HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jiang, Y. Articles by Stojilkovic, S. S Search for Related Content PubMed PubMed Citation Articles by Jiang, Y. Articles by Stojilkovic, S. S

Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA

(Requests for offprints should be addressed to S S Stojilkovic; Email: stojilks{at}mail.nih.gov)

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Soluble guanylyl cyclase is a cytosolic enzyme which catalyzes conversion of GTP to the second messenger cyclic GMP. The transcriptional regulation at the promoter levels of four soluble guanylyl cyclase subunits, termed 1, 2, ß1, and ß2, is largely unknown. In this study, we identified the transcription start site of 1-soluble guanylyl cyclase gene in rat pituitary cells and cloned the 3.5 kb 5'-promoter. Sequence analysis of this TATA-less promoter revealed the presence of several putative-binding sites for transcriptional factors, including CCAAT site at –41 to –32 and Sp1 site at –34 to –24. Transfection of pituitary cells with constructs of variable lengths confirmed the relevance of different promoter regions in the control of transcriptional activity. Among them, the –49 to + 156 region was critical for basal transcriptional activity. Electrophoretic mobility shift assay using nuclear proteins extracted from normal and immortalized pituitary cells indicated that the CCAAT/Sp1 site within the –49 to + 156 region was able to specifically interact with CCAAT-binding factor and Sp1. These two sites were partly overlapped and both of them conferred stimulatory effects. The in vivo recruitment of CCAAT-binding factor and Sp1 was confirmed by chromatin immunoprecipitation. These results indicate that the composite CCAAT/Sp1 cis-element contributes to the expression of 1-sGC subunit in resting pituitary cells.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Soluble guanylyl cyclase (sGC; E.C.4.6.1.2) catalyzes the formation of cGMP, an intracellular messenger that activates several downstream effectors, including ion channels, protein kinase G, and phosphodiesterases (Garbers et al. 1994). This signaling pathway regulates a broad spectrum of physiological processes, such as smooth muscle relaxation, platelet aggregation, and neuronal cell mobility. Based on cDNA cloning, four sGC subunits have been identified and termed 1, 2, ß1, and ß2. The human isoforms isolated from an adult brain library, termed 3 and ß3, represent the human homologues of rat and bovine 1 and ß1 (Zabel et al. 1998). The 1 and 2 subunits are interchangeable in terms of sGC activity when coexpressed with ß1, but the lß1 heterodimer is most common in mammalian tissues (Hanafy et al. 2004). The dimer activity is regulated directly at protein level. Physiologically, the most relevant mode of such regulation is mediated by binding of nitric oxide (NO) to the heme group of sGC (Humbert et al. 1990). The enzyme is also subjected to phosphorylation by protein kinase, src-like kinases, and protein kinases C and G (Pyriochou & Papapetropoulos 2005). Protein–protein interactions also contribute to the direct control of sGC activity, since documented by interaction between the chaperonin containing t-complex polypeptide subunit and ß1-sGC, which leads to inhibition of the enzyme activity (Hanafy et al. 2004).

Several reports also indicate that the regulation of sGC activity occurs indirectly, at transcriptional and post-transcriptional levels, affecting the availability of sGC subunits and the total number of functional heterodimers. For example, the steady-state sGC subunits mRNA levels are reduced by treatments with estrogens (Krumenacker et al. 2001), lipopolysaccharide (Pedraza et al. 2003), and ß-amyloid peptides (Baltrons et al. 2002). In human mesangial cells, collagen type I can also reduce the steady-state ß1-sGC mRNA levels and such reduction occurs at transcriptional level (De Frutos et al. 2005). On the other hand, the AU-rich elements identified in the 3'-untranslated region of 1- and ß1-sGC mRNAs provide sites for post-transcriptional regulation by cAMP (Kloss et al. 2004). Differential expression of mRNAs for and ß-sGC also influences the formation of functional heterodimers. For example, in pituitary cells, the mRNA levels (Budworth et al. 1999) and the protein levels (Kostic et al. 2004) for 1-sGC are lower than ß1-sGC. Consistent with these findings, the overexpression of 1-sGC alone in immortalized pituitary cells is sufficient to make several fold increase in NO-stimulated cGMP production (Kostic et al. 2004).

Changes in the mRNA expression levels of sGC subunits have also been reported in several disease models. In aortic tissue from spontaneously hypertensive rats, the vasodilator response to an NO donor was markedly attenuated compared with normotensive rats (Bauersachs et al. 1998). The detailed analysis of hypertensive animals revealed that 1- and ß1-sGC mRNA levels, as well as ß1-sGC protein levels, were reduced significantly, indicating that vasodilator dysfunction is related to the sGC gene expression (Kloss et al. 2000). In deoxycorticosterone acetate-salt model of rat hypertension, the protein expression level of ß1-sGC was also decreased in the inner medulla (Taylor et al. 2003). In addition, the protein levels of ß1-sGC were reduced in reactive astrocytes obtained from Alzheimer, Creutzfeldt–Jakob, and multiple sclerosis patients (Baltrons et al. 2004), whereas an upregulation of sGC was observed in aortas from rats with chronic heart failure (Bauersachs et al. 1999). Therefore, studies on the transcriptional regulation of sGC are not only important for understanding the mechanism of sGC gene expression, but are also clinically relevant.

However, the knowledge on promoter of sGC genes and regulation of the enzyme activity at transcriptional and post-transcriptional levels is very deficient. At the present time, the genomic organization of mouse (Sharina et al. 2000) and medaka fish (Mikami et al. 1999) 1- and ß1-sGC subunits, and human ß1-sGC subunit (Sharina et al. 2003) has been reported. Among the mammalian species, only the mouse 1-sGC promoter has been cloned, but its cis-elements have not been experimentally identified (Vazquez-Padron et al. 2004). Furthermore, the finding that 1-sGC gene for mouse has only 56 and 45% homology with predicted sequences for rat and human genes respectively (Vazquez-Padron et al. 2004), clearly indicates a need for cloning of promoter regions in other species in order to understand the variability in their structures and regulation of transcriptional activity. Here, we describe the promoter region of rat 1-sGC gene. Using pituitary tissue, we identified the transcriptional start site and cloned a 3.5 kb upstream promoter of the 1-sGC subunit. We also identified numerous binding sites for transcriptional factors by means of sequential analysis of the 1-sGC promoter region. Partly overlapping cis-elements CCAAT and Sp1 were further identified by the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), whereas the site-directed mutagenesis study indicated that this site regulates the expression of 1-sGC gene in resting cells.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Materials

Medium 199, Ham’s F12K medium, Opti-MEM, penicillin, horse serum, streptomycin, and gentamicin stock solution were purchased from Invitrogen. Fetal bovine serum was obtained from Biosource International (Camarillo, CA, USA). DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA, USA) and the nucleotide sequences are listed in Table 1 and Fig. 6. Antibodies against CCAAT-binding factor (CBF)-A (SC-13045), CCAAT/enhancer-binding protein ß (SC-746), nuclear factor-1 (NF-1) (SC-870), and Sp1 (SC-14027) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other reagents were obtained from Sigma, if not otherwise specified.

