Characterization of the human pleiotrophin gene. Promoter region and ...

Vol. 267, No. 36, Issue of December 25, pp. 26011-26016,1992 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY

Characterization of the Human Pleiotrophin Gene PROMOTERREGIONANDCHROMOSOMAL

LOCALIZATION* (Received for publication, June 19,1992)

Yue-Sheng Lis, Ruth M. Hoffman$, Michelle M. Le Beauin, Rafael Espinosa 1114, Nancy A. Jenkinsll, Debra J. Gilbert 11, Neal G. Copelandl1, and ThomasF. Deuel$** From the Departmentsof $.Medicine and **Biochemistry and Molecular Biophysics, Washington Uniuersity School of Medicine, Jewish Hospital, St. Louis, Missouri 63110, the §Department of Medicine, Section of Hematology/Oncology, The Uniuersity of Chicago, Chicago, Illinois 60637, and the IlMammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21 702

The protein (PTN) encoded by the pleiotrophin ( P T N ) gene belongs to a recently described family of heparin-binding cytokines whose expression is temporally and spatially regulated during development. We have now isolated genomic clones of the human PTN gene, characterized its promoter region, determined its transcription initiation site(s), and established functional activity of the PTN promoter. A fragment -550/+191 that contains a CAAT box, no apparent TATA box, and four consensus sites for the binding of MyoD is sufficient to provide optimal promoter activity. A serum response element is found at -559 to -568. We also haveidentified the human PTN gene on chromosome 7, band q33 and the mouse Ptn gene on chromosome 6, respectively. The data thus identify and characterize the 5’ end of the PTN gene and its promoter region, suggest potential regions that may contribute to the regulation of its transcriptional activity, and localize the PTN gene in human and mouse chromosomes.

with midkine, the productof a retinoic acid-responsive gene, Mdk (5), that is weakly mitogenic for 3T3 fibroblasts and that promotes neurite extension from PC-12 cells (6). P T N also shares 51% identity witha chick retinoic acid-induced heparin-binding protein thatalso is mitogenic for PC-12 cells (7). Based on the highly conserved sequencesand similar function, we proposed that these genes be called the PTN family of developmentally regulated genes. We now report the cloning of the 5”flanking region of the human PTN gene, the identification of its promoter activity, sites of potential cis-acting elements which may contribute to the regulation of PTN gene expression, and its transcription initiation site(s) within the promoter region. We have also localized the human PTN gene to chromosome 7, band 933, and themouse Ptn gene to chromosome 6, respectively. MATERIALSANDMETHODS

Cell Culture and RNA Isolation-Mouse embryo fibroblasts (NIH3T3, ATCC CRL1658) were grown in Dulbecco’s modified Eagle’s media with 10% calf serumand penicillin(100 units/ml)/ streptomycin (100 pg/ml) in 5% CO, at 37 “C. Total RNA was isolated by the guanidium-thiocyanate method (8). The poly(A)+ RNA fractions were isolated with oligo(dT)-cellulose (Type 7, Pbarmacia LKB Werecently cloned the cDNA that encodes an18-kDa Biotechnology Inc.). Genomic Cloning and DNA Sequencing-A human placenta geheparin-binding protein, pleiotrophin (PTN)’(l), which was originally identified asa weak mitogen for murine fibroblasts nomic library in Lambda Fix11 (Stratagene, CA) was screened by (2) and asa neurite outgrowth promoting activity in neonatal plaque hybridization (9) with a random primer-labeled (Boehringer Mannheim, GmbH) human PTN cDNAprobe(an808-bpHincII rat brain cultures (3). P T N is highly conservedamong human, fragment, which includes the 5’-untranslated region (UTR) and the rat, bovine, and mouse species (1,4),and itsgene is expressed entire coding sequence (1)). A 4.2-kb HindIII-Hind111 fragment was in ahighly restricted temporal and spatial pattern during isolated from an -18-kb fragmentwithin clone9(C9,described development (l),’ suggesting that PTN may be an important below) that hybridized with the human PTN cDNA probein Southern proteinpotentiallycontributingto a number of different blots and was subcloned into a pBluescript vector (Stratagene, CA). 55% sequence identity A 2.2-kb PstI-Sal1 fragment also was isolated from the same clone regulating systems.PTN shares nearly (C9). Smaller fragments from 4.2-kb HindIII-Hind111 and 2.2-kb PstISal1 fragments were obtained by restriction enzyme digestion, sub*This research was supported by National Institutes of Health Grants HL14147, HL31102, CA49712, and CA40046 and by a grant cloned into pBluescript, and sequenced by the dideoxy chain termiS. Biochemicals). The sefrom the Monsanto Company (to T.F. D.), by Public Health Service nation method (10) with Sequenase (U. Grant CA40046 (to M. M. L.), and by the National Cancer Institute, quences obtainedwere confirmed by sequencing both strandsof DNA with different primers. Department of Health and Human Services, under Contract N01SI Nuclease Protection-The 741-bp SacI-PstI fragment (-550 to CO-74101 with ABL (to N. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This +191, Fig. 1) in pBluescript was digested with PstI, blunt-ended with T4 DNA polymerase, and labeled with T4 polynucleotide kinase prior article must therefore be hereby marked “advertisement” in accordto digestion with Sac1 and isolation from an agarose gel. 25 pg of ance with 18 U.S.C. Section 1734 solely to indicate this fact. or 5 pg of poly(A)+ RNA obtained from The nucleotide sequence(s) reported in this paper has been submitted total human brain RNA NIH3T3 cells transfected witha CAT expression construct E741were to theGenBankTM/EMBLDataBank with accession number($ coprecipitated in ethanol in the presence of the end-labeled probe X65451. (described above), suspended in 15 pl of hybridization buffer (80% 7 Scholar of the Leukemia Society of America. The abbreviations used are:PTN, pleiotrophin; kb, kilobase pairs; formamide, 40 mM PIPES, pH 6.4,400mM NaCl, 1 mM EDTA) ( l l ) , bp, base pairs; UTR, untranslated region; nt, nucleotide; PIPES, 1,4- boiled for 10 min, and hybridized overnight at 55 “C. A human 0actin probe (278-bp AuaI-AuaI fragment containing 177 bp of the piperazinediethanesulfonic acid CAT, chloramphenicol acetyltransfirst exon (12) also was end-labeled with [y-32P]ATP andhybridized ferase; BCAT, pCATbasic vector; ESV40, pCAT control vector. apositive control. The H.-J.Yeh, I. Silos-Santiago, R. P. Guillerman, Y.-S.Li, W. Snider, with 25 pg of total human brain RNA as and T.F. Deuel, manuscript submitted. DNA/RNA hybrid was diluted into 300 p1 of S1 nuclease buffer (280

