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The integrin αv gene: Identification and characterization of the promoter region
ii
i
BB Biochi~ic~a ELSEVIER
Biochimica et Biophysica Acta 1219 (1994) 228-232
et BiophysicaA~ta
Promoter Paper
The integrin a v gene: identification and characterization of the promoter region John P. Donahue *, Nancy Sugg, Jacek Hawiger Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Received 21 March 1994
Abstract
We isolated a 15.5 kilobase pair DNA fragment that contains the 5' end of the human vitronectin receptor a subunit (a v) gene. The nucleotide sequence of the 5' flanking region, first exon and part of the first intron of the a v gene was determined. The sequence showed that the 5' end of the a v gene lies within a CpG island. The transcriptional initiation site was mapped 169 base pairs upstream of the a~ translational initiation site. The 5' flanking region of the a v gene does not contain TATA or InR transcriptional control elements but does contain four Spl binding sites, two Ets binding sites and one GATA binding site. The identified a~ gene 5' flanking region directed the expression of human growth hormone in transfected HeLa cells. Successivc deletions of the 5' flanking region demonstrated a 222 bp region that exerts a strong positive effect on a v promoter activity.
Key words: Vitronectin receptor; Integrin; a v gene; Promoter; DNA sequence
The vitronectin receptor is a m e m b e r of the integrin family of cytoadhesive cellular receptors. The vitronectin receptor a subunit (av) associates with six different /3 subunits to form receptors that are variously expressed by endothelial, osteogenic, fibroblastic, myeloid, lymphoid and epithelial cell types [2-9]. Individual receptors recognize a number of adhesive proteins that are present in plasma and extracellular matrix, including vitronectin, fibrinogen, fibronectin, and yon Willebrand factor (for review see [1]). The av/3 receptors that are expressed in the vascular and immune systems are involved in the maintenance of the structural integrity of the vascular endothelium [10-12], macrophage recognition and phagocytosis of apoptotic neutrophils and lymphocytes [13], tumor cell metastasis [14,15], and "y3-T cell activation [16]. The synthesis of the various av/3 receptors is modulated by growth factors [7,17,18], cytokines [7,19], retinoic acid [20],
The nucleotide sequence data reported in this paper have been submitted to the GenBankTM/EMBL Data Bank under accession number U07375. * Corresponding author. Fax: + 1 (615) 3437392. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 1 3 - H
vitamin D-3 [21], and phorbol esters [3]. The mechanisms involved in the regulation of a v expression in response to these various agonists are not understood. To evaluate the role of transcriptional mechanisms in controlling the expression of a v in cells, we have cloned and characterized the promoter region of the a v gene. Isolation of an at, genomic clone. A 15.5 kbp genomic clone was isolated by standard methods [22] from a human leukocyte genomic library using a 5' a v c D N A fragment [3] as probe. Following digestion of the D N A with various restriction endonucleases, a restriction map of the clone, named EMBL3-6G4, was constructed (Fig. IA). To determine if the structure of the isolated a v genomic clone was representative of the authentic av gene, a genomic Southern blot was done. A 32p-labeled D N A fragment containing the first 247 bp of the av c D N A was hybridized to a Southern blot of human genomic DNA, isolated from the K562 erythroleukemia cell line [23], that had been digcsted to completion with either HindlIl, EcoRl, HindllI and EcoRI or Hindlll and BamHI (Fig. 1B). The results of this analysis were consistent with the restriction endonuclease map of cry genomic clone, EMBL3-
J.P. Donahue et a L / Biochimica et Biophysica Acta 1219 (1994) 228-232
6G4. Likewise, genomic Southern blots of D N A isolated from primary culture-derived h u m a n umbilical vein endothelial cells ( H U V E C ) yielded identical results (data not shown). The results of genomic Southern blotting clearly demonstrated that the D N A segment captured in clone EMBL3-6G4 had not undergone rearrangement during the cloning process and is therefore an accurate representation of the 5' end of the a v gene. Nucleotide sequence of the o~,. gene 5' region. It was determined by Southern blot analysis that the region of the av genomic clone that hybridized to the a v c D N A was confined to an approximately 1400 bp HindIII/ EcoRI fragment located at the 5' end of the clone (data not shown). Consequently, the 1400 bp HindI I I / E c o R I fragment was subcloned into bacteriophage M13mp18 and M13mp19 for D N A sequence determination using the dideoxy chain termination method [24]. The D N A sequence of this fragment, which was determined from sequencing both strands, is shown in Fig. 2 beginning at the HindIII site and extending 1074 bp through the first e x o n / i n t r o n junction. The D N A sequence between residues 651 and 876 are identical to residues 1 to 226 of the a v c D N A [3]. The sequences between 692 and 874 encode the first 61 amino acid residues of the amino-terminal end of the a v protein. The sequences encoding the o~v protein are discontinued at nucleotide 876. This residue is followed by a G T dinucleotide that is typical of a 5' splice junction and indicates that this is the boundary of the first intron of the av gene. Consequently, the combined D N A sequence and Southern blot data indicate that the a v gene contains a small first exon (350 bp) followed by a large intron (a minimum of 10.4 kbp). The sequence upstream of the a v translational initiation site is 71% G + C and contains a high frequency of the dinucleotide CpG. This indicates that the 5' end of the a v gene is contained within a C p G island (for a review see [25]). Mapping the site of a,: transcriptional initiation. The site of a v transcriptional initiation within the 5' flanking region was identified by primer extension mapping of the 5' termini of a v transcripts (Fig. 3). The data indicate that transcriptional initiation occurs at nucleotides 523-527 (Fig. 2) in both H U V E C and K562 cells. Located 40 nucleotides upstream of the site of transcriptional initiation is a consensus binding site for transcription factor Spl ( C 4 8 1 C G C C C ) but a sequence resembling the T A T A element is not apparent in this region. Except for the sequence C528TCA located just downstream of the transcriptional initiation site, the nucleotide sequence encompassing this site does not have apparent homology to described Inr elements that have been shown to be involved in transcriptional initiation at many TATAless promoters (for a review see [26]). As indicated in Fig. 2, the a v promoter
229
region also contains three Spl sites in addition to the one noted above and consensus binding sites for transcription factors of the ets and G A T A families.
