, and from W134TB, a tumor producing both N-mycl and The sizes of the detected fragments are indicated on the left, and the N-myc2 RNA, were included as positive controls. The N-myc2- N-myc2-derived fragments are marked by dashes on the right. (B) specific 322-nt protected fragment and the N-mycl-specific 140-nt From top to bottom, as follows: schematic representation of the fragment resulting from partial protection of the probe are indicated N-myc2 locus, positions of CpG dinucleotides, and HpaII-MspI by arrows. restriction sites with the sizes of the fragments detected by the probe. VOL. 12, 1992 EXPRESSION OF THE WOODCHUCK N-myc2 RETROPOSON 5343 human N-myc gene, multiple transcription start sites have translated regions by using different LUCN-myc constructs been mapped in a region that extends 250 bp downstream of in transient transfection assays (our unpublished results). the TATA-like box (24, 37), suggesting that part of the Further studies are necessary to delineate the relative con- N-myc promoter might be located in the 5' region of the first tribution of transcriptional and posttranscriptional mecha- exon. However, the retroposed exon 1 sequences in N-myc2 nisms in the regulation of the N-myc2 retroposon. are devoid of promoter activity, despite their exceptionally The recent findings of functional, expressed high degree of conservation (>90%) with the woodchuck have led to the proposal that retroposition may represent a N-myc gene (N-mycl). Instead, the N-myc2 promoter is dynamic route toward evolutionary progress (6). In a simple localized in the region homologous to the 5' untranslated view, an additional functional copy of a gene may alter gene sequences of the second N-myc exon. Notably, this N-myc dosage and is not expected to be conserved unless it offers a region, significantly larger than the 5' untranslated parts of selective advantage. In this regard, the tissue expression the second c-myc and L-myc exons, contains a TATA-like pattern of a retroposon may give clues to its potential element and surrounding sequences that are well conserved function. Thus, testis-specific expression of the Pgk2 auto- in other mammalian N-myc genes. However, there is not somal retroposon in premeiotic cells is believed to compen- experimental evidence for in vivo usage of this potential sate extinction of the X-linked Pgkl gene upon X chromo- promoter in intron-containing N-myc genes. Similar to the some inactivation (27). However, in the case of the rat disruption of the first c-myc exon in some Burkitt lympho- preproinsulin genes, the retroposon and the parental gene mas that revealed a cryptic promoter in the first c-myc intron show the same expression pattern and encode highly similar (2), retroposition may have released the potential N-myc proteins (36), whereas the chicken calmodulin retroposon exon 2 promoter from a negative control, probably resulting (cCM1) (39) and the human X-linked L-myc2 gene (32) from promoter occlusion (20). present a more restricted expression pattern than the paren- When introduced into different human and murine cell tal gene. We show here that this is also the case with the lines, the N-myc2 promoter showed a constant activity that woodchuck N-myc2 retroposon. was not modulated by as much as 1 kb of upstream flanking The predicted product of the N-myc2 gene is more dis- sequences from the N-myc2 locus. In contrast, we have tantly related to that of the parental N-mycl gene than are recently demonstrated that the N-myc2 promoter can be the mammalian N-myc proteins between each other (15), efficiently activated by 5' or 3' integration of the woodchuck suggesting that it might have evolved a different function hepatitis virus enhancers (45). although it has retained full oncogenic potential. Alterna- Investigations of the tissue distribution of N-myc2 mRNA tively, N-mycl and N-myc2 proteins might have redundant in adult animals and in developing liver revealed a highly functions, and the weak expression of N-myc2 in the adult restricted expression pattern. Detectable steady-state levels brain would not dramatically increase the level of N-myc of N-myc2 transcripts were found only in brain tissues, albeit activity in this organ. However, the appearance of N-myc2 with no regionalized pattern in the organ. N-myc2 contains predates the split between woodchucks and squirrels about clusters of nonmethylated CpG doublets, and the entire 15 million years ago, and conservation of N-myc2 coding locus is constitutively unmethylated in adult tissues, like capacity over this long period of time strongly argues for other known CpG islands (5). Tissue-specific expression positive selection. The hypothesis that N-myc2 plays a role despite constitutive hypomethylation might result from a in early, specific stages of woodchuck development remains tissue-specific dosage of transcriptional trans activators. to be explored. Tissue-specific enhancers might be located further upstream or downstream of the explored region in the N-myc2 locus. ACKNOWLEDGMENTS Alternatively, tissue specificity might be achieved via a either We thank C. Trepo for providing woodchuck tumor samples and negative regulation, by regulatory elements lying H. de The for providing the pSVOALA5' and TK-LUC plasmids. outside of the promoter region or by posttranscriptional We are grateful to P. Tiollais for his constant interest in this work, mechanisms. to M. Robertson for critical reading of the manuscript, to Y. Wei for It has been shown that N-myc expression is regulated in a assistance in transfection assays, and to L. M. Da for secretarial complex manner, by both promoter activity and transcrip- assistance. tional blocking, and that the lack of N-myc expression in This work was supported in part by the National Institute of adult liver results mainly from a strong transcriptional atten- Allergy and Infectious Diseases (contract no. N01-AI-82698), by uation (46). 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