View larger version (84K): [in this window] [in a new window]   Figure 6 Delineation of nucleotides required for CBF/A and Sp1 binding. Sixty micrograms nuclear extracts from GH3 cells were incubated with ds-oligonucleotides with biotin labeling on one strand. Bound proteins were pulled down by avidin resin and analyzed by western blot. Upper panel shows the biotin-labeled strand and mutated nucleotides were indicated in bold. WT, wild type; NC, negative control (the biotin-labeled scramble ds-oligonucleotides).

Anterior pituitaries were removed from adult female Sprague–Dawley rats obtained from Tacoma Farms (Germantown, NY, USA) and dispersed as described previously (Kostic et al. 2002). Dispersed cells were cultured for 24 h in medium 199 containing Earle’s salts, sodium bicarbonate, 10% horse serum, and penicillin (100 U/ml) and streptomycin (100 µg/ml). Lacto-somatotroph GH₃ immortalized cells and MH1C1 liver cells were maintained in Ham’s F12K medium supplemented with 1.5 g/l sodium bicarbonate, 15% heat-inactivated horse serum, 2.5% fetal bovine serum, and gentamicin (100 µg/ml).

Identification of the transcription start site of 1-sGC gene

Total RNA was extracted from normal anterior pituitary cells using Trizol Reagent (Invitrogen) and mRNA was enriched by PolyATtract mRNA Isolation Systems (Promega). Identification of the transcription start site of 1-sGC gene was performed using the SMART RACE kit (Clontech) with minor modifications. Primers sGCaRT, RaceOuter, and RaceInner (Table 1) were designed according to the mRNA sequence of 1-sGC deposited in Genbank (BC085746 [GenBank] ). Primers BD SMART II A oligo, Universal Primer A Mix, and Nested Universal Primer A were provided in the kit. The sGC first strand cDNA was synthesized by the gene-specific primer sGCaRT and BD SMART II A oligo. Then PCR was conducted with the Universal Primer A Mix and the gene-specific primer RaceOuter. Nested PCR was conducted with the Nested Universal Primer A and the gene-specific primer RaceInner. The PCR product was subcloned by TOPO TA cloning kit (Invitrogen). Plasmid DNA was prepared by QIAprep Spin Miniprep kit (Qiagen) and sequenced by Veritas (Rockville, MD, USA). The distance between transcription start site and start codon was calculated based on cDNA sequence (Lorens et al. 1993, Okita et al. 2003, Salgado et al. 2004). The boundary of exons I and II was defined by aligning cDNA sequence of 1-sGC with the genomic DNA in the corresponding region retrieved from rat genome reference database (http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html).

Cloning the 5'-promoter of 1-sGC gene and generation of luciferase deletion constructs

Genomic DNA was extracted from rat anterior pituitaries by Wizard Genomic DNA purification kit (Promega). To amplify the promoter, PCR was conducted using genomic DNA as template and Pt(156)R and Pt(–3K) as primers (Table 1). The downstream primer Pt(156)R was designed according to the SMART RACE result and incorporates an XhoI site. The upstream primer Pt(–3K) was designed according to the genomic DNA sequence flanking 1-sGC gene and incorporates a KpnI site. The PCR product was digested by XhoI and KpnI sequentially, and subcloned into the XhoI/KpnI site of pGL3.Basic. The obtained promoter clone was termed pGL3.(–3K). Based on the pGL3.(–3K) sequence, primers Pt(–2K), Pt(–875), Pt(–719), Pt(–599), Pt(–516), Pt(–278), Pt(–165), and Pt(–49) were designed to prepare deletion constructs pGL3.(–2K), pGL3.(–875), pGL3.(–719), pGL3.(–599), pGL3. (–516), pGL3.(–278), pGL3.(–165), and pGL3. (–49) respectively (Table 1). In combination with Pt(156)R, these primers were able to amplify decreased length of 1-sGC promoter. Identities of these promoter constructs were confirmed by sequencing.

Reverse transcription-PCR

Total RNAs were extracted from the 19th and 40th passages of GH₃ cells and the 55th passage of MH1C1 cells using Trizol Reagent (Invitrogen). One microgram RNA was treated with amplification grade DNase I and primed with oligo(dT)₂₀ primer to synthesize cDNA using SuperScript III first-strand synthesis supermix (Invitrogen). In mocking experiments, reverse transcription was conducted in the absence of SuperScript III. Fifty microliters of the PCR mixtures contained 1 µl cDNA, 1 µl of each primer (10 µM), 3 µl H₂O, and 45 µl Platinum blue PCR SuperMix (Invitrogen). PCR was initiated with a denature step at 94 °C for 30 s, followed with 35 amplification cycles consisted of denature at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. PCR products were stored at 4 °C until use.

Transient transfection and dual-luciferase assays

One day before transfection, GH₃ and MH1C1 cells were plated in 96-well plates with a density of 0.04 million cells per well. Then 0.19 µg constructs carrying 5'-promoter of 1-sGC gene and firefly luciferase were mixed with 0.01 µg renilla luciferase construct pRL. CMV (Promega). The DNA mixture was combined with 0.6 µl lipofectamine 2000 (Invitrogen) in 50 µl OptiMEM medium according to the manufacturer’s instructions. In these studies, pRL.CMV was used as an internal control to monitor the transfection efficiency between experimental groups. Transfection mixture with lipofectamine 2000 was incubated for 20 min at room temperature and added to GH₃ cells in 100 µl OptiMEM medium. After 6-h incubation, the transfection medium was replaced with Ham’s F12K medium supplemented with 1.5 g/l sodium bicarbonate, 15% heat-inactivated horse serum, 2.5% fetal bovine serum, and gentamicin (100 µg/ml). Following 48-h incubation, medium was aspirated and cells were incubated in Ham’s F12K medium supplemented with 1.5 g/l sodium bicarbonate and 0.1% BSA for an additional 24 h. The measurement of luciferase activity was done using the Dual-Luciferase Reporter Assay System (Promega). Transfected cells were washed in 1x PBS twice and then dissolved in 5 µl passive lysis buffer (Promega). For measuring the firefly luciferase activity, 20 µl cell lysate were mixed with 50 µl Luciferase Assay Reagent II and then 50 µl Stop and Glo Reagent (Promega) were added. Luminescence signal was measured with a 5 s delay for a period of 10 s in a Mithras LB 940 mutimode reader (Berthold Technologies, Bad Wildbad, Germany).