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Pleiotrophin Gene, Promoter, and Chromosomal Loci

mM NaC1,30 mM NaAc, pH 4.4,4.5 mM ZnAcPwith sonicated salmon sperm DNA at 20 pg/ml), containing 200 units of S1 nuclease (Sigma), and incubated at 37 'C for 30 min. The digestion was terminated with 75 pl of 2.5 M NH&, 50 mM EDTA, and the protected fragments were recovered by ethanol precipitation, denatured, and analyzed by electrophoresis in a 6% polyacrylamide sequencing gel. Primer Extension Anulysis-Primer extension was carried out as described (13). An oligonucleotide primer (31 nt) corresponding to positions +120 to +90 (Fig. 1) of the human PTN genomic DNA was labeled to a specific activity of 1 x lo8 cpm/pg by using T4 polynucleotide kinase and [y-32P]ATP.The probe was precipitated with 25 pg of total human brain RNA, resuspended in hybridization buffer (as in S1 nuclease protection, above), heated at 85 "C for 15 min, and followed by incubation at 42 "C overnight. The reaction mixtures were precipitated with 2.0 volumes of ethanol and NHJc (final concentration of 2.5 M). The precipitates recovered by centrifugation were resuspended in 50 pl of 50 mM Tris, pH 8.3, 75 mM KC1,3 mM MgClz, 10 mM dithiothreitol, 0.5 mM dNTP with bovine serum albumin (100 pglml), actinomycin D (50 pglml), RNasin (800 units/ ml), and Moloney murine leukemia virus reverse transcriptase (8000 units/ml, Bethesda Research Laboratories), incubated for 1 h at 42 "C, extracted with phenol/chloroform, and precipitated with ethanol. The dried precipitate was dissolved in 6pl of 80% formamide loading buffer and analyzed on a 6% sequencing gel. Plasmids and Construction of CAT Expression Vectors"pCH110 contains the /+galactosidase gene under the control of the SV40 early promoter (14). pCAT basic vector (BCAT) lacks eukaryotic promoter and enhancer sequences. The pCAT enhancer vector (ECAT) contains anSV40 enhancer element in the BCAT plasmid backbone and the pCAT promoter vector (BSV4O) contains an SV40 promoter upstream from the CAT gene. The pCAT control vector (ESV4O) contains the SV40 promoter and enhancer sequences. The CAT expression vectors were purchased from Promega (Madison, WI). A 2175-bp Sun (blunt-ended)-PstIfragment, a 741-bp Sac1 (bluntended)-PstI fragment, a 371-bp KpnI (blunt-ended)-PstI fragment, and a 275-bp XhoI (blunt-ended)-PstI fragment were subcloned into the HindIII (blunt-ended)/PstI sites of BCAT vector to yield the PTN CAT constructs B2175, B741, B371, and B275, respectively. A 741-bp Sac1 (blunt-ended)-PstI fragment also was subcloned into XbaI (blunt-ended)/PstI sites of BCAT vector to yield PTN CAT BR741 (reverse orientation) or into the HindIII (blunt-ended)/PstI sites of ECAT vector to yield PTN CAT E741, respectively. A 1389bp HindIII-PstI fragment and 292-bp HindIII-PstI fragment were also subcloned into the HindIII/PstI sites of BCAT vector to yield the PTN CAT constructs B1389 and B292, respectively. The PTN CAT constructs designated B741 (-KP), B741 (-HP), B741 (-XP), B741 (-KH), and B741 (-KX) were created by deletion from construct B741 of a 371-bp KpnI-PstI fragment (-KP), a 292-bp HindIII-PstI fragment (-HP), a 275-bp XhoI-PstI fragment (-XP), a 79-bp KpnIHindIII fragment (-KH), and a 96-bp KpnI-XhoI fragment (-KX) (see also Fig. 4), respectively, and subsequent blunt-ending with T4 DNA polymerase and religated. The PTN CAT constructs designated B371 (-HP) andB371 (-XP) were obtained by deletion from construct B371 of a 292-bp HindIII-PstI (-HP) fragment and a 275-bp XhoIPstI (-XP)fragment (see also Fig. 4), respectively, blunt-ended, and ligated. All constructs and theirorientations were verified by multiple restriction enzyme analyses and DNA sequencing. A diagram of these constructs is shown in Fig. 4. DNA Transfection and CAT Assays-All plasmid DNA for transfection experiments was prepared by the alkaline method and purified by CsCl density gradient centrifugation. DNA was transfected into cells by the calcium phosphate coprecipitation method (15). NIH3T3 cells were seeded at 6 X 10' cells/lO-cm dish 24 h prior to transfection and were refed with fresh growth medium 4 h before transfection. For each dish, 12 pg of an appropriate CAT construct and 4 pg of the SV40 early promoter-@-galactosidaseplasmid (pCH110) were added. Following 16-18 h of transfection and two washes with serum-free media, the cells were cultured in fresh media for an additional 48 h and harvested. Cell lysates were obtained by repeated freezing and thawing in 250 mM Tris, pH8.0. CAT activity in lysates was assayed on thin layer chromatography plates as described by Gorman et al. (16). Cell lysates were first assayed for @-galactosidaseactivity (as internal control to normalize results for transfection efficiency) (17). Within each series of transfection assays, the amount of cell lysate used for assaying CAT activity was adjusted on the basis of pgalactosidase activity. The area of the chromatographs corresponding to the acetylated and non-acetylated forms of [l4C]chloramphenicol were cut and quantitated in a liquid scintillation counter. The ratio

of the acetylated form to the total acetylated and non-acetylated forms was then calculated. Human ChromosomeLocus-Human metaphase cells were prepared from phytohemagglutinin-stimulatedperipheral blood lymphocytes. The PTN probe used was the -18-kb genomic fragment (C9) in the Lambda Fix I1 phage vector. Fluorescence in situhybridization was performed as described previously (18).A biotin-labeled probe was prepared by nick translation using Bio-ll-dUTP (Enzo Diagnostics). Hybridization was detected with fluorescein-conjugated avidin (Vector Laboratories), and chromosomes were identified by staining (Sigma). with 4,6-diamidino-2-phenylindoledihydrochloride Mouse Chromosome Locus-Interspecific backcross progeny were generated by mating (C57BL/6J x Mus spretus)F1 females and C57BL/6J males as described (19). A total of 205 Nz progeny were obtained; a random subset of these N, mice wereused to map the Ptn locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (20). All blots were prepared with Zetabind nylon membrane (AMF-Cuno). The Ptn probe, an -1.5-kb EcoRI fragment of the mouse full-length cDNA? was labeled with [a-32P]dCTP,using a nick translation labeling kit (Boehringer Mannheim), washed with buffers of increasing stringency to a final concentration of 0.1 X SSCP in 0.1% SDS and a t 65 "C. Fragments of 10.0,4.9, and 1.1kb were detected in ScaI digested C57BL/6J DNA and fragments of 6.0, 4.9, and 1.3 kb were detected in ScaI-digested M. spretus DNA. The 6.0-and 1.3-kb M. spretusspecific ScaI fragments cosegregated and were typed in backcross mice. A description of the probes and restriction fragment length polymorphisms for the met proto-oncogene (Met) andT-cell receptor 0 chain (Tcrb) used to position Ptn on mouse chromosome 6 were reported previously (21,22). Recombination distances were calculated as described (23) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. RESULTS