Functional analysis of the a L, gene 5' flanking region. To demonstrate the functional activity of the identified c~v promoter region and to determine the approximate locations within this region of transcriptional regulatory elements, a series of expression plasmids were constructed that contained successive deletions in the 5' flanking region of the c~v gene fused to the human growth hormone gene (hGH). Confluent cultures of H e L a cells were cotransfected with each a v expression plasmid and the pCAT-control plasmid (control for transfection efficiency). 48 h after transfection, the concentrations of h G H in the culture medium and chloramphenicol acetyltransferase (CAT) in cell extracts were measured. The results of this analysis (Fig. 4) showed that plasmids containing 770 bp or 522 bp of a v 5' flanking sequence directed the synthesis of h G H
A S
H .-.~ E H
B
E
I
] |',
I
I
I
8.3
13.3
15.5
4.4
',
5.86.5
B
H + H
S
H +
E E BB
8.5-5.7-3.7-2.3-1.9-1.4--
Fig. 1. Restriction endonuclease map and genomic Southern blot of the 5' end of the a v gene. (A) Partial restriction endonuclease map of a v genomic clone EMBL3-6G4. Numbering of restriction endonuclease sites is in kilobase pairs. The region of the clone containing sequences complementary to the first 226 bp at the 5' end of o~v cDNA [3] is designated by the black box. The direction of transcription of the gene is indicated by the arrow. B = BamHI, E = EcoRI, H = HindlII, S = SalI. (B) Southern blot analysis of the 5' end of the a v gene. Shown is an autoradiogram of a Southern blot of 10/zg of human genomic D N A isolated from K562 cells that was digested to completion with HindlII, E c o R l , H i n d I I I / E c o R I or H i n d l I I / B a m H I and hybridized to the 32p-labelled 247 bp E c o R I / S a c I fragment of the a v cDNA [3]. The locations of l a m b d a / B s t E I l D N A size standards are shown.
J.P. Donahue et al. /Biochimica et Biophysica Acta 1219 (1994) 228-232
230
10 A G C ~
20 30 40 50 60 70 ATTTTTAAAC AACAGTCGCA CGGAAGTTCC C ~ C A AGC4~AACGTG G G T G C C C T T G
80 90 I00 ii0 CTACTCCCGT GGACGC~T AGATT~C GCTGGACCG~TC~CCGC
GATA
120
130 140 CC~CGCCC~:C A C G C C T C C T C
Spl
Spl
150 160 170 180 190 200 A G G T G C T C A G CCTGAGC4ZCT T C G T C C A G G A G C G C T G C C G C T G A C C C A G G C T C A G G A G C T G ~ C C C T G
210
220 230 240 250 260 270 280 CACAGACGCC CAGGTCTCGG GACAGGCGGC GACTGCACTC ACGGAAGTAC GCTGAGCTCT CCCCTGTAGA
290 300 310 320 330 340 350 AGGGCGCCTC TCCTCCCCCA C~6~CTC CAGCTCCACA GCAGCCTCCC GGGCCGGCTC CTCCTCCTTC
360 370 380 390 400 410 420 C A G G T C T C C T C C A G T G C C G C C G C G G C T C T C A G G C C T G A G G T G C G G C G C T C ACCCCGC4ZAG T C C C C A G C C T
430 440 450 460 470 CAGACGCTGC GTGGAGCGGC GGAGCCGGAG GGAAGCAAAG GACCGTCTGC
480 490 G C T G C T G T C C ~-~--6-~GCG
Spl
/ / / / /
CGCTCTGCGC CCCTCGTCCC TGGCGGTCGC TCCGAAGCTC AGCCCTCTTG CCTGCCCCGG AGCTGTCCCG
570 GGCTAGCCGA
580 590 600 610 620 630 GAAGAGAGCG GCCGGCAAGT TT~GCGC GCAGC4:GGCG G G C C G C G G G C A C T G G G C G C C
640 650 660 670 680 690 T C G C T ~ A G G T G G C T A C C G C T C C C G G C T T G G C G T C C C G C G C GCACTTCGC4: G A T G GCT Spl MET Ala
700 709 718 727 736 TTT C C G C C G C G G C G A C G G C T G CGC CTC GGT CCC CGC ~ CTC Phe Pro Pro A r g A r g A r g L e u A r g Leu Gly Pro A r g G l y L e u
745 754 C C G CTT CTT CTC T C G Pro L e u L e u L e u Ser
790 799 808 C T A GAC G T G G A C A G T CCT GCC Leu A s p V a l A s p Set Pro A l a
GGA CTC Gly Leu
763 772 781 C T G C T A CCT C T G TGC CGC GCC TTC A A C L e u L e u Pro L e u Cys A r g A l a Phe A s n
817 TAC T C T Tyr Ser