Electrophoretic mobility shift assay

Nuclear proteins were extracted from dispersed normal and GH₃ pituitary cells using a NucBuster Protein Extraction Kit (Novagen, Madison, WI, USA). EMSA was conducted using a DIG gel shift kit (Roche Applied Science). Briefly, equal molar of CCAAT/Sp1UP and CCAAT/Sp1DN, or MutCCAAT/Sp1UP and MutCC-AAT/Sp1DN were mixed and incubated at 95 °C for 10 min. After slowly cooling down to room temperature, double-stranded (ds) oligonucleotides formed by annealing of the respective primers were diluted with TEN buffer (10 mM Tris–Cl, 1 mM EDTA, and 0.1 M NaCl (pH 8.0)) to a final concentration of 3.85 pmol/µl. Labeling reaction was done in a final volume of 20 µl containing 1 µl ds-oligonucleotides, 9 µl water, 4 µl 5x labeling buffer, 4 µl CoCl₂ solution, 1 µl DIG-ddUTP, and 1 µl terminal deoxynucleotide transferase. The reaction mixture was incubated at 37 °C for 15 min and chilled on ice immediately. Labeling reaction was stopped by 2 µl EDTA (2 mM). The labeled ds-oligonucleotides were diluted to 0.155 pmol/µl by adding 3 µl double distilled water. For gel shift analysis, a small aliquot was further diluted to 15.5 fmol/µl. In the gel shift assay, 8 µg nuclear extract from GH₃ cells or normal pituitary cells were incubated with 4 µl binding buffer (5 mM EDTA, 50 mM (NH₄)₂SO₄, 5 mM dithiothreitol (DTT), 1% Tween 20 (v/v), 150 mM KCl, 100 mM Hepes (pH 7.6)), 1 µl poly(dI-dC), and 1 µl poly-L-lysine. Double distilled water was added to a final reaction volume of 18 µl. In the competition and supershift assays, we added unlabeled ds-oligonucleotides and antibodies in the reaction mixture respectively. After 5-min incubation at room temperature, 2 µl DIG-labeled ds-oligonucleotides were added into the reaction mixture and incubated for another 30 min. After adding 5 µl loading dye, the reaction mixture was size-fractionated in a 6% DNA retardation gel (Invitrogen) in 0.5x TBE buffer and transblotted onto a positively charged nylon membrane for 2 h using an XCell II Blot Module (Invitrogen). The membrane was then u.v.-crosslinked using a Stratalinker 2400 (Stratagene, La Jolla, CA, USA), rinsed in washing buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20 (pH 7.5)) for 5 min, and incubated for 30 min in blocking solution (1% blocking reagent in maleic acid buffer). Following incubation with anti-DIG-AP (1:10 000) in blocking solution for 30 min, the membrane was washed 2 x 15 min in washing buffer and equilibrated in detection buffer (0.1 M Tris–HCl, 0.1 M NaCl (pH 9.5)) for 5 min. Chemiluminescence signals were detected with CSPD as the substrate in X-ray film.

DNA affinity precipitation assay

Ds-probes were generated by annealing biotin-labeled upstream oligonucleotides (Fig. 6) with the respective reverse-complemented oligonucleotides. Binding mixtures contained 10 µl ds-probes (10 µM), 60 µg nuclear protein extracted from GH₃ cells, 80 µl of 5x binding buffer (5 mM EDTA, 50 mM (NH₄)₂SO₄, 5 mM DTT, 1% Tween 20, 150 mM KCl, and 100 mM Hepes (pH 7.6)), and water to 400 µl. After incubation at 4 °C for 1 h, 25 µl Tetralink tetrameric avidin resin (Promega) was added to capture biotin-labeled DNA–protein complex. Resins were washed in 1x binding buffer for five times and proteins were eluted in 25 µl Novex Tris–glycine SDS sample buffer (2x; Invitrogen). Eluted proteins were analyzed by Western blot.

Site-directed mutagenesis

The construct pGL3.(–516) was subjected to site-directed mutagenesis by the QuikChange Site-Directed kit (Stratagene) and high fidelity PfuTurbo DNA polymerase (Stratagene). Reaction mixture was composed of 5 µl of 10x reaction buffer, 20 ng DNA template, 1 µl of 10 mM dNTPs, 1 µl PfuTurbo polymerase, and 140 ng PCR primers in a final volume of 50 µl. The reaction was initiated with a denaturing step at 95 °C for 30 s, followed by PCR amplification with denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and extension at 68 °C for 6 min for a total of 16 cycles. Subsequently, the reaction mixture was cooled on ice and 1 µl DpnI was added to digest the original DNA template for 2 h at 37 °C. The PCR products were then precipitated with ethanol and used for transformation in XL1-Blue Supercompetent cells. Mutation was confirmed by DNA sequencing.

Chromatin immunoprecipitation assay

This assay was performed using ChIPs assay kit from Upstate (Waltham, MA, USA). Dispersed rat anterior pituitary normal cells and GH₃ cells were cross-linked with 1% formaldehyde and suspended in lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris–HCl (pH 8.1)). Lysates were then sonicated to shear DNA and diluted tenfold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, and 16.7 mM Tris–HCl (pH 8.1)). The resultant chromatin was immunocleared with salmon sperm DNA/protein A agarose. Subsequently, 5 µl antibody against CBF/A (SC-13045) or Sp1(SC-14027) was incubated overnight with the chromatin at 4 °C. The antibody/chromatin complex was pulled down by incubating with salmon sperm DNA/protein A agarose for 1 h. Agarose was precipitated by gentle centrifugation and washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), and 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), and 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% IGELPAL-CA630, 1% sodium deoxycholic acid, 1 mM EDTA, and 10 mM Tris–HCl (pH 8.1)), and 1x TE. Histone complex was eluted in elution buffer (1% SDS and 0.1 M NaHCO₃) and heated at 65 °C for 6 h to reverse formaldehyde cross-linking. Samples were then treated with protease K, followed by phenol extraction and ethanol precipitation. The DNA pellet was dissolved in 50 µl Tris–EDTA buffer, and then 1 µl DNA was used for PCR with 36 cycles of amplification. PCR amplification of soluble chromatin prior to immunoprecipitation was used as an input control.

