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The role of the proximal promoter GC-box in regulating basal and cAMP-dependent GTP Cyclohydrolase I gene transcription was investigated using a variety of cell lines and techniques. These studies show that the GC-box is composed of a triad of cis-elements that in vitro bind specificity proteins Sp1 and Sp3. Sp1 and Sp3 were found associated with the native proximal promoter in PC12 cells but were not recruited to the promoter during cAMP-dependent transcription. Studies using Drosophila SL2 cells showed that Sp3 occupies two sites within the GC-box and enhances transcription when acting alone and synergistically when combined with nuclear factor-Y (NF-Y) and CCAAT/Enhancer-Binding Protein (C/EBP)β, cognate binding proteins for the adjacent cAMP response element (CRE) and CCAAT-box cAMP response elements. In contrast, Sp1 bound only one site within the GC-box and did not enhance transcription unless combined with NF-Y and C/EBPβ. Studies in SL2 cells also showed that Sp1 and Sp3 do not co-occupy the GC-box, and accordingly Sp1 competes for Sp3 binding to repress Sp3-dependent transcription. In PC12 cells, complete mutation of the GC-box reduced basal but not cAMP-dependent transcription, resulting in an overall increase in the cAMP response and demonstrating that formation of this enhanceosome does not require Sp1 or Sp3. Experiments in which the GC-box was replaced with a Gal4 element and the promoter challenged with Gal4 fusion proteins support this conclusion and a role for Sp3 in maintaining high levels of basal transcription in PC12 cells. Equivalent amounts of Sp1 and Sp3 were found associated with the native proximal promoter in PC12 and Rat2 cells, which differ 10-fold in basal transcription. Similar levels of methylation of CpG dinucleotides located within the GC-box were also observed in these two cells lines. These results suggest that Sp1 and Sp3 bound to the GC-box might help to preserve an open chromatin configuration at the proximal promoter in cells which constitutively express low levels of GTP Cyclohydrolase I.
GTP cyclohydrolase I (GCH1; EC 184.108.40.206) is the first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis, the essential cofactor for monoamine and nitric oxide production and the detoxification of L-phenylalanine (Thony et al. 2000). GCH1 transcription is dynamic and can be enhanced by the second messenger cAMP in only a handful of cell types, including adrenal chromaffin cells (Abou-Donia et al. 1986), midbrain dopamine neurons (Zhu et al. 1994; Bauer et al. 2002), mesangial cells (Pluss et al. 1996), and PC12 cells (Anastasiadis et al. 1998; Kapatos et al. 2000). While this specificity implies novel signaling mechanisms, the effect of cAMP on GCH1 gene transcription is mediated entirely through the ubiquitous protein kinase A (Kapatos et al. 2007) which suggests that cAMP responsiveness is determined by the cellular complement of transcription factors made available to the GCH1 gene promoter.
Studies of the rat and human GCH1 promoters have identified the first 140 bp upstream from the transcription start sites as the minimal sequence necessary for cell type-specific cAMP-dependent transcription (Kapatos et al. 2000; Hirayama et al. 2001). Within this sequence lie a GC-box, a CRE and a CCAAT-box that are evolutionarily conserved. Both the CRE and the CCAAT-box are required for maximum basal and cAMP-dependent transcription (Kapatos et al. 2000; Kapatos et al. 2007). While the CRE binds members of the basic leucine zipper family of transcription factors, including cAMP-response element binding protein (CREB), ATF-2, c-Jun and C/EBPβ, the CCAAT-box binds the obligate heterotrimeric protein NF-Y (Kapatos et al. 2000; Hirayama et al. 2001; Sarraj et al. 2005; Wu et al. 2004; Kapatos et al. 2007). A recent examination of the endogenous GCH1 gene functioning within intact PC12 cells has confirmed these observations and also showed that cAMP treatment causes the recruitment of C/EBPβ and NF-Y along with Pol II to the proximal promoter (Kapatos et al. 2007).
