The 5′-Flanking Sequence and Regulatory Elements of the Cystatin S Gene

Biochemical and Biophysical Research Communications 261, 705–711 (1999) Article ID bbrc.1999.1072, available online at http://www.idealibrary.com on

The 59-Flanking Sequence and Regulatory Elements of the Cystatin S Gene Phyllis A. Shaw 1 and Orlando Chaparro Department of Cell Biology and Anatomy, Box 1007, Mount Sinai School of Medicine, New York, New York 10029

Received July 2, 1999

The gene encoding rat cystatin S (Cys S), a salivary gland-specific secretory protein, has CAAT and TATA boxes upstream of the inititation codon (Cox and Shaw, 1992), and contains regions that resemble those of other hormonally responsive eukaryotic genes. The 5*-flanking sequence of the rat Cys S gene has a potential CREB/AP-1 binding site (Rupp et al., 1990; Trejo et al., 1992), two potential glucocorticoid responsive elements (GREs, Drouin et al., 1989), and a possible GR/PR (glucocorticoid/progesterone) responsive element (Forman and Samuels, 1990). One of these potential GREs is adjacent to a potential AP-2 binding site, and another is typical of the glucocorticoid and progesterone receptor binding site. In this report, we have identified three regions in the 5*-flanking region of the Cys S gene that are found in salivary glandspecific genes (Ting et al., 1992) with a GT-rich region located between conserved elements II and III. Transfection experiments described in this paper suggest that a 281-bp DNA fragment from the Cys S gene promoter region with conserved elements II and III, the GT-rich region, and a possible GR/PR responsive element contains a negative regulatory element. In addition, our experiments suggest that the GT-rich region by itself is acting as a positive regulatory element. © 1999 Academic Press

Key Words: regulatory elements; cysteine proteinase inhibitor; cystatin S; submandibular gland.

Cystatins are naturally occurring cysteine proteinase which belong to an evolutionarily-related superfamily composed of three major subfamilies, the stefins (family 1), the cystatins (family 2), and the kininogens (family 3). They participate in regulating physiological processes such as intracellular catabolism of polypeptides and proteins (Barrett and Kirscke, 1981), processing of proenzymes and prohormones (Marks et al., 1 To whom correspondence should be addressed. Fax: (212) 8601174. E-mail: [email protected]. Abbreviation: cAMP, cyclic adenosine monophosphate.

1986), extracellular degradation of collagen (Etherington, 1980), bone resorption (Delaisse et al., 1984), and apoptosis (Cerretti et al., 1992; Fernandes-Alnemri et al., 1995). Cystatins have also been implicated in defense mechanisms by inhibiting the penetration and destruction of tissues by bacteria (Barrett et al., 1984; Bjorck et al., 1989) and viruses (Korant et al., 1988; Guy et al., 1991), inflammatory diseases (Mort et al., 1984), regulation and assembly of many viral proteins including those of HIV-1 (Guy et al., 1991), and cancer progression (Sloane et al., 1990). Two human diseases are known to result directly from genetic alterations of cystatins: massive cerebral hemorrhagia (Abrahamson et al., 1987) and progressive myoclonus epilepsy (Pennacchio et al., 1996). Salivary glands of mammals produce secretions important in protecting the oral cavity, salivary cystatins are thought to play a major role in this. Human salivary cystatins were purified and the salivary glandspecific cystatin, Cys S, was found to be synthesized in the acinar cells of submandibular and parotid glands (Isemura et al., 1984a,b; Minakata and Asano, 1984). The human Cys S genes form a multi-gene family with at least seven members (Saitoh et al., 1987). Homologues of the human salivary cystatins have been found in other mammalian species, including the rat (Shaw et al., 1988; Bedi, 1989). Very little information exists on regulation of cystatin genes in general, and of salivary cystatins in particular. The expression of the rat Cys S gene is tissuespecific, cell-type specific, and can be precociously induced in the submandibular and parotid glands by isoproterenol [IPR (Shaw et al., 1990; Shaw and Barka, 1989)]. That sex hormones might participate in the regulation of the Cys S gene was suggested by the finding that induction of Cys S mRNA by IPR in rat submandibular glands is more pronounced in females than in males (Shaw et al., 1990). Our recent studies of submandibular glands of ovariectomized-adrenalectomized or castrated-adrenalectomized rats suggest that steroid hormones also play a part in regulation of Cys S gene expression (Chaparro et al., 1994).

