Molecular Biology Reports 16: 255-262, 1992. 9 1992 Kluwer Academic Publishers. Printed in Belgium.

255

Nucleotide sequence of a pregnancy-specific/ member

1 glycoprotein gene family

Identification o f a f u n c t i o n a l p r o m o t e r region a n d several putative regulatory sequences

G.M. Panzetta-Dutari, 1 J.L. Bocco,2 B. Reimund, 3 A. Flury 1 & L.C. Patrito 1 1Departamento de Bioquimica Cllnica, Facultad de Ciencias Qulmicas, Universidad Nacional de Crrdoba, Suc. 16-C.C. 61, Crrdoba 5016, Argentina; 2Present address: Laboratoire de Gdn~tique Moldculaire des Eucaryotes, CNRS, U 184 de Biologie Moldculaire et de Gdnie Gdndtique de l'Institut National de la Sant~ et la Recherche M~dicale, Facult~ de Mddecine, 11 rue Humann, 67085 Strasbourg Cedex, France; 3Laboratoire de Gdndtique Moldculaire des Eucaryotes, CNRS, U 184 - INSERM, 11 rue Humann, 67085 Strasbourg Cedex, France Received 17 June 1991; accepted in revised form February 18, 1992

Key words: pregnancy-specific glycoproteins, placental protein, genomic clone, regulatory sequences

Abstract

The pregnancy-specific/31 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5' flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5' further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G + C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.

Introduction

The pregnancy-specific/31 glycoproteins (PSGs) consist of a heterogeneous group of molecules synthesized by the syncitiotrophoblast during pregnancy and have an unknown physiological function [1]. They are also found, albeit in relatively small quantities, in some normal tissues and in neoplasms of various origins [2]. Hith-

erto, molecular cloning of genomic and cDNA clones have led to the identification of at least 10 PSG genes [3-10]. Sequence comparisons reveal that the PSG family shares more than 90 ~o homology in nucleotides and more than 8 0 ~ in amino acids. In addition, proteins encoded by these genes are very similar to members of the carcinoembryonic antigen family [11] and contain repeating do-

256 mains, conserved disulfide bridges, and a/%sheet structure typical of the immunoglobulin gene superfamily [9, 12]. We have previously shown that P S G biosynthesis could be transcriptionally regulated during placental development [ 13]. At present, no information about possible regulatory sequences of this gene family has been reported. So, in order to study the regulation of the P S G gene expression, we determined the nucleotide sequence of a gene segment corresponding to a P S G family member including 1190 bases upstream of the translation initiation codon and demonstrated the promoter activity within that sequence, which did not contain a canonical TATA box or Spl boxes.

Materials and methods

Cloning of genomic sequences A human genomic library of 5 • 105 independent recombinants was prepared from partial Sau3AI digested placental D N A using the bacteriophage EMBL3 vector as described by Frischauf et al. [14].

Isolation of PSG genomic clones The library was plated and screened with minor modifications by plaque hybridization [15] using a random primer labelled full-length P S G c D N A of 1.4 kb (pBP1) previously isolated in our labor~tory [13]. A 17kb insert from one positive clone, termed SGI, was subcloned into Saildigested pBR325 and characterized by restriction endonuclease analysis. The restriction endonuclease fragments containing exons were identified by Southern blot hybridization analysis [16] using the 32p-labelled pBP1 probe. Subcloning and DNA sequence determination Various restriction fragments isolated from the genomic and c D N A clones were further subcloned into M13 mp18 or m p l 9 [17]. D N A inserts were sequenced by the dideoxynucleotide chain termination procedure of Sanger et al. [ 18] using the United States Biochemical Co. Seque-

nase kit. All sequences were determined three or more times from different subclones. Sequence data were analyzed using D N A S I S (Hitachi, Japan) software.