Previous research using in vitro footprinting and PC12 cell nuclear extracts concluded that the GCH1 proximal promoter GC-box binds members of the stimulatory protein-1 (Sp1) family of transcription factors (Kapatos et al. 2000). This same study showed that the GC-box reduces cAMP-dependent transcription conferred by the CRE and CCAAT-box cAMP-response elements on a heterologous promoter, suggesting an inhibitory role for Sp-proteins in GCH1 transcription. Sp1, Sp3, and Sp4 proteins each recognize the identical GC-rich cis-element with the same affinity through a highly conserved C-terminal zinc-finger DNA binding domain (Suske 1999; Bouwman and Philipsen 2002), yet differentially regulate gene expression through subtle differences in N-terminal activation domains (Hagen et al. 1995; Ahlgren et al. 1999). Sp1 and Sp3 are both substrates for protein kinase A and phosphorylation is reported to enhance DNA binding and trans-activation (Rohlff et al. 1997; Ge et al. 2001). Sp-proteins typically affect transcription through interactions with components of the general transcriptional machinery (Smale et al. 1990; Hoey et al. 1993; Gill et al. 1994; Saluja et al. 1998) as well as through interactions with co-activators (Ryu et al. 1999). Sp-proteins also interact with proteins known to be associated with the GCH1 promoter, including C/EBPβ (Lee et al. 1997), NF-Y (Roder et al. 1999; Borestrom et al. 2003) and ring finger protein 4 (Poukka et al. 2000).
We now present data in support of a triad model of the rat GCH1 proximal promoter GC-box in which three distinct cis-elements have the potential to bind Sp1 and Sp3 proteins. We also show that while endogenous Sp1 and Sp3 are associated with the native GCH1 proximal promoter and are important for maintaining basal transcription neither protein is recruited to the native promoter in response to cAMP or absolutely required for the cAMP response. Finally, we find no relationship between the basal rate of GCH1 transcription, the amounts of Sp1 and Sp3 protein associated with the proximal promoter and levels of CpG dinucleotide methylation within the GC-box, suggesting that Sp1 and Sp3 might also have a role in preserving an open chromatin configuration at the proximal promoter.
The construction and characterization of wild-type and mutated forms of the pGL3-based luciferase reporter construct p0.27GCH-GL3 has been reported previously (Kapatos et al. 2000). The Renilla reporter plasmid pRL-Null and the 5XGal4-luciferase construct pFR-luc were purchased (Promega, Madison WI, USA). Plasmids pPac-0, pPac-Sp1 and pPac-Sp3 were a gift from Dr Robert Tjian, UCLA, CA, USA. Plasmids pPac-NF-YA, pPac-NF-YB and pPac-NF-YC were gifts from Dr. Timothy F. Osborne, University of California, Los Angeles, USA. Gal4-Sp1(amino acids 1–603) and Gal4-Sp3(amino acids 1–527) were a gift from Dr Guntram Suske, Philipps-University, Germany. Gal4-C/EBPβ (amino acids 1–83) was a gift from Dr Peter Johnson, NCI, MD, USA. All plasmid DNA was purified by ion-exchange chromatography (Qiagen, Valencia, CA, USA).
Site-directed mutagenesis of the GC-box was performed using the technique described by Wang and Malcolm 1999. Briefly, p0.27GCH1-GL3 or p0.27GCH1-GL3-CREmtCATmt was used as template to generate either luciferase constructs or electrophoretic mobility shift assay (EMSA) probes, respectively. The forward primers for mutagenesis of the GC-box were as follows (reverse primers are the inverse compliment): mutation M1 (M1, mutation underlined) TCCAGGATTTCGGGGCGGAGGGGAGGGGCGAGCCCTT; mutation M2 (M2) CCCCTCCAGGAGGGCTTTGC GGAGGGGAGGGGC; mutation M3 (M3) CTCCAGGAGGGCGGGGCGGATTTGAGGGGCGAG; mutation M4 CTCCAGGAGGGCGGGGCGGAGGGGATTTGCGAG; mutation 1234 CCCCTCCAGGATTTCTTTGCGGATTTGATTTGCGAGC. The Gal4-GCH1-GL3 construct was generated by replacing the GC-box in the p0.27GCH1-GL3 wild-type plasmid with a single Gal4 element after first introducing restriction sites flanking the GC-box, digesting to remove the GC-box and religation of the plasmid. This construct was then used as the template for mutagenesis with forward primer (Gal4 element underlined) GCT CTT ACG CGT GCT AGC CCA ACG GAG TAC TGT CCT CCG AGT TCC TTG ACG CAA GAG GCT CGG. All mutations were confirmed by sequencing.