853 862 826 835 844 ~ CCC G A G ~ G G A A G T TAC TTC GGC TTC GCC G T G GAT T T C TTC G T G CCC A G C G l y Pro G l u G l y Ser T y r Phe Gly Phe A l a V a l A s p Phe Phe Val Pro Ser
GAG Glu
871 G C G TCT Ala Ser
884 TCGTAAGTGG
914 894 904 CCGCACTTGG AACTGGAGCT ~CCCCTCC
924 934 CCCACCGCGC GCACCCACCC
944 AGCGTTTCTC
954 CATTGGGATT
984 964 974 TCCGAGAGAT GATTTCCATT ~ATTGATT
994 1004 CCCGGCGTCT GTCTGTCTCA
1014 CCCATCCTAC
1024 CTCTCAGAGT
1034 GGTTTATATC
1064 ~AAATGAA
1044 CTGGGT6~AG
1054 GAA~TATTTG
1074 CTCCCTTTGG
Fig. 2. Nucleotide sequence of the 5' end of the human av gene. The NH2-terminal amino acid sequence of the a v subunit, encoded by the first exon of the gene, is shown underneath the nucleotide sequence. The location of the transcriptional initiation site determined by primer extension analysis of a v transcripts is designated by vertical arrows. The sequence complementary to the primer used in primer extension analysis is underlined and the GT dinucleotide located in the first intron at the 5' splice junction is double underlined. The locations of potential binding sites for transcription factors Spl, GATA and Ets are boxed and labeled.
IP. Donahue et al. / Biochimica et Biophysica Acta 1219 (1994) 228-232
AT
A517 G
CG123 I
C
T
/
r
G
T 531
'
Fig. 3. Primer extension mapping of the 5' termini of av transcripts. Shown is an autoradiogram of a 6% polyacrylamide sequencing gel. A 5' 32p-labeled oligonucleotide (32 ng; 2.4.105 dpm/ng), underlined in Fig. 2, was annealed to 5 ~zg of poly(A) + RNA isolated from HUVEC (lane 1) or K562 cells (lane 2) and extended with 400 units of M-MLV reverse transcriptase. A control reaction in which RNA was not included is shown in lane 3. Dideoxy sequencing reactions [24] of the ~ gene 5' flanking region using the same primer are shown. The region of sequence shown is complementary to the sequence shown in Fig. 2. Total RNA was isolated from confluent cultures of HUVEC, isolated and grown as described [10,27], by an SDS/acid phenol extraction method [22] and from K562 cells using the total RNA isolation kit from Promega Corporation (Madison, W], USA). Poly(A) + RNA was selected from total RNA preparations using the PolyATtract isolation system from Promega Corporation.
at levels approximately 25-fold above background levels. The deletion of a 158 bp fragment between - 5 2 2 and - 3 6 4 , which contains consensus Ets, GATA and Spl binding sites, resulted in a 1.7-fold increase in the levels of hGH in the culture medium showing that this region exerts a small negative effect on av promoter activity. In contrast, hGH levels decreased 14-fold to just 3-fold above background levels when a 222 bp
Hindlll -770
-522
Sacl -364 - 2 5 8
.............................................. t
Stul
r
Xbal
-142
+1
+170
.....................
~
[ ...........................
fragment between nucleotides - 3 6 4 and - 1 4 2 was deleted. This result demonstrates that the deleted fragment contains important positive regulatory elements for a v promoter activity. The only potential regulatory element identified within this region was a consensus Ets binding site. In conclusion, the structure and transcriptional activity of the a v gene promoter region reported herein will facilitate further study on its regulation. This research was supported by NIH grants HL30648 and HL45994. The authors wish to thank Donna Russell and Clark Tibbetts for help in DNA sequencing and for sharing their sequencing equipment and facilities, Laurence Fitzgerald for the a v cDNA clone, Stuart Orkin for the genomic library, James Forbes for the K562 cell line, and Troy Torgerson for help in preparation of the manuscript figures.