Construction of recombinant chloramphenicol acetyltransferase plasmids Fragments of the SGI clone were inserted into the polylinker ofpBL-CAT3 [ 19] upstream of the translation initiation starting site of the chloramphenicol acetyltransferase gene (CAT). The restriction fragments used were a 3.2 kb SalI/BamHI fragment, a 562bp PstI/BamHI fragment and a 215 bp StuI/BamHI fragment (the 3' end of all the fragments was the BamHI site at nucleotide + 87); as no StuI site was available in the polylinker of the vector, fill-in with Klenow enzyme was done before ligation and bacterial transformation. DNA transfection and transient expression assays COS-7 cells were grown in a Dulbecco medium supplemented with 5~o calf serum and transfected by calcium phosphate coprecipitation [20]. Briefly, approximately 10 6 cells were plated onto 9 cm tissue culture dishes 16 h before the addition of the calcium precipitate mixed with the amounts of recombinant D N A indicated in the figure legend. The precipitate was left on the cells for 20 h, thereafter the cells were refed and harvested after an additional 16 h to prepare the cellular extracts as previously described [21]. The amounts of the cell extracts to perform CAT assays [22] were normalized by the /%galactosidase activity which was expressed from the p C H l l 0 plasmid [23] cotransfected with the CAT reporter plasmids. The total amount of D N A transfected was adjusted to 14 #g per Petri dish with a double-strand carrier D N A (BlueScript).

Results

Figure 1 shows the restriction map of the SGI genomic clone and the sequencing strategy of the 5' SalI-EcoRI fragment. The nucleotide se-

257 S St

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I

A

~P

B

BSm

K

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E

II

St

St

II I I

StSm

St P

II

In

HX X

B

Ev

X B

Sm

PSt

StriPS

,Mi ,T

I

B

P

Sm~ .,. E

IEI

!

!

Fig. i. Restriction map and sequencing strategy of the SGI genomic clone. A, Restriction map and exon-intron structure. Boxes indicate coding regions. B, Sequencing strategy of the most 5' SalI-EcoRI genomic fragment. Abbreviations used are B, BamHI; E, EcoRI; Ev, EcoRV; H, HindlII; K, Xhol; P, PstI; S, Sail; Sin, SmaI; St, Sstl; X, Xbal.

quence of 3036 bases of the genomic fragment is depicted in Figure 2. It contained two exons, one intron, and 1056 nucleotides of 5' flanking sequence. The nucleotide sequence of the BP1 cDNA was also determined and its open reading frame was identical to the PSG-D clone already described [4] (data not shown). The homology between the coding regions of this genomic fragment and the BP1 was 93.7~o in nucleotides and 87.4~o in amino acids, indicating that the gene segment belongs to the PSG gene family. Oikawa et al. [7] have isolated the same gene, named PSG 5, according to the nomenclature adopted at the XVIIth Meeting of the International Society for Oncodevelopmental Biology and Medicine in Freiburg, West Germany, 1989. The sequence reported in this paper extends 1142 nucleotides 5' further upstream and differs in 10 nucleotides from that reported by Oikawa et al. [7], probably representing genetic polymorphism within the gene. Thompson etal. [24] have reported the sequence of a genomic clone including only 359 nucleotides 5' upstream of the translation initiation codon of the same gene, and have demonstrated that it has multiple transcription starting sites. The cognate cDNA has been isolated from a fetal liver cDNA library [6] and from a promyelocytic leukemia cell line cDNA

library [7] and has the domain arrangement L-N-A2-B2-C. As no regulatory sequences have been reported in this or other PSG genes, we analyzed the 5' flanking region and the first intron of the PSG 5 gene. Sequence analysis of the 1190 bases upstream of the translational start codon, revealed that it contains the consensus sequence GCCAAT (position -989) recognized by the CTF/NF 1 DNA binding proteins [25 ], the sequence CTGACCCCACCC (position - 15) highly homologous with the consensus YRRRCCNCACCC considered an upstream promoter element in the globin gene family [26], and four short (dT-dG)n repeats at positions -224, -214, -204, and -189. The stretch of 222 base pairs before the translation initiation point is purinerich (65~o) containing six purine-rich repeats (consensus PuPuPuPuPuPuGAGGPu) located between -87 and + 108 as well as an A-rich motif (position -83). Neither TATA nor TATAlike boxes, nor Spl boxes, were found in the 1056 bp upstream of the first major transcription starting point. Recently, Leslie et al. [27] have isolated a genomic clone, PSGGB, including 366 nucleotides 5' upstream of the initiation codon of the PSG 6 gene. Its nucleotide sequence shares 93~o homology with the PSG 5 gene and also

258 -1056

C G T C T C A G A A CACAACTCAC CCCACCGCCT CTCAGGTGTG AGCAACGTCC CTCCAGACAG GATCTCTGCC AATGCCTGq~f AAAA

-976

C C T C C T C T A C AGAGAGCAGC q~"GGCCAGGT CAAAACCC~C AGGACAGACC CCTGGGTATG TCAGCACATG GAGGCTGAGC

-896

CCATCATGGC CACGGAGTAA GTCGGGATGA ATCTCTCCAG CTCTGAACCC CTGC~CTGTC CCAGGC'PCCC CCTCCTGCGT

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C T C A A C G A G C CATGTCCCGC GGGGTTCCTC GGTAACTCCC CACq~FCCTCC TGCACCACAA C G G G G A A G G T GTC-GTGACCC