PC12 cells were maintained on collagen in media composed of Dulbecco's modified Eagle medium containing penicillin–streptomycin, supplemented with 5% fetal calf serum and 10% horse serum at 37°C and 10% CO2. Rat2 cells were grown in Dulbecco's modified Eagle medium containing penicillin–streptomycin and 10% fetal calf serum at 37°C and 10% CO2. SL2 cells were maintained at 24°C in air in Schneider's Drosophila SL2 media (Invitrogen, Carlsbad, CA, USA) supplemented with penicillin–streptomycin and 10% fetal bovine serum.
PC12 and SL2 cells were plated at a density of 250–300,000 cells per well in poly-D-lysine-coated 24-well plates. The next day, each well was transfected with 800 ng of DNA using Lipofectamine 2000 (Invitrogen). Cells were harvested 24–48 h later and assayed using the dual luciferase assay (Promega). In some experiments, PC12 cells were treated with 5 mmol/L 8Br-cAMP for 4 h prior to harvesting. Luciferase assays were performed in replicates of four and experiments repeated at least three times. Data were normalized across experiments relative to control values and analyzed by one-way ANOVA with post hoc Bonferroni tests to correct for multiple comparisons (PRISM, Graphpad, San Diego, CA, USA). Differences were accepted as significant at p < 0.05.
PC12 and SL2 cell nuclear extracts were prepared by plating 20 million cells on 100 mm poly-D-lysine-coated dishes. In some experiments, PC12 cells were incubated with 5 mmol/L 8Br-cAMP for 4 h prior to harvest. In others, SL2 cells were transfected with 24 μg of pPac-0, pPac-Sp1, or pPac-Sp3 and harvested 48 h later. After washing, cells were scraped into 1.5 mL of Dulbecco's phosphate-buffered saline containing protease and phosphatase inhibitors (Sigma, St Louis, MO, USA) and centrifuged at 400 g at 4°C for 5 min. Cell pellets were suspended in ice-cold Buffer A (Dignam et al. 1983; 10 mmol/L HEPES-KOH pH 7.9 at 4°C, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L NaF, 0.5 mmol/L dithiothreitol (DTT), 0.2 mmol/L phenyl methyl sulfonyl fluoride (PMSF), 10 μg/mL leupeptin, 1 μg/mL aprotinin and 10 μg/mL pepstatin A and phosphatase inhibitors), allowed to swell on ice for 10 min and centrifuged at 400 g for 5 min at 4°C. The pellet was suspended in 100 μL of Buffer C (20 mmol/L HEPES-KOH, pH 7.9 at 4°C, 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L NaF, 0.5 mmol/L DTT, 0.2 mmol/L PMSF, 10 μg/mL leupeptin, 1 μg/mL aprotinin and 10 μg/mL pepstatin A and phosphatase inhibitors), incubated on ice for 20 min and centrifuged at 4°C at 21 000 g for 10 min. The supernantant was collected and assayed for protein content (Bradford 1976).
EMSA probes for wild-type GCH1 and mutations M2, M3, and M4 were generated by PCR using as template p0.27GCH1-GL3-CREmtCATmt and primers forward −148 TAGCCCCTCCAG GAGGGC −130 and reverse −68 ACTCTGCCGGCACCG −54. For generation of probes containing mutations M1 and M1234 the forward primer was −148 TAGCCCTCCAGGAT −134. PCR products were isolated by agarose gel electrophoresis and purified using the Qiaquik Gel extraction kit (Qiagen). Probes were end-labeled with T4 polynucleotide kinase and γ32P ATP and purified using G25 spin columns. The EMSA reaction buffer contained 12.5 mmol/L HEPES-KOH, pH 7.9, 10% glycerol, 100 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 μg poly dI-dC, 5 μg acetylated BSA and 0.2 mmol/L PMSF (Kapatos et al. 2000). The order of addition on ice was reaction buffer, unlabelled competitor (250–1000×), or antibody (1 μg), nuclear extract (1 μg), and labeled probe (~10 fmoles) followed by incubation at 25°C for 20 min. Samples were loaded and run on 6% polyacrylamide gels using 0.5× tris borate EDTA running buffer. Gels were dried and either exposed to film or phosphorimager screens (Typhoon, GE Healthcare, Piscataway, NJ, USA). Phosphorimage screens were scanned and the relative optical density of bands determined using ImageQuant software (Molecular Dynamics).