References [1] Hynes, R.O. (1992) Cell 69, 11-25. [2] Cheresh, D.A. (1987) Proc. Natl. Acad. Sci. USA 84, 6471-6475. [3] Suzuki, S., Argraves, W.S., Arai, H., Languino, L.R., Pierschbacher, M.D. and Ruoslahti, E. (1987) J. Biol. Chem. 262, 14080-14085. [4] Pytela, R., Pierschbacher, M.D. and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. USA 82, 5766-5770. [5] Suzuki, S., Argraves, W.S., Pytela, R., Arai, H., Krusius, T., Pierschbacher, M.D. and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. USA 83, 8614-8618. [6] Krissansen, G.W., Elliot, M.J., Lucas, C.M., Stommski, F.C., Berndt, M.C., Cheresh, D.A., Lopez, A.F. and Burns, G.F. (1990) J. Biol. Chem. 265, 823-830. [7] Bates, R.C., Rankin, LM., Lucas, C.M., Scott, J.L., Krissansen, G.W. and Burns, G.F. (1991) J. Biol. Chem. 266, 18593-18599.
p(Zv~$0GH po¢, 692GH
.................. ..............
231
:F--4
po¢,534GH
~
p ~ 312GH
~-~
p~GH
r
I 50 Percentage
1OO
150
200
H u m a n Growth H o r m o n e
Fig. 4. Analysis of promoter activity of the a v gene 5' flanking region. HeLa cells were cotransfected with 1 #g of each o~v expression plasmid or parent plasmid p4)GH and 1 /xg of the pCAT-Control plasmid (Promega Corporation) using Lipofectase Reagent (GIBCO BRL, Gaithersberg, MD, USA). 48 h after transfection the concentrations of human growth hormone (hGH) in the culture medium and chloramphenicol acetyltransferase (CAT) in cell extracts were determined. Growth hormone was measured by a radioimmunoassay procedure [28] and CAT was measured as described [29]. Expression of CAT was used as a control for variation in transfection efficiency. Data are presented as the mean _+S.E. of four independent determinations expressed as a percentage of the hGH concentration determined for transfections with pav940GH. Growth hormone levels measured in pav940GH transfections varied from 21 to 84 ng/ml. Various lengths of 5' flanking sequence were inserted in pqSGH by polymerase chain reaction mediated synthesis of specific genomic segments or by using pre-existing restriction endonuclease sites. The XbaI site at + 170 was inserted in the av genomic clone by oligonucleotide directed mutagenesis [30]. The locations of other restriction endonuclease sites used in the construction of the av expression plasmids and potential Spl ( I ) , GATA ( n ) and Ets (¢) binding sites are indicated.
232
J.P. Donahue et al. / Biochimica et Biophysica Acta 1219 (19941 228-232
[8] Busk, M., Pytela, R. and Sheppard, D. (1992) J. Biol. Chem. 267, 5790-5796. [9] Moyle, M., Napier, M.A. and McLean, J.W. (1991) J. Biol. Chem. 266, 19650-19658. [10] Chen, C.S. and Hawiger, J. (1991) Blood 77, 2200-2206. [11] Charo, I.F., Bekeart, L.S. and Phillips, D.R. (1987) J. Biol. Chem. 262, 9935-9938. [12] Lampugnani, M.G., Resnati, M., Dejana, E., Marchisio, P.C. (1991) J. Cell Biol. 112, 479-490. [13] Savill, J., Dransfield, 1., Hogg, N. and Haslett, C. (1990) Nature (Lond.) 343, 170-173. [14] Shaughnessy, S.G., Lafrenie, R.M., Buchanan, M.R., Podor, T.J. and Orr, F.W. (1991) Am. J. Pathol. 138, 1535-1543. [15] Felding-Habermann, B., Muelller, B.M., Romerdahl, C.A. and Cheresh, D.A. (1992) J. Clin. Invest. 89, 2018-2022. [16] Wang, A.M., Doyle, M.V. and Mark, D.F. (1989) Proc. Natl. Acad. Sci. USA 86, 9717-9721. [17] Ignotz, R.A., Heino, J. and Massague, J. (19891 J. Biol. Chem. 264, 389-392. [18] De Nichilo, M.O. and Burns, G.F. (1993) Proc. Natl. Acad. Sci. USA 90, 2517-2521. [19] Defilippi, P., Truffa, G., Stefanuto, G., Altruda, F., Silengo, L. and Tarone, G. (1991) J. Biol. Chem. 266, 7638-7645.
[20] Dedhar, S., Robertson, K. and Gray, V. (1991) J. Biol. Chem. 266, 21846-21852. [21] Medhora, M.M., Teitelbaum, S., Chappel, J., Alvarez, J., Mimura, H., Ross, F.P. and Hruska, K. (1993) J. Biol. Chem. 268, 1456 1461. [22] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. [23] Lozzio, B.B., Lozzio, C.B., Bamberger, E.G. and Feliu, A.S. (19811 Proc. Soc. Exp. Biol. Med. 166, 546-550. [24] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. [25] Bird, A. (19861 Nature (Lond.) 321, 209-213. [26] Weis, L. and Reinberg, D. (1992) FASEB J. 6, 3300-3309. [27] Gimbrone Jr., M.A. (1976) in Progress Hemostasis and Thrombosis (Spaet, T.H., ed.), Vol. 3, pp. 1 28, Grune and Stratton, New York. [28] Nichols Institute, San Juan Capistrano, CA, USA. [29] Neumann, J.R., Morency, C.A. and Russian, K. (1987) Biotechniques 5, 444. [30] Zoller, M.J. and Smith, M. (1983) Methods Enzymol. 100, 468-500.