Isolation of the Genomic Clones of the HumanPTN GemTo obtain genomic fragments of the PTN gene, approximately lo6 bacteriophage from an amplified human genomic library were screened. Three different recombinant clones that hybridized to thehuman PTN cDNA probe were identified. One clone (C9) was analyzed in detail and used in this study. C9 contained a 4.2-kb HindIII fragment that hybridized to the 5'-UTR of the PTN cDNA (see Fig. 1).This HindIIIfragment corresponds to a similar sized fragment that was detected earlier in Southern blotting of HindIII-digested human genomic DNA (data notshown). This fragment contains 250 bp of the 5'-UTR from previously isolated human PTN cDNA clones (1)followed by a large (-16 kb) intervening sequence (data notshown). Identification of the 5'-Flanking Regwn-To further characterize the human P T N genomic clones and to identify the promoter region of this gene, we next identified a 2.0-kb SalIPstI fragment that hybridizes to an oligonucleotide (27 nt, -86 to -60) corresponding to the 5' end of the HindIII fragment and determined its sequences (Fig. 1). Several notable features were observed in the 5"flanking sequences. A CCAAT box potentially able to mediate the effects of CTF/ NF1 (24) was found 91 nt upstream from the major transcriptioninitiation site. No apparent TATA boxwas present between the CCAAT box and the major transcription initiation site. However, a TAATAA sequence (-101 to -106) was observed and may be able to serve as analternative functional TATA box. A consensus sequence (YAYTCYYY)forbinding of the product of the initiator element gene (25) was identified at position -9 to -16 (Fig. 1).Consensus or near-consensus potential cis-acting elements for MyoD (26) and AP1 (27) also were observed and are indicated in Fig. 1. Interestingly, Y.-S. Li, R. M. Hoffman, and T. F. Deuel, unpublished results.

Pleiotrophin Promoter, Gene, and

Chromosomal Loci

26013

FIG. 1. DNA sequence of the 5’ region of human PTN gene. a, restriction endonuclease cleavage map of the genomic fragmentof the human PTN gene is shown. A, AccI; E , E c o R V ; H , HindIII; K, KpnI; P, PstI; S , Sad; and X , XhoI. b, portion of the 5“flanking region of human PTN gene sequence. The major 5’ start site of human PTN as determined by S1 nuclease mapping and primer extension is indicated by closed triangks and designated as +l. The 5’-flanking sequences are given negatiue numbers, while sequences in exon 1 are given positive numbers. The CCAAT box is underlined. The consensus sequence of initiation elements is more heavily underlined. Potential binding sites for AP1, MyoD, and GT1 are ,respectively, marked as A, 0,and -en and a serum response element is boxed. An arrow underscores the 31-nt primer used for primer extension.

we also located a sequence corresponding to the serum response element (CC(A)6GG)(28) at nt -559 to -568, which may account forour previous results that Ptnis up-regulated by the platelet-derived growth factor (29). Gel mobility shift assays were used to confirm that this sequence binds to a protein in Hela cell nuclei with properties identical to the serum response f a ~ t o r However, .~ sequences for the retinoic acid response element (30) were not observed. The potential trans-acting factor-binding sites aresummerized in Fig. 5. Transcription Initiation Sites-Ribonuclease protection andprimer extension assays were used to determine the transcription startsite(s) (see “MaterialsandMethods”). Both analyses yielded several products, suggesting multiple initiation sites withinthe PTN promoter. As shown in Fig. 2, the major transcription start site is located at position +1 (187 nt in RNase protection (Fig. 2a) and 120 nt in primer extension (Fig. 2b), respectively). The products of RNase protection and primer extension were readily detected in RNA prepared from human brain or NIH3T3 cells transfected with a CAT construct (E741) containing sequences -550 to +I91 (see Fig. 1). The products were not seen when tRNA was used as anegative control. However, several additional faintsignals were also observed in RNase protectionand primer extension (Fig. 2) analyses with both RNA preparations. The intensity of the bands observed suggested one major and perhaps several minor start sites. The most frequently used start site was designated as +l. Functional Analysis of the PTN Promoter-We attempted to establish the functional activity of the human PTN promoter. The 741-bp (-550 to +191) fragment was placed in front of the promoterless CAT reporter gene both in the native transcriptional and in the reverse orientation (see “Materials and Methods,” above). When the construct containing the promoter in its native orientation was transfected into NIH3T3 cells, a high level of CAT activity was detected

‘Y.- S. Li and T. F. Deuel, unpublished observations.