Data transformation and statistical analysis

Promoter activities were expressed in firefly luciferase activity normalized against renilla l uciferase activity in transfected GH₃ and MH1C1 cells. The normalization was conducted to minimize the potential problems of variations in transfection efficiency among groups. Data presented were expressed as mean ± S.E.M. and analyzed using Student’s t-test. The differences were considered significant at P<0.05.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Identification of 1-sGC transcription start site

Primer extension is a common method used to map the transcription start site. However, premature termination and intrinsic terminal transferase activity of reverse transcriptase make this method ineffective in some experiments (Gong & Ge 2000). Since SMART RACE can reduce these problems (Gong & Ge 2000), it was used in this study to identify the transcription start site of 1-sGC gene. Pituitary mRNA was transcribed to cDNA with a gene-specific primer sGCaRT in the presence of BD SMART II A oligonucleotides and 5'-CDS primer. Amplification of cDNA with primers Universal Primer A Mix and RaceOuter produced a specific band with a size about 600 bp. Using primers Nested Universal Primer A and RaceInner, a specific band with a size around 400 bp was obtained (data not shown) and subcloned into the TOPO TA cloning vector. Sequencing of five clones revealed that they possessed different 5'-ends (data not shown). The most upstream 5'-end, located at 287 bp upstream of the start codon, was assigned as position + 1 (Fig. 1). The respective clone contains the longest 5'-untranslated region (UTR) among other rat 1-sGC cDNA sequences (BC085746 [GenBank] , NM_017090 [GenBank] ) and gene sequence (AF327645 [GenBank] ) deposited in the Genbank. Alignment between the cDNA sequence and the genome sequence of 1-sGC gene indicated, it has a 183 bp exon I and a 356 bp exon II. In exon I, two nucleotides were different between cDNA sequence in this report and that in rat genome, possibly reflecting the presence of single nucleotide polymorphism (Fig. 1). The start codon is located in the exon II (Fig. 1).

View larger version (45K): [in this window] [in a new window]   Figure 1 Nucleotide sequence of the exons I and II of 1-sGC gene from rat anterior pituitary. The identified transcriptional start site is underlined and denoted as + 1. Nucleotides in exon I that mismatch with rat genome sequence were shown in italic bold. The position of the transcriptional start site previously reported (BC085746) is indicated by an arrow and the translational start codon in exon II is shown in capital bold.

Based on the genomic sequence in the rat genome reference database and the identified transcription start site, primers Pt(–3K) and Pt(156)R were designed to amplify the 5'-promoter of 1-sGC gene. These primers are located at –3529 to –3502 and 137 to 156 respectively. The obtained 3.067 kb fragment was cloned into pGL3.Basic vector and sequenced (Gen-Bank accession: DQ141223 [GenBank] ). Sequence analysis of the promoter fragment revealed the presence of several putative cis-elements, including Sp1, CCAAT, Wilms’ tumor, activator protein-1, and retinoid X-receptor (Fig. 2). Interestingly, no TATA box was found in the promoter. To identify region(s) critical for the transcription activity of 1-sGC, promoter constructs with different 5'-ends were prepared and the transfection was conducted in GH₃ and MH1C1 cells. GH₃ cells were derived from rat pituitary, express 1-sGC mRNA, and thus represent a good experimental cell model (Kostic et al. 2004); whereas MH1C1 is a valuable control model because of the lack of 1-sGC mRNA expression.

View larger version (94K): [in this window] [in a new window]   Figure 2 Nucleotide sequence of the promoter region of rat 1-sGC gene. Putative cis-elements are shown in bold, and annotated under the sequence. Non-mammalian originated elements were manually removed. The solid arrow indicates exon I.

View larger version (35K): [in this window] [in a new window]   Figure 3 Promoter activity of 1-sGC deletion constructs. (A) Expression of 1-sGC mRNAs in GH3 immortalized pituitary and MH1C1 liver cells. PCRs were conducted using 1-sGC specific primers and ß-actin specific primers in the absence (–) or presence (+ ) of reverse transcription (RT). (B) Transient transfection analysis of deletion constructs. GH3 and MH1C1 cells were cotransfected with renilla luciferase construct pRL-CMV and firefly luciferase reporter constructs containing different size promoter fragments. Promoter activities for each construct were presented as a ratio of relative light units between firefly luciferase and renilla luciferase. Results are means ± S.E.M. (n = 8).

Characterization of CCAAT/Sp1 transcription-binding sites

The most proximal –49 region contains the putative CCAAT/Sp1 cis-element (Fig. 2). To investigate the role of CCAAT/Sp1 site in basal promoter activity of 1-sGC, DNA–protein interaction was studied by EMSA. The synthesized oligonucleotides, CCAAT/Sp1UP and CCAAT/Sp1DN, located between –50 and –18 (Table 1), encompassed the CCAAT/Sp1 element completely. The annealed ds-oligonucleotide was named dsCCAAT/Sp1. Using DIG-labeled dsCCAAT/Sp1 and nuclear extract from GH₃ cells, EMSA revealed the presence of specific band in addition to the free probe (Fig. 4A, lane 1). The specific band was eliminated by the competition of unlabeled dsCCAAT/Sp1 in a dose-dependent manner (Fig. 4A, lanes 2–4). Competition assay was also conducted using mutated dsCCAAT/Sp1. Analysis done by a transcription element search system (http://www.cbil.upenn.edu/tess/) indicated that this mutation renders the dsCCAAT/Sp1 loss of pattern of CCAAT and Sp1 elements. In this case, the competition with mutated dsCCAAT/Sp1 did not change the signal of the specific band (Fig. 4A, lanes 5–7). When nuclear proteins from normal anterior pituitary cells were used, one strong shifted band was observed (Fig. 4B, lane 1). This band was reduced by unlabeled dsCCAAT/Sp1 in a dose-dependent manner (Fig. 4B, lanes 2–4), but was not affected when the core sequence of dsCCAAT/Sp1 was mutated (Fig. 4B, lanes 5–7).