i
BB Biochi~ic~a ELSEVIER
Biochimica et Biophysica Acta 1219 (1994) 228-232
et BiophysicaA~ta
Promoter Paper
The integrin a v gene: identification and characterization of the promoter region John P. Donahue *, Nancy Sugg, Jacek Hawiger Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Received 21 March 1994
Abstract
We isolated a 15.5 kilobase pair DNA fragment that contains the 5' end of the human vitronectin receptor a subunit (a v) gene. The nucleotide sequence of the 5' flanking region, first exon and part of the first intron of the a v gene was determined. The sequence showed that the 5' end of the a v gene lies within a CpG island. The transcriptional initiation site was mapped 169 base pairs upstream of the a~ translational initiation site. The 5' flanking region of the a v gene does not contain TATA or InR transcriptional control elements but does contain four Spl binding sites, two Ets binding sites and one GATA binding site. The identified a~ gene 5' flanking region directed the expression of human growth hormone in transfected HeLa cells. Successivc deletions of the 5' flanking region demonstrated a 222 bp region that exerts a strong positive effect on a v promoter activity.
Key words: Vitronectin receptor; Integrin; a v gene; Promoter; DNA sequence
The vitronectin receptor is a m e m b e r of the integrin family of cytoadhesive cellular receptors. The vitronectin receptor a subunit (av) associates with six different /3 subunits to form receptors that are variously expressed by endothelial, osteogenic, fibroblastic, myeloid, lymphoid and epithelial cell types [2-9]. Individual receptors recognize a number of adhesive proteins that are present in plasma and extracellular matrix, including vitronectin, fibrinogen, fibronectin, and yon Willebrand factor (for review see [1]). The av/3 receptors that are expressed in the vascular and immune systems are involved in the maintenance of the structural integrity of the vascular endothelium [10-12], macrophage recognition and phagocytosis of apoptotic neutrophils and lymphocytes [13], tumor cell metastasis [14,15], and "y3-T cell activation [16]. The synthesis of the various av/3 receptors is modulated by growth factors [7,17,18], cytokines [7,19], retinoic acid [20],
The nucleotide sequence data reported in this paper have been submitted to the GenBankTM/EMBL Data Bank under accession number U07375. * Corresponding author. Fax: + 1 (615) 3437392. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0 1 6 7 - 4 7 8 1 ( 9 4 ) 0 0 1 1 3 - H
vitamin D-3 [21], and phorbol esters [3]. The mechanisms involved in the regulation of a v expression in response to these various agonists are not understood. To evaluate the role of transcriptional mechanisms in controlling the expression of a v in cells, we have cloned and characterized the promoter region of the a v gene. Isolation of an at, genomic clone. A 15.5 kbp genomic clone was isolated by standard methods [22] from a human leukocyte genomic library using a 5' a v c D N A fragment [3] as probe. Following digestion of the D N A with various restriction endonucleases, a restriction map of the clone, named EMBL3-6G4, was constructed (Fig. IA). To determine if the structure of the isolated a v genomic clone was representative of the authentic av gene, a genomic Southern blot was done. A 32p-labeled D N A fragment containing the first 247 bp of the av c D N A was hybridized to a Southern blot of human genomic DNA, isolated from the K562 erythroleukemia cell line [23], that had been digcsted to completion with either HindlIl, EcoRl, HindllI and EcoRI or Hindlll and BamHI (Fig. 1B). The results of this analysis were consistent with the restriction endonuclease map of cry genomic clone, EMBL3-
J.P. Donahue et a L / Biochimica et Biophysica Acta 1219 (1994) 228-232
6G4. Likewise, genomic Southern blots of D N A isolated from primary culture-derived h u m a n umbilical vein endothelial cells ( H U V E C ) yielded identical results (data not shown). The results of genomic Southern blotting clearly demonstrated that the D N A segment captured in clone EMBL3-6G4 had not undergone rearrangement during the cloning process and is therefore an accurate representation of the 5' end of the a v gene. Nucleotide sequence of the o~,. gene 5' region. It was determined by Southern blot analysis that the region of the av genomic clone that hybridized to the a v c D N A was confined to an approximately 1400 bp HindIII/ EcoRI fragment located at the 5' end of the clone (data not shown). Consequently, the 1400 bp HindI I I / E c o R I fragment was subcloned into bacteriophage M13mp18 and M13mp19 for D N A sequence determination using the dideoxy chain termination method [24]. The D N A sequence of this fragment, which was determined from sequencing both strands, is shown in Fig. 2 beginning at the HindIII site and extending 1074 bp through the first e x o n / i n t r o n junction. The D N A sequence between residues 651 and 876 are identical to residues 1 to 226 of the a v c D N A [3]. The sequences between 692 and 874 encode the first 61 amino acid residues of the amino-terminal end of the a v protein. The sequences encoding the o~v protein are discontinued at nucleotide 876. This residue is followed by a G T dinucleotide that is typical of a 5' splice junction and indicates that this is the boundary of the first intron of the av gene. Consequently, the combined D N A sequence and Southern blot data indicate that the a v gene contains a small first exon (350 bp) followed by a large intron (a minimum of 10.4 kbp). The sequence upstream of the a v translational initiation site is 71% G + C and contains a high frequency of the dinucleotide CpG. This indicates that the 5' end of the a v gene is contained within a C p G island (for a review see [25]). Mapping the site of a,: transcriptional initiation. The site of a v transcriptional initiation within the 5' flanking region was identified by primer extension mapping of the 5' termini of a v transcripts (Fig. 3). The data indicate that transcriptional initiation occurs at nucleotides 523-527 (Fig. 2) in both H U V E C and K562 cells. Located 40 nucleotides upstream of the site of transcriptional initiation is a consensus binding site for transcription factor Spl ( C 4 8 1 C G C C C ) but a sequence resembling the T A T A element is not apparent in this region. Except for the sequence C528TCA located just downstream of the transcriptional initiation site, the nucleotide sequence encompassing this site does not have apparent homology to described Inr elements that have been shown to be involved in transcriptional initiation at many TATAless promoters (for a review see [26]). As indicated in Fig. 2, the a v promoter
229
region also contains three Spl sites in addition to the one noted above and consensus binding sites for transcription factors of the ets and G A T A families.