(Fig. 3, B741), whereas this fragment did not drive detectable CAT activity when it was placed upstream of the reporter gene in the reverse orientation (+191/-550) (Fig. 3, BR741). These experiments demonstrate that the -550/+191 fragment contains a functional promoter element that is sufficient to drive the expression of a reportergene in the absence of an exogenous enhancer. When the SV40 enhancer was placed 3’ to the CAT gene in the same construct, a 14-fold increase in the CAT activity (Fig. 3, E741) wasobserved; the PTN promoter activity in this fragment is -90% of that driven by the native SV40 promoter activity when assayed in NIH3T3 cells. Theseresultsfurther establish that the -550/+191 fragment contains functional promoter activity. Similar experiments with transient transfections of Hela cells also demonstrated functional PTN promoter activity (data notshown). To define upstream elements within the promoter region of the human PTN gene that may influence transcription, the activity of various promoter constructs which include -1984/ +191, -1197/+191, -550/+191, -180/+191, -101/+191, and -84/+191 of 5”flanking sequences were assayed after transfection into NIH3T3 cells (Fig. 4). The maximum activity was found in a fragment that contained sequences from -550 to +191 (B741).Since this profile of promoter fragment/CAT expression activities (Fig. 4) was reproducible in several independent experiments, the CAT activity of B741 (-5501 +191) was arbitrarily assigned as 100%and used as areference for analysis of other constructs. Because deletion from -180 to +191 (B741(-KP)) resultsin near totalloss of CAT activity, the sequences spanning -180 to +191 (construct 3371) appear to be essential for full constitutive activity of the human PTN promoter in the transient expression systems used in these studies. Several additional mutations were also made within the -550/+191 fragment and tested in the transient expression system (Fig. 4). Nearly half of the maximum promoter activity in the human PTN gene is maintained in fragments -550/


26014

b

310 -

-

281 271 234 -

194

1 I8

-

-1 2

3 2 5 6 7 8

I

2 34

FIG.2. Analysis of the transcription initiation sites. a, S1 nuclease protection. 25 pg of total human brain RNA (lane 5), 5 pg of poly(A)+RNA isolated from NIH3T3 cells transfected with a CAT construct E741 (lane 7), and 25 pgof yeast tRNA (lane 4 ) were hybridized to a 32Pend-labeled probe (lane I ) extending from -550 to +187 of the human PTN gene. The protectedproducts were analyzed in a 6% denatured sequencing gel. Size markers are 32Pendlabeled @X174Hue111 fragments (BRL) (lane 3 ) and a sequencing marker (lane 8). The number with an arrow indicates the position of the major protected product. A major 187-bp fragment is consistently protected from S1 nuclease digestion, corresponding to nucleotide positions +1 (see Fig. 1).An 177-bp protected band (lane 7) was also predicted when 25 pg of total human brain RNA was hybridized to a 32Pend-labeled human 8-actin probe (lane 2, positive control). b, primer extension analysis. An oligonucleotide primer (+120 to +90) end-labeled with [y3*P]ATPwas annealed to 25 pg of total human brain RNA (lane 2) or yeast tRNA (lane I ) a t 42 “C overnight and was extended with reverse transcriptase a t 42 “C for 1 h (13). The extended products were analyzed in a6% sequencing gel. Size markers are 32P end-labeled @X174Hue111 fragments (lane 3, BRL) and a sequencing ladder (lane 4 ) . The number with an arrow indicates the extended band position.

*

0.03

1.0

0.02

140

1.1

* 0

-!AC-CAM

9.8

Rel. Ac.

FIG.3. Promoter activity of the human PTN gene. CAT assay of cell lysates from NIH3T3 cells transiently transfected with human PTN promoter constructs. 12 pg of reporter constructs and 4 pg of SV40-LacZ (pCH110) were transfected into thecells by calciumphosphate precipitation. Cells were harvested and CAT activity was determined as described under “Materials and Methods.” CAT activities were related to B741 activity (Rel. Ac.) and represent the means of three independent experiments. A representative experiment is shown. Ac-Cam, acetylated-chloramphenicol; CAM, nonacetylated-chloramphenicol.

FIG.4. Functional analysis of the human PTN promoter activities and the regulatory element. PTN promoter deletion CAT constructs were used in transient transfection assays inNIH3T3 cells. The line diagram a t the top is a restriction map of the 2.1-kb fragment whose sequence is presented in Fig. 1. Exon 1 is presented by an open box. The line diagrams below the map show the PTN fragments placed upstream from CAT reporter gene. A restriction map of the promoter is depicted. Plasmid construction, transfection assays, and determination of CAT activity were carried out as described under “Materials and Methods.” CAT activities were related to the activity of construct B741 (Rel. Ac.) and represent the means k S.D. of several independentexperiments (n).A representative experiment is shown.

-101 (B741(-HP)), -550/-84 (B741(-XP)), -180/+191 (B371), -101/+191 (B292),and -84/+191 (B275)) respectively (Fig. 4), suggesting that more than one site of initiation of transcription may be functionally active, a suggestion consistent with results of S1 protection and primer extension analysis. However, the presence of the additional DNA at the 5’ end (+1/+191) in some but not all constructs may affect CAT translatability and the stability of CAT mRNA. Thus, the CAT activities measured may not accurately reflect relative promoter strength. We also surveyed the PTN gene 5’ upstream region for additional regulatory elements, using different portions of the PTN gene linked 5‘ to the CAT reporter gene. Construct B1389 (-1197/+191), which includes the 648-bp sequence from -1197 to -550 in the PTNgene, had 7-fold lower activity than that of B741 (-550/+191) (Fig. 4), whereas constructs withdeletions of this 648-bp fragment (-1197/-550) had significant increases in activity (data notshown). The significance of this region has not been further analyzed. Chromosomal Localization-We then used the C9 clone to localize the human PTN gene by fluorescence in situ hybridization of abiotin-labeled PTN probe to normal human metaphase chromosomes. Hybridization of the C9 probe resulted in specific labeling only of chromosome 7 (Fig. 6). Specific labeling of 7q33 was observed on one (two cells),two (seven cells), three (10 cells), or all four (six cells) chromatids of the chromosome 7 homologues in the 25 cells examined. Similar results were obtained in a second hybridization experiment using this probe. Thus, the human PTN gene is localized to chromosome 7, band q33. The murine chromosomal location of the Ptn locus was determined by interspecific backcross analysis using progeny derived from matings of ((C57BL/6J X M. spretus)F, X C57BL/6J) mice. This interspecific backcross mapping panel has been typed for over 950loci that are well distributed among all the autosomes as well as the X chromosome (19). C57BL/6J and M. spretus DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization for informative Ptn restriction fragment length polymorphisms using the 1.5-kb P t n cDNA probe (see “Materials

Pleiotrophin Gene, Promoter, and Chromosomal Loci Potenlial MyoD Binding Sites

API API GTI SRE TGAATAA TGATTAA TGTGGTATG CC(A),GG

5‘+W?V” .‘.1737 731

,1406 ,1400 ,1279 .I271

,568

459

lnlllatof Elemenl

b

-

CAATTG

CAGGTG CAATTG

CATCTG

26015 Major Transcription hitlation Sl:e

%

; ; ;c

.

”

A A- A 490 4 8 5 ,454 ,449 .30k 299 2 5 7 252

I 3‘ .95 .91 .I6 .9

FIG. 5. Potential trans-acting factor-binding sites in the 5”flanking region of the PTN gene. Nucleotides are numbered relative to the major transcription initiation site (+I).

6

86 70

11

3

4

6 FIG. 6. A and B, insitu hybridization of a biotin-labeled PTN probe to human metaphasecells from phytohemagglutinin-stimulated peripheral blood lymphocytes. A , counterstained with 4,6-diamidino2-phenylindoledihydrochloride;B, detection of the probe with rhodamine isothiocyanate-conjugated avidin. The chromosome 7 homologues are identified with arrows; specific labeling was observed a t 7q33. C, partial karyotype of a chromosome 7 homologue illustrating specific labeling a t 7q33 (arrow).

and Methods”). Twocosegregating 6.0- and 1.3-kb M. spretusspecific ScaI fragments were used to follow the segregation of the Ptn locus in backcross mice. The mapping results indicated that Ptn is located in the proximal region of mouse chromosome 6 linked to Met and Tcrb. 180 mice were analyzed for Met, Ptn, and Tcrb and are shown in the segregation analysis (Fig. 7). The ratios of the total number of mice exhibiting recombinant chromosomes to the totalnumber of mice analyzed for each pair of loci and the most likely gene order are: centromere-Met-l7/183-Ptn-7/181-Tcrb. The recombination frequencies (expressed as genetic distances in centimorgans the standard error) are Met-9.3 f 2.2-Ptn3.9 f 1.4-Tcrb.

+

DISCUSSION

These results establish the initial characterization of the

PTN gene. We cloned the humanPTN gene promoter region, established sites of transcription initiation,identified regulatory regions within its 5”flanking sequences that may be important for PTN gene expression, and determined the location of the Ptngene in mouse and human chromosomes. The sequence analysis of the promoter region failed to identify a TATA box. Interestingly, no TATA box has been found in the 5”flanking region of Mdk, a member of the Ptn gene family (1,31) nor in the Wnt-2 gene, which is activated during early development in mouse (32). Also, p18, a phosphoprotein which is highly expressed in fetal tissues in the developing mouse, lacks an identifiable TATA box within its promoter region (33). The failure to identify a typical TATA box may suggest a feature of genes that become activated in the later stages of embryonic development and suggests that many genes that are influenced by eithertimeorspatial constraints in development may respond to different mechanisms of regulation. In the PTN gene, a CCAAT box was identified 91 ntupstream from the major cap site. Functional activity of this site has not been tested. A second feature of the analysis of the 5”flanking region

T

t 9.3

I

Met 7q31

FIG.7. Ptn maps to the proximal region of mouse chromosome 6. Ptn was mapped on mouse chromosome 6 by interspecific backcross analysis. The segregation patterns of Ptn and flanking genes in 180 backcross animals that were typed for all loci are shown at thetop of the figure. For individual pairs of loci, up to 183 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/ 6J X M.spretus) F, parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at thebottom of each column. A partial chromosome 6 linkage map showing the location of Ptn in relation ot linked genes is shown at thebottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes are shown to the right. References for the map positions of human loci can be obtained from GDB, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD). of the PTN gene was the failure to detect potentialbinding sites for known general transcription factors in theimmediate promoter region, although two AP1 and one GT1 sites were found in the distal 5”promoter region (Fig. 5). It was also observed by primerextension and S1 nuclease protection experiments that transcription was initiated from one major and several minor start sites. While none of the features of the 5’-flanking region of the PTN gene established the mechanisms of regulation for its expression, there appears tobe an unusualcomplexity to the regulatory elements which in turn may be required for the apparent diversity of influence that expression of the gene appears tohave. Although the -550/-84 and -84/+191 fragments have independentpromoteractivityin transient expression systems,optimal expression of the PTN gene

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Acknowledgments-We thank Yogesh D. Patel, B. Cho, M. Barnappears to require the coordinate interaction of both fragments. Finally, the -1197/-550 fragment containing a si- stead, and D. Swing for excellent technical assistance. lencer region upstream from the promoter also may play a REFERENCES key role inregulating PTN geneexpression in uiuo. The 1. Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M. Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250,16901 precise mapping of the sequence element in this fragment 1694 may provide further understanding of the restricted expres2. Mine;, P. G., Li, Y.-S., Hoffman, R. M., Kodner, C. M., Siegel, N. R., and Deuel, T. F. (1989) Biochem. Biophys. Res. Commun. 1 6 5 , 1096-1103 sion pattern of the PTN gene. 3. Rauvala, H. (1989) EMBO J. 8 , 2933-2941 4. Tezuka, K. I., Takeshita, S., Hakeda, Y., Kumegawa, M., Kikuno, R., and The possible linkage of the Ptngene with previously idenHashimoyo-gotoh, T. (1990) Biochem. Biophys. Res. Commun. 173,246tified mouse mutations was examined by aligning our inter251 5. Kadomatsu, K., Mineko, T., and Muramatsu, T. (1988) Biochem. Biophys. specific linkage map of chromosome 6 with the composite Res. Commun. 151,1312-1318 linkage map (compiled from GBASE,a computerized database 6. Muramatsu, H., and Muramatsu, T. (1991) Biochem. Biophys. Res. Commun. 177,652-658 maintained at The Jackson Laboratory, Bar Harbor, ME), 7. Raulais, D., Lagente-Chevallier, O., Guettet, C., Duprez, D., Courtis, Y., which reportsthemaplocation of many unclosedmouse and Vigny, M. (1991) Biochem. Biophys. Res. Commun. 1 7 4 , 708-715 8. Chomczynski, P., and Scchi, N. (1987) Anal. Biochem. 162,156-159 mutations. In this analysis, themouse P t n map was found in 9. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Moleculnr Cloning: a the vicinity of the hop-sterile (hop) mutation. Homozygous Cold Spring Laboratory Manwl, 2nd ed., Cold Spring Harbor Laboratory, Harbor, NY hop mice display a neurological defect observed as a charac- 10. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 teristic hopping gait, preaxial polydactyly of both fore and 11. Fay?!or_q,,J., Treisman, R., and Kamen, R. (1980) Methods Enzymol. 6 5 , hind feet, scoliosis, hydrocephalus, and male sterility (34). ‘Il F 1 4 Y N , S. Y., Gunning, P., Eddy, R., Ponte, P., Leavitt, J., Shows, T., and 12. Preliminary results from in situ hybridization analysis have %edes, L. (1985) Mol. Cell. Biol. 5 , 2720-2723 established high levels of P t n gene expression in spinal cord, 13. Hohn, P. A., Popescu, N. C. Hanson, R. D., Salvesen, G., and Ley, T. J. (1989) J. Biol. Chem. 264: 13412-13419 brain, choroid plexus, and bone in developing mouse embryos. 14. Lee, F., Hall, C., Ringold, G., Dobson, D., Luh, J., and Jacob, P. (1984) Expression of the Ptn gene thus correlates in time and in Nucleic Acids Res. 12,4191-5001 15. Graham R., and Van der Ed, A. (1973) Virology 52,452-467 location during embryogenesis with the development of the 16. Gorman: C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2 , organs potentially defection in the hopmouse. The results of 1044-1051 17. Herbomel, R., Bowrachot, B., and Yaniv, M. (1984) Cell 3 9 , 653-662 in situ hybridization did not include testis. In a preliminary 18. Rowle J. D., Diaz, M. 0..Espinosa, R., Patel, Y. D., van Melle, E., Ziemin, s.,i+.a1llon-Miller, P., Lichter, P., Evans, G. A., Kersey, J. D., Ward, D. analysis, Southern blots of DNA from homozygous and hetC.,Domer, P. H., and Le Beau, M. M. (1990) Proc. Natl. Acad. Sci. erozygous hop mice using a mouse P t n cDNA probe failed to U. S. A. 87,9358-9362 demonstrate differences in hybridization to SacI- or BglII- 19. Copeland, N.G., and Jenkins, N. A. (1991) Trends Genet. 7 , 113-118 20. Jenkins, N. A., Copeland, N. G., Taylor, B. A,, and Lee, B. K. (1982) J. digested fragmentswiththesesamples(datanotshown). Virol. 4 3 , 26-36 21. Re nier, D. C., Kozak, C. A., Kingsley, D. M., Jenkins, N. A,, Copeland, N. gene of the hopmouse were Thus, gross alterations in the Ptn Lan don, W. Y . , and Morse, H. C., I11 (1989) J. Virol. 63,367&3682 not observed. In themouse, P t n is linked to the Met and Tcrb 22. Siracusa, &. D., Jenkins, N. A,, and Copeland, N. G. (1991) Genetrcs 1 2 7 , 169-179 genes on chromosome 6 (Fig. 7). The humanMET and TCRB 23. Green. E. L. (1981) Genetics and Probabilitv in Animal Breedinz- Exmri. 77-113, Macmillan, New Yorkgenes are localized to 7q31 and 7q35, respectively, indicating Yamamoto, K. R., and Tjian, R. (1985) Cell 42,559-572 24. Jo~~~!f?.pkl; that thislinkage group isconserved. The localization of PTN 25. Roy, A. L., Meisterernst, M., Pognonec, P., and Roeder, R. G.(1991) Nature 354. 245-248 to 7q33 support the results of genetic linkage analysis in the 26. Lassar,’ B., buskin, J. N., Lockshon, D., Davis, R. L., Apone, S., mouse which established that the murinegene is flanked by Hauschka, S. D., and Weintraub, H. (1989) Cell 58,823-831 27. Lee, W., Mitchell, P., and Tjian, R.(1987) Cell 4 9 , 741-752 the Met andTcrb loci. 28. Treisman, R. (1986) Cell 46,567-574 The extraordinarylevel of conservation of P T N in different 29. Li. Y.-S.. Gurrierl. M.. and Deuel. T. F. (1992) . . Biochem. BioDhvs. . - Res. 184,427-432 species and its apparent involvement in many diverse cellular 30. deCommun. The, H., Vivanco-Ruiz, M. D.M., Tiollais, T., Stunnenberg, H., and Dejean, A. (1990) Nature 343,177-180 processes suggests roles of PTN inprocesses that controlcell 31. Matsubara S., Tomomura M., Kadomatsu, K., and Muramatsu, T. (1990) growth and differentiation. The identification of the active J. Biol. &hem. 2 6 5 , 944i-9443 32. Smith, R., Peters, G., and Dickson, C. (1988) EMBO J. 7,1013-1022 promoter region and 5’ upstream flanking sequences of the 33. Luo, X. N., Arcasoy, M. O., Brickner, H. E., Mistry, S., Schechter, A. D., human PTN gene and the chromosomal loci of this gene in and Atweh, G. F. (1991) J.Biol. Chem. 266,21004-21010 M. C. (1989) in Genetic Variants and Strains of the Laboratory mouse and in human may continue to provide clues to other 34. Green, Mowe (Lyon, M. F., and Searle, A. G., eds), p. 164, Oxford University Press, New York functional roles of this gene. ~~

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