View larger version (67K): [in this window] [in a new window]   Figure 4 Analysis of protein complexes formed between putative CCAAT/Sp1 oligonucleotides and nuclear extracts from (A) GH3 and (B) normal pituitary cells. Eight micrograms nuclear extracts were incubated with DIG-labeled putative CCAAT/Sp1 oligonucleotides in the absence of competitor (lane 1) and in the presence of different folds of unlabeled CCAAT/Sp1 oligonucleotides (lanes 2–4) or unlabeled mutated CCAAT/Sp1 oligonucleotides (lanes 5–7). Horizontal arrows indicate specific binding.

To identify the transcriptional factors bound to the CCAAT/Sp1 site, the supershift analysis was conducted. In this experiment, antibodies against CBF/A, CCAAT/enhancer-binding protein, NF-1, and Sp1 were used. Three of these transcriptional factors, CBF/A, CCAAT/enhancer-binding protein, and NF-1, can bind with CCAAT element (Mantovani 1999). Using nuclear extract from GH₃ cells, antibody against CBF/A reduced the specific signal of shifted bands, indicating the presence CBF/A in the CCAAT/Sp1–nuclear protein complex (Fig. 5A, lane CBF/A). On the other hand, when antibody against Sp1 was used, a strong supershift band was observed (Fig. 5A, lane Sp1). Antibodies against CCAAT/enhancer-binding protein and NF-1 did not change the shifted bands (Fig. 5A, lanes C/EBP and NF-1). When labeled mutated dsCCAAT/Sp1 was used in EMSA, no shifted band was observed, indicating that the binding sites for Sp1 and CBF/A were overlapping (Fig. 5A, lane 6). Similar results were observed when nuclear extracts from the normal cells from rat pituitary were used in the assay (Fig. 5B). Furthermore, antibodies against CBF/A and Sp1 have additive effects on the supershift result (Fig. 5B, lane CBF/A+ Sp1), indicating CBF/A and Sp1 can independently bind to the CCAAT/Sp1 element.

View larger version (60K): [in this window] [in a new window]   Figure 5 Identification of transcription factors binding with CCAAT/Sp1 oligonucleotides in (A) GH3 and (B) normal pituitary cells. Eight micrograms nuclear extracts were incubated with DIG-labeled putative CCAAT/Sp1 oligonucleotides in the absence of antibody (laneControl) orinthepresenceof different antibodies (lanes 2–5 in panel A and lanes 2–6 in panel B). Shifted bands are indicated with arrows. To confirm that binding sites for CBF/A and Sp1 are overlapping, 8 µg nuclear extracts were also incubated with DIG-labeled mutated CCAAT/Sp1 oligonucleotides (lane 6 in panel A). C/EBP, CCAAT/enhancer-binding protein; NF-1, nuclear factor-1.

To precisely delineate nucleotides required for CBF/A and Sp1 binding, DNA affinity precipitation assay was conducted using a series of mutated ds-oligonucleotides, termed mutants 1–8 (Fig. 6, upper panel). The upstream oligonucleotides of wild type has the same sequence as primer CCAAT/Sp1UP, located at –50 to –18, and mutant 5 in this experiment has the same sequence as the above-mentioned primer MutCCAAT/ Sp1UP. When Sp1 antibody was used, signals in mutants 5–8 were weaker than that in the wild type (Fig. 6, panel Sp1), indicating that the region between –35 and –26 was important for Sp1 binding. On the other hand, when CBF/A antibody was used, signals in mutants 3–6 were much weaker than that in the wild type (Fig. 6, panel CBF/A), indicating that the region between –39 and –30 was important for CBF binding. Clearly, the CCAAT and Sp1 cis-elements are partly overlapping because mutation in the region between –35 and –30 reduced the affinity for both CBF/A and Sp1 binding (Fig. 6).

Inhibition of transcription by mutation of CCAAT/Sp1 element

To clarify the potential role of CCAAT and Sp1 cis-element in basal promoter activity of 1-sGC gene, mutagenesis analysis was conducted. The CCAAT and Sp1 sites in construct pGL3.(–516) was mutated to pGL3.(–516Mut3), pGL3.(–516Mut5), pGL3. (–516Mut7) according to the sequences of mutant 3, 5, and 7 respectively. Mutant 3 has a reduced affinity to CBF/A but not to Sp1, whereas mutant 7 has a reduced affinity to Sp1 but not to CBF/A (Fig. 6). Mutant 5 has the weakest affinity to both CBF/A and Sp1 (Fig. 6). Comparing with the promoter activity of pGH3.(–516), all three mutant constructs had a weaker promoter activity, demonstrating that both CCAAT and Sp1 cis-elements were stimulatory to the basal activity of 1-sGC promoter (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Figure 7 Effects of CCAAT/Sp1 site mutation on the promoter activity of 1-sGC in GH3 cells. Construct pGL3-sGC(–516) was used to prepare mutation constructs pGL3-sGC(Mutant 3), pGL3-sGC(Mutant 5), and pGL3-sGC(Mutant 7). GH3 cells were transfected with renilla luciferase construct pRL-CMV and different firefly luciferase reporter constructs. Promoter activity for each construct was presented as the ratio of relative light units between firefly luciferase and renilla luciferase. Results are means ± S.E.M. (n = 8) and asterisks indicate significant difference when compared with the wild-type pGL3-sGC(–516; P<0.05).