Functional analysis of the a L, gene 5' flanking region. To demonstrate the functional activity of the identified c~v promoter region and to determine the approximate locations within this region of transcriptional regulatory elements, a series of expression plasmids were constructed that contained successive deletions in the 5' flanking region of the c~v gene fused to the human growth hormone gene (hGH). Confluent cultures of H e L a cells were cotransfected with each a v expression plasmid and the pCAT-control plasmid (control for transfection efficiency). 48 h after transfection, the concentrations of h G H in the culture medium and chloramphenicol acetyltransferase (CAT) in cell extracts were measured. The results of this analysis (Fig. 4) showed that plasmids containing 770 bp or 522 bp of a v 5' flanking sequence directed the synthesis of h G H
A S
H .-.~ E H
B
E
I
] |',
I
I
I
8.3
13.3
15.5
4.4
',
5.86.5
B
H + H
S
H +
E E BB
8.5-5.7-3.7-2.3-1.9-1.4--
Fig. 1. Restriction endonuclease map and genomic Southern blot of the 5' end of the a v gene. (A) Partial restriction endonuclease map of a v genomic clone EMBL3-6G4. Numbering of restriction endonuclease sites is in kilobase pairs. The region of the clone containing sequences complementary to the first 226 bp at the 5' end of o~v cDNA [3] is designated by the black box. The direction of transcription of the gene is indicated by the arrow. B = BamHI, E = EcoRI, H = HindlII, S = SalI. (B) Southern blot analysis of the 5' end of the a v gene. Shown is an autoradiogram of a Southern blot of 10/zg of human genomic D N A isolated from K562 cells that was digested to completion with HindlII, E c o R l , H i n d I I I / E c o R I or H i n d l I I / B a m H I and hybridized to the 32p-labelled 247 bp E c o R I / S a c I fragment of the a v cDNA [3]. The locations of l a m b d a / B s t E I l D N A size standards are shown.
J.P. Donahue et al. /Biochimica et Biophysica Acta 1219 (1994) 228-232
230
10 A G C ~
20 30 40 50 60 70 ATTTTTAAAC AACAGTCGCA CGGAAGTTCC C ~ C A AGC4~AACGTG G G T G C C C T T G
80 90 I00 ii0 CTACTCCCGT GGACGC~T AGATT~C GCTGGACCG~TC~CCGC
GATA
120
130 140 CC~CGCCC~:C A C G C C T C C T C
Spl
Spl
150 160 170 180 190 200 A G G T G C T C A G CCTGAGC4ZCT T C G T C C A G G A G C G C T G C C G C T G A C C C A G G C T C A G G A G C T G ~ C C C T G
210
220 230 240 250 260 270 280 CACAGACGCC CAGGTCTCGG GACAGGCGGC GACTGCACTC ACGGAAGTAC GCTGAGCTCT CCCCTGTAGA
290 300 310 320 330 340 350 AGGGCGCCTC TCCTCCCCCA C~6~CTC CAGCTCCACA GCAGCCTCCC GGGCCGGCTC CTCCTCCTTC
360 370 380 390 400 410 420 C A G G T C T C C T C C A G T G C C G C C G C G G C T C T C A G G C C T G A G G T G C G G C G C T C ACCCCGC4ZAG T C C C C A G C C T
430 440 450 460 470 CAGACGCTGC GTGGAGCGGC GGAGCCGGAG GGAAGCAAAG GACCGTCTGC
480 490 G C T G C T G T C C ~-~--6-~GCG
Spl
/ / / / /
CGCTCTGCGC CCCTCGTCCC TGGCGGTCGC TCCGAAGCTC AGCCCTCTTG CCTGCCCCGG AGCTGTCCCG
570 GGCTAGCCGA
580 590 600 610 620 630 GAAGAGAGCG GCCGGCAAGT TT~GCGC GCAGC4:GGCG G G C C G C G G G C A C T G G G C G C C
640 650 660 670 680 690 T C G C T ~ A G G T G G C T A C C G C T C C C G G C T T G G C G T C C C G C G C GCACTTCGC4: G A T G GCT Spl MET Ala
700 709 718 727 736 TTT C C G C C G C G G C G A C G G C T G CGC CTC GGT CCC CGC ~ CTC Phe Pro Pro A r g A r g A r g L e u A r g Leu Gly Pro A r g G l y L e u
745 754 C C G CTT CTT CTC T C G Pro L e u L e u L e u Ser
790 799 808 C T A GAC G T G G A C A G T CCT GCC Leu A s p V a l A s p Set Pro A l a
GGA CTC Gly Leu
763 772 781 C T G C T A CCT C T G TGC CGC GCC TTC A A C L e u L e u Pro L e u Cys A r g A l a Phe A s n
817 TAC T C T Tyr Ser