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C A G G A C A G T C AGCTGGGCAG AGAAGAGAGG ACATCAAAGA TGGTCAGGGA GAACAGGAGA A A C C C T G A G C TCCAGCTCAG

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CTGCCAGACC CCAGGGAGCC ATAGAGTGAC GAAGAACTGT CCTCTTGACT CTGGGACTCA CAGCCCCCAC TGAGCAACCA

-576

G C A C A G G C C T C~TCC~CCCAA GAGGCATGAG AGATTCACCC TGCACACCTC CAGGAAGGAC AGI~"~VTCCTCT GGGACACCCA

-496

GGTCCTGAAG GTCCATCC,~T GGAAGCTGCA GCCAAGCTAG ACACGATTAG AGAAAGGAAG GGTCCCT~.CTC ACCAGGCAAC

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A C A C A G C T C A CCCACAGCAC AATGGGGCAT CACTGTGACA GGGACCCGTT GCAGCTTCAG C C T C C T G C A C TGAAGGGGAG

-336

A G C C A G A T G G GGATGAAAGA GGGCAAAGGG CGTGAATGTG CATCCCGACC CAGAGCCATG GGGACAGCAG GAGGCTGAGG

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CCCAGGACTC TGCTTGCCCA ACCTGCGGGG TATGTGTGTG A ~ G T G T G G G

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TCTGTGTGTG T C T C T T C T G T GTGTGTG'PGT

CTGCACAAAG TGTGGTTGAG G,~'FTGGTGAA AGAATCACTG CTGAAAAATG CAGAGGCCTC CACAA~"~fCCC A G G G A C C T G A

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65 i !45

C A G C C G T G C T CAGGAAGTq~ CTGGATCCTA GGCTCAGCTC CACAGAGGAG AACACGCAGG C G C A G A G A C C ATGGGGCCCC M G P T C T C A G C C C C TCCC~GCACA C A G C A C A ~ A CCTGGAAGGG GGTCCTGCTC ACAG~-~AGG AGAGAAC"/*PC CTGGGAGAGG

4 225

A C A G G A G G A G SAAGAAGAGT GAC!"7~_GA~'/'~=GGGTCTCCTGG AGAGGATGGG GTTCTAAAAA ATAAAAGAAG CCAGCACT'I~

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G G G A G G A T G A GGTGGGTGGA TTATGAGATC AGGAGTTCAA GGTCAGTCCT C,CC-AACACAG T G A A G C C C T G TCTCTACTAA

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465

C A T G A A C C C G GGAGGCAGAA ATTGCAGCGA GCCGAGATAG CACCACTGCA CGCCAGCCTG GGTGACAGTA CGAGACTCTG

545

TCTCAAAIGkA ATAAAAAGAA AAAAP~GAAA AAAGAAAAAA A A G G A A A G A A CTCTCTGTTG- GAGCC~/GGA_T AGC~2TAAAAT

625

A C A C C A G A G A GGGACAGAGG TCAAAACTGG AAAGTCATAT TGAACCAGAA ~'FGqTAAGAG G T A G G A A A T C "I~fAAGTGTTC

705

TGTq'~TCCTG ATTAATCATC AGGGGCCACG ACATTTTGAA AAATGATAAT AATAACTATA T C A G A T G A C A CTTCTAATAA

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G T C C T C A C A G AGCTCACGCT GTCATGGGGA GGAAGGCAGA CATGCAAAGA GA~CTAGAAT GTGAGGTCAG G T G T T G A C A A IliHil " * * "" * * * GAACCCTGGA GGGAGCAGAG CAGGGA/~GG TCAGAAAGC-G AAGACCCAGG GTCTCTGAAG G A C G T G T C A G G A A A G A A G T C

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TAAGGATGCC CTGATGTGAG CAGGACCTGA GGGCAGTGq'T GGGGGGGGGG TGCCGTGTGG A C C C C T G G G G A A G A G G A T T C

2 185

CATACAAAAA TGCCAAGGTC AGAAGq~3TTG AAGGAATAGG CGTCACGCTG CTGACCTTGA C C T A G T A G G A C A G T A G G A C A

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CACAGGTCTC A A T A T T G A C T G G T G C ~ C T