Recruitment of Sp1 and CBF/A to the promoter of 1-sGC gene in vivo

To investigate whether both Sp1 and CBF/A can be recruited to the CCAAT/Sp1 element in vivo, ChIP was conducted in GH₃ and normal pituitary cells. Four primer pairs were designed to amplify different regions of 1-sGC promoter. Primers IPt(–3K)UP and IPt(–3K)DN were used to amplify a 115 bp fragment located 3 kb upstream of CCAAT/Sp1 site, and primers IPt(–1K)UP and IPt(–1K)DN were used to amplify a 138 bp fragment located 1 kb upstream of CCAAT/Sp1 site (Table 1). On the other hand, primer pairs IPt(–165)UP and IPt(–18)DN, IPt(–63)UP, and IPt(59)DN cover the CCAAT/Sp1 region (Fig. 8, lower panel). PCR products were only analyzed in a qualitative manner. In GH₃ chromatin, all these primers amplified a specific product with the predicted size using the input DNA as the template (Fig. 8A, Input). When the chromatin was immunoprecipitated by antibody against the CBF/A, no PCR product was found using first primer pairs (Fig. 8A, CBF/A, lane 1), and only a weak signal appeared using second primer pairs (Fig. 8A, CBF/A, lane 2). In agreement with the presence of CBF/A binding in the region between –41 and –32, third and fourth primer pairs generated specific PCR products (Fig. 8A, CBF/A, lanes 3 and 4). When the chromatin was immunoprecipitated with antibody against Sp1, a strong PCR amplification was also observed using primer pairs covering the CCAAT/ Sp1 element (Fig. 8A, Sp1, lanes 3 and 4). No or weak PCR amplification was detected using primer pairs distal to the CCAAT/Sp1 element (Fig. 8A, Sp1, lanes 1 and 2). The PCR amplification was specific because no amplicon was observed when chromatin was precipitated with immunoglobulin G as the negative control (Fig. 8A, IgG). Similar results were observed in normal pituitary cells (Fig. 8B).

View larger version (59K): [in this window] [in a new window]   Figure 8 Recruitment of CBF/A and Sp1 transcription factors to the 1-sGC promoter in (A) GH3 and (B) normal pituitary cells. Soluble chromatin from GH3 cells or normal cells was precipitated with antibodies against CBF/A, Sp1, or preimmune rabbit immunoglobulin (IgG). The DNA size marker is labeled M. DNA regions analyzed in PCR (lanes 1–4) are schematically represented at the bottom. The predicted overlapping binding site for CBF/A and Sp1 is indicated by vertical dotted line.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

The sequence analysis indicated that the cloned promoter of rat 1-sGC subunit belong to the TATA-less promoters. The promoter of mouse 1-sGC (Vazquez-Padron et al. 2004) and human ß1-sGC (Sharina et al. 2003) subunit genes also lacks a TATA box. In general, the TATA-like element is crucial for the accurate transcription initiation in the protein-coding gene (Smale 1997). Consistently, our results also revealed the presence of different transcription initiation sites among different clones. However, a special scenario exists in medaka fish, where only a single transcriptional start site was identified in different tissues despite the absence of functional TATA-like element in the promoter of 1-sGC (Yamamoto & Suzuki 2002).

Our transient transfection experiments confirmed that the cloned promoter of 1-sGC subunit exhibits low activity in resting cells and identified elements that could contribute to it. The maximal transcription activity was obtained when a 599 bp promoter fragment was inserted in the luciferase construct. In another study, it was shown that the transcriptional activity increased twofold when a 1.6 kb mouse 1-sGC promoter sequence was inserted in the promoterless construct and expressed in NIE-115 cells, whereas the transcriptional activity increased 4.6-fold when a 1.4 kb mouse ß1-sGC promoter sequence was inserted (Sharina et al. 2000). Our deletion analysis demonstrated that a putative inhibitory element might exist in the region between –2 and –3 kb. In this region, several putative cis-elements were identified, which can bind with transcription factors, including the glucocorticoid receptor, hepatocyte NF-1, GATA, retinoid X receptor, and CCAAT/enhancer-binding protein. Notably, glucocorticoid receptor can interact with corepressors, such as nuclear receptor corepressor and silencing mediator of retinoid and thyroid receptors, and reduce gene expression (Wang et al. 2004).

The expression of 1-sGC mRNA is cell specific. In mice, the highest expression level of 1-sGC mRNAs was observed in lung, and the lowest levels in liver, muscle, and ileum (Mergia et al. 2003). In the present study, RT-PCR was unable to detect 1-sGC mRNA in a liver-derived cell line MH1C1. Furthermore, the activities of promoters more proximal to –2 kb in MH1C1 cells were much weaker than that in GH₃ cells, indicating that the lack of 1-sGC expression in MH1C1 cells may partly result from reduced proximal promoter activity. However, the elements and the 3.5 kb region so far reported are not enough to achieve the cell-specific expression of 1-sGC, since the promoter activity of pGL3.(–3K) was similar in different cell lines. We have examined the possible involvement of DNA methylation and histone modifications in transcriptional regulation of 1-sGC mRNA. Treatment with DNA demethylating reagent 5-azacytidine or histone deacetylase inhibitor trichostatin A did not change the 1-sGC mRNA level in GH₃ cells (data not shown), indicating that the epigenetic regulation does not contribute to the expression of 1-sGC mRNA in GH₃ cells.

The coexpression of 1- and ß1-sGC subunits is required to obtain a functional enzyme, implying that the expression of both subunits is coordinated. As a matter of fact, the expression of 1 and ß1 subunits is tightly coregulated (Nedvetsky et al. 2002). However, variable mechanisms may account for coregulation of two subunits in different animal models. In medaka fish, 1- and ß1-sGC genes are 986 bp apart and organized in tandem in the genome. The expression of both subunits is directly coordinated by the 5'-upstream region of 1-sGC subunit (Mikami et al. 1999). This is not the case with mammalian species. According to the National Center for Biotechnology Information database, 1 and ß1 subunits are separated by 60 and 43 kb in mouse and human respectively. In mouse, the extended region comprises 2% of the total chromosomal length (Sharina et al. 2000). In rat, genes for 1- and ß1-sGC subunits have been mapped to chromosome 2 and linked to known quantitative trait locus markers of salt-sensitive hypertension in the Dahl rats (Azam et al. 1998), and two subunits are separated by 19 kb (NCBI data).

The coordination of transcriptional regulation of two subunits in mammals may be achieved by sharing some common transcription factor-binding sites, such as CCAAT box. Proteins that can bind with CCAAT box include CBF, NF-1, CCAAT/enhancer-binding protein, and CCAAT displacement protein (Mantovani 1999). Furthermore, CBF is composed of three subunits, CBF/ A, CBF/B, and CBF/C, which are required for DNA binding. The binding of CBF with CCAAT box can result in the disruption of nucleosome and transcription activation (Coustry et al. 2001). In the promoter of ß1-sGC gene in human, the CCAAT box can bind with CBF and enhance promoter activity (Sharina et al. 2003). In this study, we also identified the CCAAT box in 1-sGC promoter at region between –50 and –18, which can interact with the transcription factor CBF. Moreover, the region between –39 and –30 is most critical for this interaction. Disruption of the CBF–CCAAT interaction resulted in twofold decrease in promoter activity. Therefore, CCAAT box conferred the stimulatory effect on both 1- and ß1-sGC promoter activities. The in vivo binding of CBF with this segment was confirmed by ChIP assay, providing a mechanism to coordinate the gene expression of 1- and ß1-sGC subunits. The binding of CCAAT displacement protein to this site was not examined, because it normally acts as an inhibitory factor (Nishio & Walsh 2004).