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In order to characterize the regulatory elements in the Cys S gene promoter, we have sequenced the gene (Cox and Shaw, 1992). This paper describes the sequence of 1911 bp of 59-flanking Cys S DNA and tissue culture experiments analyzing DNA fragments containing salivary gland-specific conserved elements and a GT-rich region from the 59-flanking sequence of the Cys S gene for transcriptional regulation. MATERIALS AND METHODS Materials. Materials and their sources were: T 4 DNA ligase, DNA polymerase I (Klenow fragment), and restriction enzymes, New England Biolabs; [ 32P] ATP (3000 Ci/mmole), New England Nuclear (NEN); Sequenase Kits, U.S. Biochemical Corporation; luciferase assay kit, DNA maxi-prep kits, and pGEM vectors, Promega; luminescent b-galactosidase chemiluminescent kit, Clontech; protein Assay Kit, Bio-Rad; fetal bovine serum (FBS), Hyclone; OptiMem I, GIBCO BRL; Dulbecco’s Modification of Eagle’s Medium (DMEM), Cellgro. Preparation of riboprobe and screening of genomic library. The P-labeled Cys S riboprobe was generated from an SP6 plasmid containing the Cys S cDNA (clone 1a, Shaw et al., 1988). A 2.7 kb Cys S genomic clone was obtained by hybridizing a rat liver Eco R I genomic library (Sargent et al., 1979) with the Cys S-specific 32Plabeled riboprobe. This 2.7 kb fragment was then subcloned into the Eco R I site of pGEM-3, named pG3-7, and was sequenced as described below. 32

DNA sequence. Double-stranded pG3-7 DNA was prepared using the Promega maxi-prep kit. The sequence of the 59-flanking region of the rat Cys S gene was determined by the dideoxy chain-termination method (Sanger et al., 1977) using the Sequenase kit. Primers were used for sequencing the 2.7 kb Cys S genomic DNA: Cys S P1, 59-GGACAGAGGGCAAAGATAGG-39; Cys S P4, 59-GATTTGTGTCAGATAC TGTC-39; Cys S P5, 59-ATTAATATTTAAGTATATAC-39; GL-1, 59-TGTATC TTATGGTACTGTAA-39. Sequence of pG3-7 revealed that it contained 2.7 kb of the rat Cys S gene, representing 1911 bp of 59-flanking region (Fig. 1), 301 bp of exon 1 (all of exon 1), and 488 bp of intron 1. The 59-flanking region of the Cys S gene was analyzed for transcription factor binding sites using the 1995 updated Signal Scan Program (Prestridge, 1991) of the BioInformatics and Molecular Analysis Section at NIH (http://www.bimas.dcrt.nih. gov/molbio/signal/). Oligonucleotides. Sense GT oligo (GT-S), 59-TACGAGCTCTGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTACGCGTTC-39, and antisense GT oligo (GTA), 59-TGAACGCGTAAACACACACACACACACACACACACACACACACACACACACACACACACACACACAAACACAGAGCTCGT-39 were synthesized by GENSET Corp. (La Jolla, Calif.). Sense and antisense oligos containing conserved elements II and III were also synthesized by GENSET: GT-II,III sense (GTII,III-S), 59-TACGAGCTCTGAGGGAGTGAGTGAATGTGTTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTACAAGTGACCCTAACGCGTTCA-39; and GT-II,III antisense (GTII,III-A), 59-TGAACGCGTTAGGGTCACTTGTAAACACACACACACACACACACACACACACACACACACACACACACACACACACACAAACACATTCACTCACTCCCTCAGAGCTCGTA-39. These oligos were annealed and restricted with Mlu I for subcloning into the pGl2-CMV-luc vector previously restricted with Sma I and Mlu I. Subcloning of the correct sequences was confirmed by restriction analysis with Sac I and Mlu I, and by sequencing those clones that contained inserts of the correct size. In vitro analyses of the effect of the GT-containing fragments on CMV promoter activity. A 281 bp DNA fragment containing conserved elements II and III and the GT repeat region (GT 27), released