853 862 826 835 844 ~ CCC G A G ~ G G A A G T TAC TTC GGC TTC GCC G T G GAT T T C TTC G T G CCC A G C G l y Pro G l u G l y Ser T y r Phe Gly Phe A l a V a l A s p Phe Phe Val Pro Ser
GAG Glu
871 G C G TCT Ala Ser
884 TCGTAAGTGG
914 894 904 CCGCACTTGG AACTGGAGCT ~CCCCTCC
924 934 CCCACCGCGC GCACCCACCC
944 AGCGTTTCTC
954 CATTGGGATT
984 964 974 TCCGAGAGAT GATTTCCATT ~ATTGATT
994 1004 CCCGGCGTCT GTCTGTCTCA
1014 CCCATCCTAC
1024 CTCTCAGAGT
1034 GGTTTATATC
1064 ~AAATGAA
1044 CTGGGT6~AG
1054 GAA~TATTTG
1074 CTCCCTTTGG
Fig. 2. Nucleotide sequence of the 5' end of the human av gene. The NH2-terminal amino acid sequence of the a v subunit, encoded by the first exon of the gene, is shown underneath the nucleotide sequence. The location of the transcriptional initiation site determined by primer extension analysis of a v transcripts is designated by vertical arrows. The sequence complementary to the primer used in primer extension analysis is underlined and the GT dinucleotide located in the first intron at the 5' splice junction is double underlined. The locations of potential binding sites for transcription factors Spl, GATA and Ets are boxed and labeled.
IP. Donahue et al. / Biochimica et Biophysica Acta 1219 (1994) 228-232
AT
A517 G
CG123 I
C
T
/
r
G
T 531
'
Fig. 3. Primer extension mapping of the 5' termini of av transcripts. Shown is an autoradiogram of a 6% polyacrylamide sequencing gel. A 5' 32p-labeled oligonucleotide (32 ng; 2.4.105 dpm/ng), underlined in Fig. 2, was annealed to 5 ~zg of poly(A) + RNA isolated from HUVEC (lane 1) or K562 cells (lane 2) and extended with 400 units of M-MLV reverse transcriptase. A control reaction in which RNA was not included is shown in lane 3. Dideoxy sequencing reactions [24] of the ~ gene 5' flanking region using the same primer are shown. The region of sequence shown is complementary to the sequence shown in Fig. 2. Total RNA was isolated from confluent cultures of HUVEC, isolated and grown as described [10,27], by an SDS/acid phenol extraction method [22] and from K562 cells using the total RNA isolation kit from Promega Corporation (Madison, W], USA). Poly(A) + RNA was selected from total RNA preparations using the PolyATtract isolation system from Promega Corporation.
at levels approximately 25-fold above background levels. The deletion of a 158 bp fragment between - 5 2 2 and - 3 6 4 , which contains consensus Ets, GATA and Spl binding sites, resulted in a 1.7-fold increase in the levels of hGH in the culture medium showing that this region exerts a small negative effect on av promoter activity. In contrast, hGH levels decreased 14-fold to just 3-fold above background levels when a 222 bp
Hindlll -770
-522
Sacl -364 - 2 5 8
.............................................. t
Stul
r
Xbal
-142
+1
+170
.....................
~
[ ...........................
fragment between nucleotides - 3 6 4 and - 1 4 2 was deleted. This result demonstrates that the deleted fragment contains important positive regulatory elements for a v promoter activity. The only potential regulatory element identified within this region was a consensus Ets binding site. In conclusion, the structure and transcriptional activity of the a v gene promoter region reported herein will facilitate further study on its regulation. This research was supported by NIH grants HL30648 and HL45994. The authors wish to thank Donna Russell and Clark Tibbetts for help in DNA sequencing and for sharing their sequencing equipment and facilities, Laurence Fitzgerald for the a v cDNA clone, Stuart Orkin for the genomic library, James Forbes for the K562 cell line, and Troy Torgerson for help in preparation of the manuscript figures.
References [1] Hynes, R.O. (1992) Cell 69, 11-25. [2] Cheresh, D.A. (1987) Proc. Natl. Acad. Sci. USA 84, 6471-6475. [3] Suzuki, S., Argraves, W.S., Arai, H., Languino, L.R., Pierschbacher, M.D. and Ruoslahti, E. (1987) J. Biol. Chem. 262, 14080-14085. [4] Pytela, R., Pierschbacher, M.D. and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. USA 82, 5766-5770. [5] Suzuki, S., Argraves, W.S., Pytela, R., Arai, H., Krusius, T., Pierschbacher, M.D. and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. USA 83, 8614-8618. [6] Krissansen, G.W., Elliot, M.J., Lucas, C.M., Stommski, F.C., Berndt, M.C., Cheresh, D.A., Lopez, A.F. and Burns, G.F. (1990) J. Biol. Chem. 265, 823-830. [7] Bates, R.C., Rankin, LM., Lucas, C.M., Scott, J.L., Krissansen, G.W. and Burns, G.F. (1991) J. Biol. Chem. 266, 18593-18599.
p(Zv~$0GH po¢, 692GH
.................. ..............
231
:F--4
po¢,534GH
~
p ~ 312GH
~-~
p~GH
r
I 50 Percentage
1OO
150
200
H u m a n Growth H o r m o n e
Fig. 4. Analysis of promoter activity of the a v gene 5' flanking region. HeLa cells were cotransfected with 1 #g of each o~v expression plasmid or parent plasmid p4)GH and 1 /xg of the pCAT-Control plasmid (Promega Corporation) using Lipofectase Reagent (GIBCO BRL, Gaithersberg, MD, USA). 48 h after transfection the concentrations of human growth hormone (hGH) in the culture medium and chloramphenicol acetyltransferase (CAT) in cell extracts were determined. Growth hormone was measured by a radioimmunoassay procedure [28] and CAT was measured as described [29]. Expression of CAT was used as a control for variation in transfection efficiency. Data are presented as the mean _+S.E. of four independent determinations expressed as a percentage of the hGH concentration determined for transfections with pav940GH. Growth hormone levels measured in pav940GH transfections varied from 21 to 84 ng/ml. Various lengths of 5' flanking sequence were inserted in pqSGH by polymerase chain reaction mediated synthesis of specific genomic segments or by using pre-existing restriction endonuclease sites. The XbaI site at + 170 was inserted in the av genomic clone by oligonucleotide directed mutagenesis [30]. The locations of other restriction endonuclease sites used in the construction of the av expression plasmids and potential Spl ( I ) , GATA ( n ) and Ets (¢) binding sites are indicated.
232
J.P. Donahue et al. / Biochimica et Biophysica Acta 1219 (19941 228-232
[8] Busk, M., Pytela, R. and Sheppard, D. (1992) J. Biol. Chem. 267, 5790-5796. [9] Moyle, M., Napier, M.A. and McLean, J.W. (1991) J. Biol. Chem. 266, 19650-19658. [10] Chen, C.S. and Hawiger, J. (1991) Blood 77, 2200-2206. [11] Charo, I.F., Bekeart, L.S. and Phillips, D.R. (1987) J. Biol. Chem. 262, 9935-9938. [12] Lampugnani, M.G., Resnati, M., Dejana, E., Marchisio, P.C. (1991) J. Cell Biol. 112, 479-490. [13] Savill, J., Dransfield, 1., Hogg, N. and Haslett, C. (1990) Nature (Lond.) 343, 170-173. [14] Shaughnessy, S.G., Lafrenie, R.M., Buchanan, M.R., Podor, T.J. and Orr, F.W. (1991) Am. J. Pathol. 138, 1535-1543. [15] Felding-Habermann, B., Muelller, B.M., Romerdahl, C.A. and Cheresh, D.A. (1992) J. Clin. Invest. 89, 2018-2022. [16] Wang, A.M., Doyle, M.V. and Mark, D.F. (1989) Proc. Natl. Acad. Sci. USA 86, 9717-9721. [17] Ignotz, R.A., Heino, J. and Massague, J. (19891 J. Biol. Chem. 264, 389-392. [18] De Nichilo, M.O. and Burns, G.F. (1993) Proc. Natl. Acad. Sci. USA 90, 2517-2521. [19] Defilippi, P., Truffa, G., Stefanuto, G., Altruda, F., Silengo, L. and Tarone, G. (1991) J. Biol. Chem. 266, 7638-7645.
[20] Dedhar, S., Robertson, K. and Gray, V. (1991) J. Biol. Chem. 266, 21846-21852. [21] Medhora, M.M., Teitelbaum, S., Chappel, J., Alvarez, J., Mimura, H., Ross, F.P. and Hruska, K. (1993) J. Biol. Chem. 268, 1456 1461. [22] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. [23] Lozzio, B.B., Lozzio, C.B., Bamberger, E.G. and Feliu, A.S. (19811 Proc. Soc. Exp. Biol. Med. 166, 546-550. [24] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. [25] Bird, A. (19861 Nature (Lond.) 321, 209-213. [26] Weis, L. and Reinberg, D. (1992) FASEB J. 6, 3300-3309. [27] Gimbrone Jr., M.A. (1976) in Progress Hemostasis and Thrombosis (Spaet, T.H., ed.), Vol. 3, pp. 1 28, Grune and Stratton, New York. [28] Nichols Institute, San Juan Capistrano, CA, USA. [29] Neumann, J.R., Morency, C.A. and Russian, K. (1987) Biotechniques 5, 444. [30] Zoller, M.J. and Smith, M. (1983) Methods Enzymol. 100, 468-500.