Our results also demonstrated that Sp1 is involved in the transcription regulation of 1-sGC subunit gene. In this case, the region between –35 and –26 is important for the DNA–protein interaction. A mutant with reduced affinity to Sp1 exhibited lower promoter activity than wild type. As the founding member of the family of zinc finger transcription factors, Sp1 participates in a wide variety of physiological processes, including the cell-cycle regulation and hormonal activity (Chu & Ferro 2005). In the NO/sGC signaling pathways, Sp1 is important for the basal activity of endothelial (Wu 2002) and neuronal (Saur et al. 2002, Bachir et al. 2003) NO synthases. Moreover, it is also involved in the regulation of human type I protein kinase G gene expression (Sellak et al. 2002). Together with the findings presented here, these observations indicated the importance of Sp1 in the NO/sGC signaling.

The binding sites for CBF and Sp1 are overlapping in the region between –35 and –30, which implies the cooperation between CBF and Sp1 in the transcriptional regulation of 1-sGC subunit gene. In our experiments, mutation of the overlapping region resulted in the highest reduction in the promoter activity. In the promoter of rat fatty acid synthase, an insulin-responsive element (IRE) was composed of a CCAATelement and a Sp1 element with 10 bp in distance. Using IRE as the probe, three kinds of DNA–protein complex were formed: IRE:Sp1, IRE:CBF, and IRE:Sp1:CBF. The cooperation of Sp1 and CBF increased the stability of DNA–protein complex (Roder et al. 1997). Because the distance between CBF and Sp1 seems to be critical for the synergism of CBF and Sp1 (Alimov et al. 2005), their relationship in the control of 1-sGC subunit gene expression remains to be proven.

	Acknowledgements

 
We are thankful to Chon-Hwa Tsai-Morris for helpful suggestions.

	Funding

This research was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	References

Top Abstract Introduction Materials and methods Results Discussion References

 
Alimov AP, Park-Sarge OK, Sarge KD, Malluche HH & Koszewski NJ 2005 Transactivation of the parathyroid hormone promoter by specificity proteins and the nuclear factor Y complex. Endocrinology 146 3409–3416.[Abstract/Free Full Text]

Azam M, Gupta G, Chen W, Wellington S, Warburton D & Danziger RS 1998 Genetic mapping of soluble guanylyl cyclase genes: implications for linkage to blood pressure in the Dahl rat. Hypertension 32 149–154.[Abstract/Free Full Text]

Bachir LK, Garrel G, Lozach A, Laverriere JN & Counis R 2003 The rat pituitary promoter of the neuronal nitric oxide synthase gene contains an Sp1-, LIM homeodomain-dependent enhancer and a distinct bipartite gonadotropin-releasing hormone-responsive region. Endocrinology 144 3995–4007.[Abstract/Free Full Text]

Baltrons MA, Pedraza CE, Heneka MT & Garcia A 2002 Beta-amyloid peptides decrease soluble guanylyl cyclase expression in astroglial cells. Neurobiology of Disease 10 139–149.[CrossRef][ISI][Medline]

Baltrons MA, Pifarre P, Ferrer I, Carot JM & Garcia A 2004 Reduced expression of NO-sensitive guanylyl cyclase in reactive astrocytes of Alzheimer disease, Creutzfeldt-Jakob disease, and multiple sclerosis brains. Neurobiology of Disease 17 462–472.[CrossRef][ISI][Medline]

Bauersachs J, Bouloumie A, Mulsch A, Wiemer G, Fleming I & Busse R 1998 Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression, and in superoxide anion production. Cardiovascular Research 37 772–779.[Abstract/Free Full Text]

Bauersachs J, Bouloumie A, Fraccarollo D, Hu K, Busse R & Ertl G 1999 Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation 100 292–298.

Budworth J, Meillerais S, Charles I & Powell K 1999 Tissue distribution of the human soluble guanylate cyclases. Biochemical and Biophysical Research Communications 263 696–701.[CrossRef][ISI][Medline]

Chu S & Ferro TJ 2005 Sp1: regulation of gene expression by phosphorylation. Gene 348 1–11.[CrossRef][ISI][Medline]

Coustry F, Hu Q, de Crombrugghe B & Maity SN 2001 CBF/NF-Y functions both in nucleosomal disruption and transcription activation of the chromatin-assembled topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in chromatin is dependent on the promoter structure. Journal of Biological Chemistry 276 40621–40630.[Abstract/Free Full Text]

De Frutos S, Saura M, Griera M, Rivero-Vilches FJ, Zaragoza C, Rodriguez-Puyol D & Rodriguez-Puyol M 2005 Differential regulation of soluble guanylyl cyclase expression and signaling by collagens: involvement of integrin-linked kinase. Journal of the American Society of Nephrology 16 2626–2635.[Abstract/Free Full Text]

Garbers DL, Koesling D & Schultz G 1994 Guanylyl cyclase receptors. Molecular Biology of the Cell 5 1–5.[ISI][Medline]

Gong B & Ge R 2000 Using the SMART cDNA system to map the transcription initiation site. Biotechniques 28 846–852.[ISI][Medline]

Hanafy KA, Martin E & Murad F 2004 CCTeta, a novel soluble guanylyl cyclase-interacting protein. Journal of Biological Chemistry 279 46946–46953.[Abstract/Free Full Text]

Humbert P, Niroomand F, Fischer G, Mayer B, Koesling D, Hinsch KD, Gausepohl H, Frank R, Schultz G & Bohme E 1990 Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method. European Journal of Biochemistry 190 273–278.[ISI][Medline]

Kloss S, Bouloumie A & Mulsch A 2000 Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase. Hypertension 35 43–47.[Abstract/Free Full Text]