by Afl II/Xho I (Afl II cuts at 21011 and Xho I cuts at 2730 from the transcription start-site of the Cys S gene) digestion from pGL2-2 kb (pGL2 vector plus 2 kb of the 59 flanking region of the Cys S gene) and end-filled, was subcloned into the Hind III site of the pGL2-CMV vector containing the luciferase gene (pGL2-CMV-luc) to test for its effect on CMV promoter activity. Oligos containing GT 27 (65 bp) alone, and oligos containing GT 27 plus conserved elements II and III (92 bp) were annealed, treated with Mlu I, and cloned into the Sma I/Mlu I site of the pGL2-CMV luciferase vector (pGL2-CMV-luc). These four constructs (pGL2-CMV-luc, 281 bp-pGL2-CMV-luc, 92 bp-pGL2-CMV-luc, 65 bp-pGL2-CMV-luc, see Fig. 2) were transfected for 3 hrs (Lipofectin to DNA ratio of 2:1) into a rat submandibular gland cell line, A5, (Dr. Bruce Baum, NIH) in OptiMem. A5 cells were co-transfected with pCMV-b-galactosidase in order to correct for transfection efficiency. The transfected cells were washed and allowed to recover for 24 hrs in DMEM containing 10% FCS. After transfection, the cells were washed with phosphate buffered saline (PBS), treated with 13 Promega lysis buffer, and kept at room temperature for 5 min. Lysed cells were scraped from the plates, transferred to Eppendorf tubes, and centrifuged 20 sec at 4 oC at 14,000 rpm. 20 ml of cell lysate were added to 100 ml of Promega luciferase assay buffer. Luminescence of the samples was measured for 20 sec (LKB 1251 Luminometer). The luminescent b-galactosidase (b-gal) assay was performed on 50 ml of cell lysate mixed with 200 ml of Clontech b-gal assay buffer. The samples were gently mixed, kept at room temperature for 1 hr at which time 300 ml of Clontech chemiluminescent accelerator were added; exactly 15 sec later, the b-gal samples were read in a luminometer for 5 sec. The data presented are corrected for transfection efficiency using b-gal [light units (LU)/mg protein], and were analyzed by the Friedman (Exact) Test on median values for each experiment for each group, and by the Student’s t-test for independent samples.

RESULTS Characterization of the 59-Flanking Region of the Cys S Gene Our studies have suggested that hormonal and/or neuronal regulation play a part in the expression of the Cys S gene (Chaparro et al., 1994; Chaparro et al., 1997). Hormonal regulation of gene transcription is typically mediated by DNA sequences located in the 59-flanking, untranslated exonic, intronic, or 39 untranslated regions of specific genes. In order to understand the mechanisms regulating Cys S gene expression, we sequenced the corresponding rat gene (Cox and Shaw, 1992), and now report the sequence of 1.9 kb of its 59-flanking DNA (Fig. 1). It contains CAAT (248) and TATA (228) boxes upstream of the initiation codon, as well as several possible regulatory sequences that resemble those identified for other hormonally responsive eukaryotic genes. The sequence 59-TGACATCA-39 located at position 21777 in the 59-flanking region of the Cys S gene is a potential CREB/AP 1 binding site (Rupp et al., 1990; Trejo et al., 1992). Two potential identical glucocorticoid receptor binding sites are located at positions 21487 and 2187 (59-TGTGAT-39; Drouin et al., 1989); there is a potential AP-2 binding site (2194, TGGGGA; Imagawa et al., 1987) adjacent to the putative glucocorticoid receptor binding site located at position 2187. In addition, another potential glucocorticoid/progesterone receptor responsive ele-