About 5–10 μg of protein was loaded onto 4–12% acrylamide gels and electrophoresed using morpholino propane sulfonic acid running buffer (NuPAGE, Invitrogen). Proteins were transferred to nitrocellulose using a semi-dry apparatus. Membranes were blocked using dry milk and incubated overnight with a 1: 500 dilution of rabbit antibodies directed against Sp1 (Upstate, Lake Placid, NY, USA), Sp3 or Sp4 (both from Santa Cruz, Santa Cruz, CA, USA). Membranes were washed, incubated with a 1: 25,000 dilution of horseradish peroxidase-coupled goat anti-rabbit, developed using a chemiluminescent horseradish peroxidase substrate (Pierce, Rockford, IL, USA) and simultaneously exposed to film. To insure equal loading of nuclear proteins in experiments using PC12 cells membranes were stripped and re-probed using an antibody to TFIID (Santa Cruz).
The chromatin immunoprecipitation (ChIP) assay was performed as described (West et al. 2004) with minor modifications (Kapatos et al. 2007). Briefly, 20 million PC12 cells or 10 million Rat2 cells were plated on poly-D-lysine-coated 100 mm dishes. The next day cells were treated with 5 mmol/L 8Br-cAMP for 1 h or directly cross-linked by addition of serum-free media containing 1% fresh formaldehyde for 10 min at 25°C. Cells were washed with phosphate-buffered saline and cross-linking terminated by the addition of 0.125 M glycine for 10 min. All further procedures were performed at 4°C in the presence of protease and phosphatase inhibitors. Cells were scraped into phosphate-buffered saline and collected by centrifugation. Cell pellets were suspended in 1 mL of lysis buffer and incubated for 10 min at 4°C. DNA was fragmented by sonicating to an average size of 400 bp. Cell debris was pelleted, supernatant harvested and 5% set aside as input DNA. After reversing cross-links the DNA content of input DNA was determined and equal amounts of DNA were then diluted 10-fold with dilution buffer and pre-cleared by the addition of rabbit IgG and salmon sperm DNA/BSA blocked Protein A-agarose. Supernatants were then divided into 4 parts, each receiving 5 μg of antibody directed against either the large subunit of RNA polymerase II, (Santa Cruz) Sp1 or Sp3. Anti-Green Fluorescent Protein (GFP; Santa Cruz) or normal rabbit immunoglobulin G (IgG; Santa Cruz) served as negative controls. Immune complexes were collected and washed sequentially with low-salt, high-salt, LiCl, and finally TE. Immune complexes and input DNA were incubated with RnaseA, overnight at 65°C to reverse cross-links and then digested with Proteinase K. DNA was purified using the Qiaquick PCR Purification system (Qiagen). Samples were analyzed by real-time quantitative PCR (Roche LightCycler, Mannheim, Germany) using QuantiTect SYBR-Green PCR reagent (Qiagen). PCR primers amplified either the proximal (forward −46 GCCGCGCCTCTCTTTTTATG −27; reverse −146 TGTGCAACTGCGGGGTTTAG −167) or distal (forward −5396 TCACTGGCTCATGTACTGAATG −5374; reverse −5439 GAC-CTGCTTCCTCTCAATACAG −5417) sequence of the rat GCH1 promoter. All samples were run in triplicate and experiments repeated at least three times. Standard curves for each PCR product were generated by plotting Ct versus serial dilutions of pooled input DNA. The enrichment for Pol II, Sp1, and Sp3 was calculated by first subtracting values for non-specific immunoprecipitation and then dividing by input DNA. Data were normalized relative to control and analyzed by one-way ANOVA with post hoc t-tests. Differences were accepted as significant at p < 0.05. In experiments comparing PC12 and Rat2 cells sample data were expressed relative to the PC12 standard curve of input DNA.
Total RNA was isolated from control or 4 h 8Br-cAMP-treated cultures of PC12 or Rat2 cells. RNA was treated with DNase I as recommended by the supplier (Qiagen, RNeasy) and 25–150 ng of RNA was reverse transcribed (Qiagen, Omniscript) in a reaction primed with random hexamers. 10–20% of this reaction served as template for quantitative real-time RT-PCR analysis using Quanti-Tect SYBR-Green PCR reagents (Qiagen), to amplify either rat GCH1 (forward, caagggataccaggagacca; reverse, tctcgtcatggtcctcatca) or β-actin (forward, gtcgtaccactggcattgtg; reverse, ctctcagctgtggtggtgaa) cDNA. Control reactions minus reverse transcriptase were included for each primer pair and quantity of RNA. Standard curves for each transcript were generated using serial dilutions of pooled RNA from control samples and distinct reverse transcription reactions. GCH1 mRNA abundance were expressed relative to that of β-actin.