Kloss S, Srivastava R & Mulsch A 2004 Down-regulation of soluble guanylyl cyclase expression by cyclic AMP is mediated by mRNA-stabilizing protein HuR. Molecular Pharmacology 65 1440–1451.[Abstract/Free Full Text]

Kostic TS, Tomic M, Andric SA & Stojilkovic SS 2002 Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells. Journal of Biological Chemistry 277 16412–16418.[Abstract/Free Full Text]

Kostic TS, Andric SA & Stojilkovic SS 2004 Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells. Molecular Endocrinology 18 458–470.[Abstract/Free Full Text]

Kraft PJ, Haynes-Johnson D, Bhattacharjee S, Lundeen SG & Qiu Y 2004 Altered activities of cyclic nucleotide phosphodiesterases and soluble guanylyl cyclase in cultured RFL-6 cells. International Journal of Biochemistry and Cell Biology 36 2086–2095.[CrossRef][ISI][Medline]

Krumenacker JS, Hyder SM & Murad F 2001 Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus. PNAS 98 717–722.[Abstract/Free Full Text]

Lorens JB, Nerland AH, Aasland R, Lossius I & Male R 1993 Expression of growth hormone genes in Atlantic salmon. Journal of Molecular Endocrinology 11 167–179.[Abstract]

Mantovani R 1999 The molecular biology of the CCAAT-binding factor NF-Y. Gene 239 15–27.[CrossRef][ISI][Medline]

Mergia E, Russwurm M, Zoidl G & Koesling D 2003 Major occurrence of the new alpha2beta1 isoform of NO-sensitive guanylyl cyclase in brain. Cellular Signalling 15 189–195.[CrossRef][ISI][Medline]

Mikami T, Kusakabe T & Suzuki N 1999 Tandem organization of medaka fish soluble guanylyl cyclase alpha1 and beta1 subunit genes. Implications for coordinated transcription of two subunit genes. Journal of Biological Chemistry 274 18567–18573.[Abstract/Free Full Text]

Nedvetsky PI, Kleinschnitz C & Schmidt HH 2002 Regional distribution of protein and activity of the nitric oxide receptor, soluble guanylyl cyclase, in rat brain suggests multiple mechanisms of regulation. Brain Research 950 148–154.[CrossRef][ISI][Medline]

Nishio H & Walsh MJ 2004 CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription. PNAS 101 11257–11262.[Abstract/Free Full Text]

Okita K, Nobuhisa I, Takizawa M, Ueno M, Kimura N & Taga T 2003 Genomic organization and characterization of the mouse ELYS gene. Biochemical and Biophysical Research Communications 305 327–332.[CrossRef][ISI][Medline]

Pedraza CE, Baltrons MA, Heneka MT & Garcia A 2003 Interleukin-1 beta and lipopolysaccharide decrease soluble guanylyl cyclase in brain cells: NO-independent destabilization of protein and NO-dependent decrease of mRNA. Journal of Neuroimmunology 144 80–90.[CrossRef][ISI][Medline]

Pyriochou A & Papapetropoulos A 2005 Soluble guanylyl cyclase: more secrets revealed. Cellular Signalling 17 407–413.[CrossRef][ISI][Medline]

Roder KH, Wolf SS, Beck KF & Schweizer M 1997 Cooperative binding of NF-Y and Sp1 at the DNase I-hypersensitive site, fatty acid synthase insulin-responsive element 1, located at –500 in the rat fatty acid synthase promoter. Journal of Biological Chemistry 272 21616–21624.[Abstract/Free Full Text]

Salgado MC, Meton I, Egea M & Baanante IV 2004 Transcriptional regulation of glucose-6-phosphatase catalytic subunit promoter by insulin and glucose in the carnivorous fish, Sparus aurata. Journal of Molecular Endocrinology 33 783–795.[CrossRef]

Saur D, Seidler B, Paehge H, Schusdziarra V & Allescher HD 2002 Complex regulation of human neuronal nitric-oxide synthase exon 1c gene transcription. Essential role of Sp and ZNF family members of transcription factors. Journal of Biological Chemistry 277 25798–25814.[Abstract/Free Full Text]

Sellak H, Yang X, Cao X, Cornwell T, Soff GA & Lincoln T 2002 Sp1 transcription factor as a molecular target for nitric oxide- and cyclic nucleotide-mediated suppression of cGMP-dependent protein kinase-Ialpha expression in vascular smooth muscle cells. Circulation Research 90 405–412.[Abstract/Free Full Text]

Sharina IG, Krumenacker JS, Martin E & Murad F 2000 Genomic organization of alpha1 and beta1 subunits of the mammalian soluble guanylyl cyclase genes. PNAS 97 10878–10883.[Abstract/Free Full Text]

Sharina IG, Martin E, Thomas A, Uray KL & Murad F 2003 CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line. PNAS 100 11523–11528.[Abstract/Free Full Text]

Smale ST 1997 Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochimica et Biophysica Acta 1351 73–88.[Medline]

Taylor TA, Pollock JS & Pollock DM 2003 Down-regulation of soluble guanylyl cyclase in the inner medulla of DOCA-salt hypertensive rats. Vascular Pharmacology 40 155–160.[CrossRef][ISI][Medline]

Vazquez-Padron RI, Pham SM, Pang M, Li S & Aitouche A 2004 Molecular dissection of mouse soluble guanylyl cyclase alpha1 promoter. Biochemical and Biophysical Research Communications 314 208–214.[CrossRef][ISI][Medline]

Wang Q, Blackford JA Jr, Song LN, Huang Y, Cho S & Simons SS Jr 2004 Equilibrium interactions of corepressors and coactivators with agonist and antagonist complexes of glucocorticoid receptors. Molecular Endocrinology 18 1376–1395.[Abstract/Free Full Text]

Wu KK 2002 Regulation of endothelial nitric oxide synthase activity and gene expression. Annals of the New York Academy of Sciences 962 122–130.[Abstract/Free Full Text]

Yamamoto T & Suzuki N 2002 Promoter activity of the 5'-flanking regions of medaka fish soluble guanylate cyclase alpha1 and beta1 subunit genes. Biochemical Journal 361 337–345.[CrossRef][ISI][Medline]

Zabel U, Weeger M, La M & Schmidt HH 1998 Human soluble guanylate cyclase: functional expression and revised isoenzyme family. Biochemical Journal 335 51–57.

Received 29 August 2006
Accepted 12 September 2006

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal