THE JOURNAL OF BIOLOGICAL C H E M I S T R Y Vol. 267, No. 36, Issue of December 25, pp. 26011-26016,1992 Printed in U.S.A.

Characterization of the Human Pleiotrophin Gene PROMOTER REGION AND CHROMOSOMAL LOCALIZATION*

(Received for publication, June 19,1992)

Yue-Sheng Lis, Ruth M. Hoffman$, Michelle M. Le Beauin, Rafael Espinosa 1114, Nancy A. Jenkinsll, Debra J. Gilbert 11, Neal G. Copelandl1 , and Thomas F. Deuel$** From the Departments of $.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 (PTN) gene belongs to a recently described family of heparin-binding cytokines whose expression is tem- porally and spatially regulated during development. We have now isolated genomic clones of the human PTN gene, characterized its promoter region, deter- mined its transcription initiation site(s), and estab- lished functional activity of the PTN promoter. A frag- ment -550/+191 that contains a CAAT box, no appar- ent TATA box, and four consensus sites for the binding of MyoD is sufficient to provide optimal promoter ac- tivity. A serum response element is found at -559 to -568. We also have identified 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 pro- moter region, suggest potential regions that may con- tribute to the regulation of its transcriptional activity, and localize the PTN gene in human and mouse chro- mosomes.

We recently cloned the cDNA that encodes an 18-kDa heparin-binding protein, pleiotrophin (PTN)’ (l), which was originally identified as a weak mitogen for murine fibroblasts (2) and as a neurite outgrowth promoting activity in neonatal rat brain cultures (3). PTN is highly conserved among human, rat, bovine, and mouse species (1,4), and its gene is expressed in a highly restricted temporal and spatial pattern during development (l),’ suggesting that PTN may be an important protein potentially contributing to a number of different regulating systems. PTN shares nearly 55% sequence identity

*This research was supported by National Institutes of Health Grants HL14147, HL31102, CA49712, and CA40046 and by a grant from the Monsanto Company (to T. F. D.), by Public Health Service Grant CA40046 (to M. M. L.), and by the National Cancer Institute, Department of Health and Human Services, under Contract N01- 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 article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number($ X65451.

7 Scholar of the Leukemia Society of America. The abbreviations used are: PTN, pleiotrophin; kb, kilobase pairs;

bp, base pairs; UTR, untranslated region; nt, nucleotide; PIPES, 1,4- piperazinediethanesulfonic acid CAT, chloramphenicol acetyltrans- ferase; BCAT, pCAT basic vector; ESV40, pCAT control vector.

H.-J.Yeh, I. Silos-Santiago, R. P. Guillerman, Y.-S. Li, W. Snider, and T. F. Deuel, manuscript submitted.

with midkine, the product of a retinoic acid-responsive gene, Mdk (5), that is weakly mitogenic for 3T3 fibroblasts and that promotes neurite extension from PC-12 cells (6). PTN also shares 51% identity with a chick retinoic acid-induced hepa- rin-binding protein that also is mitogenic for PC-12 cells (7). Based on the highly conserved sequences and 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 the mouse Ptn gene to chromosome 6, respectively.

MATERIALS AND METHODS

Cell Culture and RNA Isolation-Mouse embryo fibroblasts (NIH3T3, ATCC CRL1658) were grown in Dulbecco’s modified Ea- gle’s media with 10% calf serum and 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 frac- tions were isolated with oligo(dT)-cellulose (Type 7, Pbarmacia LKB Biotechnology Inc.).

Genomic Cloning and DNA Sequencing-A human placenta ge- nomic library in Lambda Fix11 (Stratagene, CA) was screened by plaque hybridization (9) with a random primer-labeled (Boehringer Mannheim, GmbH) human PTN cDNA probe (an 808-bp HincII fragment, which includes the 5’-untranslated region (UTR) and the entire coding sequence (1)). A 4.2-kb HindIII-Hind111 fragment was isolated from an -18-kb fragment within clone 9 (C9, described below) that hybridized with the human PTN cDNA probe in Southern blots and was subcloned into a pBluescript vector (Stratagene, CA). A 2.2-kb PstI-Sal1 fragment also was isolated from the same clone (C9). Smaller fragments from 4.2-kb HindIII-Hind111 and 2.2-kb PstI- Sal1 fragments were obtained by restriction enzyme digestion, sub- cloned into pBluescript, and sequenced by the dideoxy chain termi- nation method (10) with Sequenase (U. S. Biochemicals). The se- quences obtained were confirmed by sequencing both strands of DNA with different primers.