Genomic DNA was isolated from 5 × 106 PC12 and Rat2 cells (Zymo Research, Orange, CA, USA). 500 ng of PC12 and Rat2 cell DNA was then treated with bisulfite to convert unmethylated cytosines to uracils (Zymo Research). Bisulfite-treated GCH1 promoter DNA was amplified by methylation-specific PCR using primers designed by MethylPrimer Express software v1.0 (Applied Biosystems, CA, USA) to amplify a 288 bp product encompassing the GC-box, CRE and CCAAT-box (forward, −182 ATTTGAGGGTTGTTTTGTGTAA −161; reverse + 86 ACACCCRAA AATACTACCAAA + 107). PCR products were directly cloned into the pGEM-T vector (Promega), transformed into Top10 bacteria and DNA mini-preps prepared from 10 colonies for each cell type. DNA was sequenced using the M13 forward primer and analyzed by comparison to the sequence of untreated DNA.
A bioinformatics analysis of the entire 142 bp of the GCH1 proximal promoter (transcription element search system; Schug and Overton, 1997) determined that the three Sp-protein binding sites with the highest probability scores are all clustered within the GC-box footprint (Fig. 1a). These cis-elements will be referred to as Site I (−134 to −125), Site II (−130 to −121) and Site III (−124 to −114) and form the basis of the triad model of the GCH1 GC-box (Fig. 1a). Sites I and III are located at either ends of the GC-box and do not overlap whereas Site II is located in the center of the GC-box and overlaps Sites I and III (Fig. 1a and b). Because Sites I and III do not overlap, Sites I and III can potentially be occupied simultaneously by Sp-proteins (Fig. 1a and b). Since Sites I and III overlap with Site II, however, steric hindrance between binding proteins would prevent occupation of Site II if either Site I or III are bound (Fig. 1a and b). Similarly, because Site II overlaps Sites I and III, occupation of Site II would block protein binding to Sites I and III (Fig. 1a and b).
Before experiments were begun on the role of the GC-box and cognate binding proteins in GCH1 transcription it was imperative to identify which members of the Sp-protein immediate family are expressed by PC12 cells. Moreover, it was critical to know whether incubation with 8Br-cAMP alters Sp-protein levels, as is known to occur in this cell line following treatment with phorbol esters (Papanikolaou and Sabban 2000) or nerve growth factor (Sobue et al. 2005). Western blotting of nuclear extracts from control PC12 cells and PC12 cells treated for 4 h with 5 mmol/L 8Br-cAMP detected both Sp1 and Sp3 proteins but not Sp4 (Fig. 2a). Although a single Sp1 protein of approximately 100 kDa was observed, three different Sp3 proteins were found; one large abundant protein of approximately 100 kDa and two smaller less abundant proteins of approximately 77 and 73 kDa. Incubation with 8Br-cAMP did not alter the abundance of Sp1 or Sp3 proteins or result in the appearance of Sp4. We have therefore focused our attention on Sp1 and Sp3 proteins.
Another essential early step towards understanding the role played by the GC-box in GCH1 transcription was to use the ChIP technique to determine whether endogenous Sp1 and Sp3 proteins are localized to the native GCH1 proximal promoter under basal conditions and, like C/EBPβ and NF-Y (Kapatos et al. 2007), are recruited in response to cAMP. Sheared DNA from untreated PC12 cells was immunoprecipitated using antibodies specific to the large subunit of Pol II, Sp1, Sp3, and control IgG. GCH1 proximal promoter DNA was detected by PCR using primers that amplify a 141 bp product (−167 to −27) which includes the GC-box, CRE and CCAAT-box (Fig. 2b). A 65 bp product from the distal promoter (−5439 to −5375) located 5.4 kb upstream from the cap site was also amplified and served as a gene-specific control (Fig. 2b). Gel analysis showed that amplifications of proximal but not distal promoter DNA resulted in PCR products of the correct size for Pol II, Sp1, and Sp3 immunoprecipitations whereas no product was observed for the IgG control (Fig. 2b). Both Sp1 and Sp3 are therefore specifically associated with the proximal GCH1 promoter active within PC12 cells. Quantitative real-time PCR analysis of ChIP samples subsequently revealed that Sp1 and Sp3 are not recruited to the proximal promoter in response to cAMP treatment, although the amount of Pol II was increased by more than twofold indicating enhanced gene transcription (Fig. 2c).