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FIG. 1. Sequence of 59-flanking region of the rat Cys S gene. The DNA sequence includes 1911 bp 59 of the transcription start site and 50 bp past the transcription start site. The CCAAT box is located at position 248; the TATA box is located at 228. Three potential glucocorticoid responsive elements are located at positions 21487, 2753, and 2187, and an adjacent AP-2 binding site is located at 2194. A potential CREB/AP-1 binding site is located at position 21777. Three salivary gland-specific conserved elements are located at 2711, 2865, and 2947. A GT-rich region is identified beginning at 2924.

ment, 59-TGTTCT-39, is located at position 2753 (Strawhecker et al., 1989; Forman and Samuels, 1990).

Interestingly, the 59-flanking region of the gene contains three regions that are common to all known sequenced salivary gland-specific genes (Table 1). In the

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Common Elements in Human, Macaque, Rat, and Mouse Salivary Gland-Specific Genes Gene

I

II

hAMY1C rCys S hCST 1

2849 TTTCCTACC 2711 ......... 248 .C....G..

2802 AGAGTCCCTG 2865 ..T.A....A 2173 C-......CA

hCST 2 hCSTP1 hPRP I hPRP II macPRP mPRP mPsp

248 248 2212 2212 2936 2400 2697

2173 2183 2183 296 215 216

.C....G.A .C.....AT .A....... CA....C.. .......G. .A......T .G.......

N.S. ....-....C. ........AA ........A. ...A-...A. ..T.G..... ...A....A.

III 2784 2947 2245 2178 2245 2167 2167 2129 2173 2671

TGAGGGATGC .......GTG AA...ACG.A* --.....GAG N.S. A......CGG ........A. ........A. .A......T. .....T.A.G* .......ATG

Note. A total of 1911 bp of 59-flanking sequence of the rat Cys S (rCys S) gene was compared with 251 bp of the human salivary amylase gene (hAMY1C, Ting et al., 1992), 1 kb each of the human proline-rich proteins (hPRPs I and II, Kim and Maeda, 1986), macaque proline-rich protein gene (macPRP, MnP4, Lin et al., 1991), mouse proline-rich protein gene (mPRP, Ann and Carlson, 1985), and the parotid secretory protein gene (mPsp, Shaw and Schibler, 1986). Approximately 360 bp of the 59-flanking region of the human salivary cystatin genes CST1 and CSTP1 and 100 bp of the human CST2 gene were compared (Saitoh et al., 1987). N.S., not sequenced; *, reverse orientation; ., identity.

rat Cys S gene they are: I, 2711 TTTCCTACC; II, 2865 AGTGACCCTA; III, 2947 TGAGGGAGTG. The arrangement of these three regions in the 59-flanking DNA of salivary gland-specific genes is shown in Table 2. These three regions are arranged in the same order in all the salivary gland cystatins in which 59-flanking sequence is known, that is III, II, I. The 59-flanking region of the rat Cys S gene also contains a GT-rich region of 27 GT repeats that are located between conserved elements II and III; this GT repeat region is flanked by TTT. Analyses of Cys S Promoter DNA Fragment Containing Conserved Elements II and III and GT 27 We began our analyses of the DNA promoter region of the Cys S gene in transfection experiments designed

TABLE 2

Arrangement of Common Elements in the 59-Flanking Region of Rat, Human, Mouse, and Macaque Salivary Gland-Specific Genes Gene Rat Cys S Human cystatin, CST1 Human cystatin, CST2 Human cystatin, CSTP1 Human amylase Human proline-rich protein, hPRP I Human proline-rich protein, hPRP II Macaque proline-rich protein, macPRP Mouse proline-rich protein, mPRP Mouse parotid secretory protein, mPsp Note. N.S., not sequenced.

Element III III N.S. III I I I I I I

II II N.S. II II II II III II III

I I I I III III III II III II

to determine the effect of the salivary gland-specific promoter conserved elements II and III, and the GT repeats from the Cys S promoter region on the high efficiency promoter, CMV using the luciferase reporter gene. A5 cells were transfected for three hours with four Cys S promoter constructs: 1) pGL2-CMV-luc vector (CMV promoter alone), 2) 281 bp-pGL2-CMV-luc containing conserved elements II and III, GT 27, and GR/PR placed 59 of the CMV promoter, 3) 92 bp-pGL2CMV-luc containing conserved elements II and III and GT 27 placed 59 of the CMV promoter, or 4) 65 bp-pGL2CMV-luc containing only GT 27 placed 59 of the CMV promoter (Fig. 2). In all experiments, pCMV-b-gal was co-transfected to correct for transfection efficiency; the data were analyzed and expressed as light units (LU) per microgram of protein (Fig. 3). Analyses of these data by the Friedman (Exact) Test on median values for each of three experiments for each group gave a p value of 0.0382. The data were then analyzed by the Student’s t-test for independent samples to determine the significance of the differences in the transcriptional activation of the CMV promoter by domains II, III and GT 27. The activity of the CMV promoter with the 281 bp fragment containing conserved elements II and III, GT 27, and a potential GR/PR (281 bp-CMV-luc construct) upstream was statistically significantly (p 5 0.0001) reduced compared to the construct containing the CMV promoter alone. The transcriptional activity of the 281 bp-CMV construct is approximately 27% that of the construct containing only the CMV promoter. In contrast, the 65 bp-CMV-luc construct containing only GT 27 showed a statistically significant 33% increase in transcriptional activity of the CMV promoter (p 5 0.05). The transcriptional activitiy of the 92

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DISCUSSION Our sequencing of the 59-flanking region of the rat Cys S gene revealed that it contains three potential glucocorticoid receptor binding sites, two of which are identical to one found in the pro-opiomelanocortin (POMC) gene (Drouin et al., 1989); one of these two sites is located adjacent to a potential AP-2 binding site. The other potential glucocorticoid binding site is typical of a glucocorticoid/progesterone binding site (Forman and Samuels, 1990). In addition, there is a potential cyclic AMP responsive element, the CREB/ AP-1-like binding site. Previous data indicated that IPR induced Cys S mRNA in submandibular glands of adult rats; this induction was greater in adult female than in adult male animals (Shaw et al., 1990). More recent data indicate that that steroid hormones may also play a part in the sexually dimorphic regulation of the gene (Chaparro et al., 1994). In light of these data, and the results that are reported in this paper, the elements identified in the 59-flanking region of the rat Cys S gene are likely to be involved in regulating its expression. Induction of the Cys S gene by the b-adrenergic agonist, IPR, can be blocked by the b-adrenergic antagonist propranolol and by specific b 1-antagonists (metaprolol, atenolol), indicating that the effect of IPR

FIG. 2. Constructs that were transfected into A5 cells as described under Materials and Methods. 1. pGL2-CMV-luc vector (CMV promoter alone). 2. 281-bp pGL2-CMV-luc containing conserved elements II and III, GT 27, and GR/PR placed 59 of the CMV promoter. 3. 92-bp pGL2-CMV-luc containing conserved elements II and III and GT 27 placed 59 of the CMV promoter. 4. 65-bp pGL2CMV-luc containing GT 27 placed 59 of the CMV promoter.

bp-CMV-luc construct containing only the conserved elements II, III, and GT 27 was 21% increased compared to the CMV promoter alone, however this increase was not statistically significant (p 5 0.26).

FIG. 3. Analyses of transfection experiments using Cys S promoter fragments containing salivary gland-specific conserved elements II and III and GT 27. Transfections were performed as described under Materials and Methods. The data have been normalized versus b-galactosidase activity and are expressed as light units/microgram of protein. The LU/mg protein for 281-bp CMVluc equals 0.043 1 0.005 (S.E.M); LU/mg protein for 92-bp CMV-luc equals 0.193 1 0.027 (S.E.M.); 65-bp CMV-luc equals 0.211 1 0.021 (S.E.M.); LU/mg protein for CMV-luc equals 0.159 1 0.012 (S.E.M.); no vector equals 0.033 1 0.008 (S.E.M.).

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is mediated by the b 1-adrenoreceptor/cAMP pathway (Bedi, 1993). Transcription factor AP-1 directly transduces protein kinase C (PKC)-mediated signals (Lee et al., 1987) and indirectly transduces cAMP-dependent protein kinase A (PKA)-mediated intracellular signals (Boutillier et al., 1991). AP-2 transduces both cAMPdependent PKA- and PKC-mediated signals (Imagawa et al., 1987). The Cys S gene contains a potential AP-1 binding site (CREB/AP 1 site) at position 21777 in its 59-flanking region, and a potential AP-2 binding site at 2194 that is adjacent to a potential glucocorticoid receptor binding site at 2187. The identification of potential AP-1 and AP-2 binding sites in the 59-flanking region of the Cys S gene, suggests the possibility that both the PKA- and PKC-mediated transduction pathways could be operative in regulation of Cys S gene expression. One of the most interesting characteristics of the 59-flanking region of the Cys S gene is the presence of three short elements that are present in other salivary gland-specific genes. Ting et al. (1992) were the first to describe the presence of these elements in the proximal promoter regions of the human salivary amylase gene and the proline-rich protein (PRP; Kim and Maeda, 1986) genes, as well as the mouse parotid secretory protein (mPsp; Shaw and Schibler, 1986), and mouse proline-rich protein (mPRP; Ann and Carlson, 1985) genes. We extended the search to other salivary glandspecific genes to include the salivary cystatins, and find that these elements are present in all known sequenced salivary gland-specific genes, including human, macaque, mouse, and rat (Table 1). Importantly, the arrangement of these elements is different (compared to the amylase gene) in the cystatin genes in which sequence is known (Table 2). The arrangement is the same for the salivary gland-specific cystatin genes (III, II, I). Whether or not this different arrangement has any regulatory function in gene expression or tissue specificity remains to be investigated. The 59-promoter region of the Cys S gene has another striking characteristic; it has 27 GT repeats flanked by TTT located between the common salivary glandspecific elements II and III. The repetitive sequence of (GT) n is highly dispersed in eukaryotic genomes (yeast, fish, amphibians, insects and mammals). (GT) n repeats have been identified in several genes including actin, histone, immunoglogulin, globin, atrial natriuretic factor, somatostatin, growth hormone, and prolactin (Naylor and Clark, 1990). The distribution of (GT) n blocks in rodent and human genes shows striking similarities with almost 50% of the (GT) n sequences in intronic regions, whereas 20% of them are either present at the 59 or the 39 flanking regions of the genes (Stallings et al., 1991). It has been proposed that these repetitive (GT) n sequences have the potential to adopt Z-DNA structures and inhibit (Naylor and Clark, 1990)

or enhance transcriptional activity, and selectively bind nuclear proteins (Epplen et al., 1996). The data presented in this paper suggest that the GT-rich region in the 59-flanking DNA of the Cys S gene is transcriptionally active. Furthermore, our data also suggest that a larger DNA fragment from the Cys S gene promoter region with conserved elements II, III, the GT-rich region and a putative GR/PR binding site contains a negative regulatory element. Future experiments will focus on characterizing these putative regulatory regions of the Cys S gene in vivo in transgenics, and identifying transcription factors that participate in its regulation. ACKNOWLEDGMENTS This work was supported by Grant DE08714 to P.A.S. From the National Institute of Dental Research, National Institutes of Health. The authors thank Yaneth Castellanos, Jian Luo, and Hong Wang for technical assistance.

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