SI Nuclease Protection-The 741-bp SacI-PstI fragment (-550 to +191, Fig. 1) in pBluescript was digested with PstI, blunt-ended with T4 DNA polymerase, and labeled with T4 polynucleotide kinase prior to digestion with Sac1 and isolation from an agarose gel. 25 pg of total human brain RNA or 5 pg of poly(A)+ RNA obtained from NIH3T3 cells transfected with a CAT expression construct E741 were coprecipitated in ethanol in the presence of the end-labeled probe (described above), suspended in 15 pl of hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4,400 mM NaCl, 1 mM EDTA) ( l l ) , boiled for 10 min, and hybridized overnight at 55 “C. A human 0- actin probe (278-bp AuaI-AuaI fragment containing 177 bp of the first exon (12) also was end-labeled with [y-32P]ATP and hybridized with 25 pg of total human brain RNA as a positive control. The DNA/RNA hybrid was diluted into 300 p1 of S1 nuclease buffer (280

26011

26012 Pleiotrophin Gene, Promoter, and Chromosomal Loci

mM NaC1,30 mM NaAc, pH 4.4,4.5 mM ZnAcP with 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 polynu- cleotide 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 6 pl 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) con- tains an SV40 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)-PstI fragment, a 741-bp Sac1 (blunt- ended)-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 1389- bp 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 KpnI- HindIII 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) and B371 (-XP) were obtained by deletion from construct B371 of a 292-bp HindIII-PstI (-HP) fragment and a 275-bp XhoI- PstI (-XP) fragment (see also Fig. 4), respectively, blunt-ended, and ligated. All constructs and their orientations 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 trans- fection 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-@-galactosidase plasmid (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, pH 8.0. CAT activity in lysates was assayed on thin layer chromatography plates as described by Gorman et al. (16). Cell lysates were first assayed for @-galactosidase activity (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 p- galactosidase 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 Chromosome Locus-Human metaphase cells were pre- pared from phytohemagglutinin-stimulated peripheral blood lympho- cytes. The PTN probe used was the -18-kb genomic fragment (C9) in the Lambda Fix I1 phage vector. Fluorescence in situ hybridization was performed as described previously (18). A biotin-labeled probe was prepared by nick translation using Bio-ll-dUTP (Enzo Diagnos- tics). Hybridization was detected with fluorescein-conjugated avidin (Vector Laboratories), and chromosomes were identified by staining with 4,6-diamidino-2-phenylindoledihydrochloride (Sigma).

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 were used to map the Ptn locus (see text for details). DNA isolation, restriction enzyme diges- tion, agarose gel electrophoresis, Southern blot transfer, and hybrid- ization 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 strin- gency to a final concentration of 0.1 X SSCP in 0.1% SDS and at 65 "C. Fragments of 10.0,4.9, and 1.1 kb 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. spretus- specific 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) and T-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 MAD- NESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution pat- terns.

RESULTS

Isolation of the Genomic Clones of the Human PTN Gem- To obtain genomic fragments of the PTN gene, approximately lo6 bacteriophage from an amplified human genomic library were screened. Three different recombinant clones that hy- bridized to the human 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 HindIII fragment corresponds to a similar sized fragment that was detected earlier in Southern blotting of HindIII-digested human ge- nomic DNA (data not shown). 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 not shown).

Identification of the 5'-Flanking Regwn-To further char- acterize the human PTN genomic clones and to identify the promoter region of this gene, we next identified a 2.0-kb SalI- PstI 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 no- table 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 transcrip- tion initiation site. No apparent TATA box was present between the CCAAT box and the major transcription initia- tion site. However, a TAATAA sequence (-101 to -106) was observed and may be able to serve as an alternative functional TATA box. A consensus sequence (YAYTCYYY)for binding 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 Gene, Promoter, and Chromosomal Loci 26013

FIG. 1. DNA sequence of the 5’ re- gion of human PTN gene. a, restric- tion endonuclease cleavage map of the genomic fragment of the human PTN gene is shown. A, AccI; E, EcoRV; 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 neg- atiue numbers, while sequences in exon 1 are given positive numbers. The CCAAT box is underlined. The consen- sus sequence of initiation elements is more heavily underlined. Potential bind- ing sites for AP1, MyoD, and GT1 are marked as A, 0, and -en , respectively, 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 re- sponse element (CC(A)6GG) (28) at nt -559 to -568, which may account for our previous results that Ptn is 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 are summerized in Fig. 5.