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CCACCTCCq~CATCACTTT ~ S L TTq'CCGAGGG GAAGGATGq'T V S E G K D V C%2GATGGACC TCTACCATTA L M D L Y H Y A G A A A C A G T A TAq~TCCAATG E T V Y S N TAAAG~:GAGG TGAT~GGACT I K|~ G DIP T GGGTGq~GGG GACAGGGGTC

AAGTCACGAT Q V T I cq'I~CTGGCT L A G AAATATATAT N I Y AAGACGCAGG E D A G ~ATACC~--~ L Y ~ GCCTGGACTG

1905

TTATGTCCCA TGCTGCGG~T TGGGCATq~TA GTGCAGGACA CACACAGAGG AGACAAATq'T C A A C A G A T C A G A A T T C

TAAACTTC~G L N F W CTTCTACq~PG L L L CATTACATCA I T S CATCCCTGCT A S L L AGAGGACT~ R G V Aq~TCTACTT

GAACCTGCCT N L P TCCACAATTT V H N L TATGTAGTAG I V V GATCCAGAAT I Q N CTGGATAT~'T T G Y F CACACACACA

ATCACTGCTC I T A GCCTCAGAAT P Q N ACGGTCAAAT D G Q I GTCACCCGGG V T R CACCTTCAAC T F N GGAq'FGTCAG

TGAAGCCCTG E A L ACATCTGGTA Y I W y GGGCCTGCAT G P A ATCCTACACC S Y T AGTGA~TCCA

CCACCCAAAG P P K CAAAGGACAA K G Q ACACTGGACG u T G R q'TACACATCA L H I CATGATCCCT

T G C C T G T G T C CCTCTCTGCA

Fig. 2. Partial nucleotide sequence of the 5' SalI-EcoRI SGI genomic fragment. The sequence starts 1190 nucleotides upstream of the initiation codon. Sequences containing exons are underlined by the cognate amino acid sequence (one letter code). Consensus splice site sequences are boxed. Sequences of potential regulatory significance are indicated as follows: closed triangles, NF1 consensus binding site; open triangles, upstream promoter element (UPE) homology; horizontal arrows, (dT-dG), short repeats; overline, A-rich motif; thin underlines, purine-rich repeats; dots, SV40 enhancer core consensus sequence; squares, SV40 enhancer octamer motif; dashed lines, NFIII octamer binding element; thick underline, core of the consensus glucocorticoid responsive element; open circles, AP1 homology; asterisks, ATF homology; double underlines, simple repeat nucleotide sequences (dAdC)10-(dTdG)14. Vertical arrows indicate the major transcription starting site as deduced from Thompson et al. [24]. The R G D sequence is boxed.

259 o n the other hand, the putative regulatory sequences found in the first intron (Fig. 2) included six sequences homologous to the SV40 enhancer core consensus sequence (positions: +247, +321, +425, +610, +652, and +676), one homologous to the SV40 enhancer octamer motif (position + 986) [29], the octamer element recognized by the transcription factor NFIII of the adenovirus2 (nucleotide +746) [30], the sequence TGATTAA (position +713) corresponding to the consensus sequence TGANT(C/A)A recognized by the AP1 transcription complex [31], and the sequence TGAGGTCA (+ 1006) highly homologous to the consensus ATF binding site (TGACGTCA) [32]. A hexanucleotide sequence, TGTTCT, corresponding to the core binding site for the glucocorticoid receptor complex, was found at position + 700. The surrounding sequence was in good agreement with the sequence involved in the glucocorticoid receptor binding activity of the mouse mammary tumor virus gene located 98 nucleotides upstream of its transcriptional starting site [33]. We also found a region of simple sequence DNA (dAdC)10 interrupted by an AT in the middle (posi-

does not contain any obvious TATA or GC boxes. Furthermore, analysis of the 5' controlling region of the human CEA gene [28] revealed the absence of a TATA or GC box in the 200 base pairs upstream of the major transcription starting point. Sequence comparison among the 5' region of PSG 5, PSG 6 and CEA genes (Fig. 3) revealed the presence of similar purine- and A-rich motifs at near identical positions, as well as the conservation of the (dT-dG), repeats in the PSG 6 gene. In order to define the promoter activity of the 5' flanking region analyzed, we carried out transient transfection experiments with chimeric plasmids containing the bacterial chloramphenicol acetyltransferase gene driven by different 5' flanking DNA fragments from the SGI genomic clone. Figure 4 shows that neither the largest 3.6 kb nor the 562 bp SGI 5' flanking fragment has the ability to induce the CAT gene activity. But, surprisingly, the smallest DNA fragment, which is a derivative from the other chimeric plasmids, encompassing the nucleotides -123 to + 87, functioned as a strong promoter for the reporter gene in the COS-7 cell line (Fig. 4).

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Fig. 3. Schematic comparison of the 5' upstream region of the PSG 5, PSG 6, and CEA genes. Dotted boxes represent the 5' end of the leader peptide domain; open boxes, the (dT-dG)n repeats; overline closed boxes, A rich motives; underline closed boxes, purine-rich repeats. The translation initiation codon (ATG) and the furthest 5' upstream transcription starting sites are indicated.

260

Fig. 4. COS-7 cells were transfected with (12 or 2/~g) ofpBL-CAT reporter plasmid [19] in which the indicated 5' flanking D N A restriction fragments from the SGI genomic clone (Figure 1) were inserted upstream of the CAT gene. After 32 h of transfection, the cells were harvested and assayed for CAT activity, as described in the section Materials and methods.

tion + 1262), followed directly by a poly(dTdG)14 interrupted by a TT at the center (position + 1303). These types of sequences have been reported at similar positions in some other carcinoembryonic antigen gene family members as elements probably involved in the regulation of gene expression or as recombination sites [34]. Like other PSG members, this gene codes for a polypeptide which has the tripeptide RGD (Arg-Gly-Asp) in its N-terminal domain, suggesting a biological role of these proteins in the cell-recognition phenomena [35].

Discussion

In this study, we isolated and characterized a genomic clone corresponding to the PSG 5 gene family member including 1056 nucleotides upstream of the first major transcription starting point and demonstrated the promoter activity within that sequence. The most striking finding in the analysis was the lack of TATA or TATA-like box element. Certainly, the absence of this sequence is exceptional in view of the fact that most eukaryotic RNA polymerase II transcribed genes do have the consensus sequence [36]. Nevertheless, genes such as housekeeping genes [37], Thy-1 [38], TGFfll [39] and the chicken progesterone receptor gene [40] do not have TATA elements but do have G + C-rich regions 5' to the

transcriptional starting site, and often have Splbinding domains [41]. Furthermore, as more promoter sequences became available, a further set of genes that has only the initiator element as the major sequence requirement to control transcription, has become evident [42 and references therein]. Many of this type of promoters are not constitutively active but are regulated in a differentiation- and development-dependent fashion [43]. The CAT assays results showed that the PSG-5 gene contains a functional promoter activity included between nucleotides -123 and + 87, although the analysis of the 5' flanking SGI sequences indicated that it has neither TATA box, G + C-rich regions, nor Spl boxes. However, it is very suggestive that the PSG 5 sequence encompassing the transcriptional start site is highly homologous with the initiator element found in the globin gene family [26] and in the terminal deoxynucleotidyl transferase gene [43]. In view of these results and due to the sequence conservation between different PSG genes, it is tempting to propose that the initiator is a major cis-acting element that regulates the transcriptional start in PSG and CEA family related genes. Conservation of the purine repeats among the 5' flanking region of the three PSGlike genes so far sequenced, suggests that they may be also important in the control of the PSG gene transcription. Sequences 5' further up-

261 stream to the -471 position are sufficient to abolish the promoter activity in the COS-7 cells. We have no experimental evidence in this work about the specific SGI sequence element involved in this effect. However, the sequence including four (dTdG)n short repeats, also contained in the PSG 6 clone at similar positions, is a good candidate that could function as a silencer, abolishing the expression in cells where the PSG genes are not normally expressed as in COS-7 cell line. This observation is reinforced by the fact that a similar element has been found in several viral enhancers and transcriptional control sequences and has been implicated in the regulation of gene expression [44]. Among the consensus sequences found in the first intron, the presence of the octamer elements and the SV40 enhancer core consensus sequence is remarkable, since they have been identified in a variety of ubiquitous and tissue-specific genes [45]. The systematic mutagenesis of the PSG 5 functional promoter region and also of the putative regulatory sequences identified here should provide new insights into the molecular mechanisms governing the expression of both the PSG and CEA genes during normal and tumor development.

Acknowledgements We are grateful to Dr. Roberto Staneloni for help and advice in the preparation of the genomic library and thank Dr. C. K6dinger for critical reading of the manuscript. This work was supported by grants from CONICOR and CONICET. Both G.P.D. and J.L.B. thank CONICOR and CONICET for the fellowships granted.

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