Transcription Initiation Sites-Ribonuclease protection and primer extension assays were used to determine the transcription start site(s) (see “Materials and Methods”). Both analyses yielded several products, suggesting multiple initiation sites within the 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 a negative control. However, several additional faint signals were also observed in RNase protection and primer extension (Fig. 2) analyses with both RNA preparations. The intensity of the bands observed suggested one major and perhaps sev- eral 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 pro- moter. 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 con- taining 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 frag- ment contains a functional promoter element that is sufficient to drive the expression of a reporter gene 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) was observed; the PTN promoter activity in this fragment is -90% of that driven by the native SV40 promoter activity when assayed in NIH3T3 cells. These results further establish that the -550/+191 fragment contains functional promoter activity. Similar ex- periments with transient transfections of Hela cells also dem- onstrated functional PTN promoter activity (data not shown).

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 trans- fection 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 in- dependent experiments, the CAT activity of B741 (-5501 +191) was arbitrarily assigned as 100% and used as a reference for analysis of other constructs. Because deletion from -180 to +191 (B741(-KP)) results in near total loss of CAT activity, the sequences spanning -180 to +191 (construct 3371) ap- pear 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

310 - 2 8 1 - 2 7 1 -

234 -

194 -

1 I8 --

Pleiotrophin Gene, Promoter, and Chromosomal Loci

b

1 2 3 2 5 6 7 8 I 2 3 4

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 pg of yeast tRNA (lane 4 ) were hybridized to a 32P end-labeled probe (lane I ) extending from -550 to +187 of the human PTN gene. The protected products were analyzed in a 6% denatured sequencing gel. Size markers are 32P end- labeled @X174 Hue111 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 32P end-labeled human 8-actin probe (lane 2, positive control). b, primer extension analysis. An oligonucleotide primer (+120 to +90) end-labeled with [y3*P]ATP was annealed to 25 pg of total human brain RNA (lane 2) or yeast tRNA (lane I ) at 42 “C overnight and was extended with reverse transcriptase at 42 “C for 1 h (13). The extended products were analyzed in a 6% sequencing gel. Size markers are 32P end-labeled @X174 Hue111 fragments (lane 3, BRL) and a sequencing ladder (lane 4) . The number with an arrow indicates the extended band position.

* * 0 - !AC-CAM

0.03 1.0 0.02 140 1.1 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 the cells by calcium- phosphate precipitation. Cells were harvested and CAT activity was determined as described under “Materials and Methods.” CAT ac- tivities were related to B741 activity (Rel. Ac.) and represent the means of three independent experiments. A representative experi- ment is shown. Ac-Cam, acetylated-chloramphenicol; CAM, non- acetylated-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 in NIH3T3 cells. The line diagram at 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 de- scribed 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 independent experiments (n). A representative experiment is shown.

-101 (B741(-HP)), -550/-84 (B741(-XP)), -180/+191 (B371), -101/+191 (B292), and -84/+191 (B275)) respec- tively (Fig. 4), suggesting that more than one site of initiation of transcription may be functionally active, a suggestion con- sistent 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 rela- tive 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 PTN gene, had 7-fold lower activity than that of B741 (-550/+191) (Fig. 4), whereas constructs with deletions of this 648-bp fragment (-1197/-550) had significant increases in activity (data not shown). The signif- icance 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 hybrid- ization of a biotin-labeled PTN probe to normal human metaphase chromosomes. Hybridization of the C9 probe re- sulted 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 ex- periment 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 950 loci 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 hybridi- zation for informative Ptn restriction fragment length poly- morphisms using the 1.5-kb P t n cDNA probe (see “Materials

Pleiotrophin Gene, Promoter, and Chromosomal Loci 26015

Potenlial MyoD Binding Sites lnlllatof Elemenl Major Transcription

API API GTI SRE b hitlation Sl:e

TGAATAA TGATTAA TGTGGTATG CC(A),GG CAATTG CAGGTG CAATTG CATCTG c;;; % . 5‘ +W?V” - ”

A A A- I 3‘ .1737 .‘731 ,1406 ,1400 ,1279 .I271 ,568 459 490 4 8 5 ,454 ,449 .30k 299 2 5 7 2 5 2 .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).

FIG. 6. A and B, in situ hybridization of a biotin-labeled PTN probe to human metaphase cells from phytohemagglutinin-stimulated peripheral blood lymphocytes. A , counterstained with 4,6-diamidino- 2-phenylindoledihydrochloride; B, detection of the probe with rho- damine isothiocyanate-conjugated avidin. The chromosome 7 homo- logues are identified with arrows; specific labeling was observed at 7q33. C, partial karyotype of a chromosome 7 homologue illustrating specific labeling at 7q33 (arrow).

and Methods”). Two cosegregating 6.0- and 1.3-kb M. spretus- specific ScaI fragments were used to follow the segregation of the Ptn locus in backcross mice. The mapping results indi- cated 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 total number of mice analyzed for each pair of loci and the most likely gene order are: centromere-Met-l7/183-Ptn-7/181-Tcrb. The re- combination frequencies (expressed as genetic distances in centimorgans + the standard error) are Met-9.3 f 2.2-Ptn- 3.9 f 1.4-Tcrb.

DISCUSSION

These results establish the initial characterization of the PTN gene. We cloned the human PTN gene promoter region, established sites of transcription initiation, identified regula- tory regions within its 5”flanking sequences that may be important for PTN gene expression, and determined the location of the Ptn gene 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 phos- phoprotein 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 either time or spatial constraints in development may respond to different mecha- nisms of regulation. In the PTN gene, a CCAAT box was identified 91 nt upstream 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

86 70 6 11 3 4

T 6

t Met 7q31

9.3 I

FIG. 7. Ptn maps to the proximal region of mouse chromo- some 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 the top of the figure. For individual pairs of loci, up to 183 animals were typed (see text). Each column represents the chromosome iden- tified 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 chro- mosome is listed at the bottom of each column. A partial chromosome 6 linkage map showing the location of Ptn in relation ot linked genes is shown at the bottom 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 main- tained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

of the PTN gene was the failure to detect potential binding sites for known general transcription factors in the immediate promoter region, although two AP1 and one GT1 sites were found in the distal 5”promoter region (Fig. 5). It was also observed by primer extension 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 to be an unusual complexity to the regulatory elements which in turn may be required for the apparent diversity of influence that expression of the gene appears to have. Although the -550/-84 and -84/+191 frag- ments have independent promoter activity in transient expression systems, optimal expression of the PTN gene

26016 Pleiotrophin Gene, Promoter, and Chromosomal Loci

appears to require the coordinate interaction of both frag- ments. Finally, the -1197/-550 fragment containing a si- lencer region upstream from the promoter also may play a key role in regulating PTN gene expression in uiuo. The precise mapping of the sequence element in this fragment may provide further understanding of the restricted expres- sion pattern of the PTN gene.

The possible linkage of the Ptn gene with previously iden- tified mouse mutations was examined by aligning our inter- specific linkage map of chromosome 6 with the composite linkage map (compiled from GBASE, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME), which reports the map location of many unclosed mouse mutations. In this analysis, the mouse P t n map was found in the vicinity of the hop-sterile (hop) mutation. Homozygous hop mice display a neurological defect observed as a charac- teristic hopping gait, preaxial polydactyly of both fore and hind feet, scoliosis, hydrocephalus, and male sterility (34). Preliminary results from in situ hybridization analysis have established high levels of P tn gene expression in spinal cord, brain, choroid plexus, and bone in developing mouse embryos. Expression of the Ptn gene thus correlates in time and in location during embryogenesis with the development of the organs potentially defection in the hop mouse. The results of in situ hybridization did not include testis. In a preliminary analysis, Southern blots of DNA from homozygous and het- erozygous hop mice using a mouse P t n cDNA probe failed to demonstrate differences in hybridization to SacI- or BglII- digested fragments with these samples (data not shown). Thus, gross alterations in the Ptn gene of the hop mouse were not observed. In the mouse, P tn is linked to the Met and Tcrb genes on chromosome 6 (Fig. 7). The human MET and TCRB genes are localized to 7q31 and 7q35, respectively, indicating that this linkage group is conserved. The localization of PTN to 7q33 support the results of genetic linkage analysis in the mouse which established that the murine gene is flanked by the Met and Tcrb loci.

The extraordinary level of conservation of PTN in different species and its apparent involvement in many diverse cellular processes suggests roles of PTN in processes that control cell growth and differentiation. The identification of the active promoter region and 5’ upstream flanking sequences of the human PTN gene and the chromosomal loci of this gene in mouse and in human may continue to provide clues to other functional roles of this gene.

Acknowledgments-We thank Yogesh D. Patel, B. Cho, M. Barn- stead, and D. Swing for excellent technical assistance.

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