Chromatin immunoprecipitation is not necessarily informative about the binding of a protein to its cis-element and cannot confirm that Sp1 and Sp3 are actually bound to the GCH1 GC-box DNA in vivo. EMSA was therefore used to determine whether the GC-box binds the Sp1 and Sp3 proteins found in PC12 nuclear extracts and ChIP assays. Control PC12 cells and PC12 cells treated with 5 mmol/L 8Br-cAMP for 4 h were also compared in these experiments to determine whether a direct assay of protein-DNA interactions can detect phosphorylation-induced changes in Sp-protein binding. The probe used for EMSA spanned from position −148 to −54 and contained the wild-type GC-box but mutated forms of the CRE and CCAAT-box.
Initial experiments with short film exposure times at −80°C revealed two protein-DNA complexes, a major bottom band and minor top band (data not shown). Application of the more sensitive and linear phosphorimaging technique to EMSA revealed six protein-DNA complexes (complexes 1–6) all of which were competed away with excess probe and did not appear to be altered by 4 h treatment with 8Br-cAMP (Fig. 3a). Densitometric analysis of these six protein-DNA complexes indicated that complex 1 contains 5%, complex 2 contains 10%, complex 3 contains 25%, complex 4 contains 40%, complex 5 contains 10% and complex 6 contains 10% of total binding activity (Fig. 3d). Additional studies showed that complexes 1–5 but not complex 6 were competed by a consensus but not mutated Sp1 element (Fig. 3b), demonstrating that Sp-proteins are components of at least five of the six complexes. EMSA super-shift assays are notoriously difficult to interpret when there are multiple complexes present. Nonetheless, EMSA using antibodies directed against Sp1 and Sp3 (Fig. 3c) combined with densitometric analysis (Fig. 3e) allowed definitive identification of Sp1 in complex 4, Sp3 in complexes 3 and 5 and tentative identification of Sp3 in complexes 1 and 2. Based upon these results and the fraction of total binding made up by each complex we estimate that Sp1 comprises approximately 40% and Sp3 upwards of 50% of the GC-box binding proteins found in PC12 nuclear extracts. Treatment of cells with 8Br-cAMP did not modify the pattern of super-shifted complexes (Fig. 3c). Complex 6 was not shifted by antibodies to either Sp1 or Sp3 and was not studied further.
The complexity of the GC-box, expression of Sp1 and multiple forms of Sp3 and the EMSA binding pattern make it very difficult to study the role of Sp1 and Sp3 in GCH1 transcription in PC12 cells. Drosophila SL2 cells do not express the Sp family of transcription factors (Courey and Tjian 1988) and thus offer a cellular milieu to establish a basic understanding of Sp1 and Sp3 function at the GCH1 promoter. Western blots of nuclear extracts were prepared from SL2 cells transfected with vector driving Sp1 or Sp3 expression under the control of the Drosophila β-actin promoter (Fig. 4a). SL2 cells transfected with the empty pPac-0 vector contained no endogenous proteins that react with the antibodies to Sp1 and Sp3 used here. Moreover, Sp1 and Sp3 expressed by SL2 cells were of similar size (~100 kDa) and abundance, with no truncated forms of Sp3 present (Fig 4a). EMSA using nuclear extracts prepared from transfected SL2 cells also clearly showed that there are no endogenous proteins capable of binding to the GC-box (Fig. 4b, see pPac-0) and that the GC-box specifically recruits Sp1 and Sp3 (Fig. 4b). However, the patterns of Sp1 and Sp3 binding to the GC-box are different. Sp1 overwhelmingly formed a single complex whereas Sp3 formed two complexes in approximately equal abundance, one similar in size to that produced by Sp1 and the other significantly larger. Super-shift assay using antibodies to Sp1 and Sp3 confirmed that Sp1 is responsible for the single complex while Sp3 forms both the slow migrating large and fast migrating small complexes (Fig. 4b). Competition experiments subsequently determined that Sp1 and Sp3 binding is displaced by increasing concentrations of unlabeled probe and that the slow migrating Sp3 complex is displaced at lower probe concentrations than is the fast migrating complex, suggesting that Sp3 binds with lower affinity to a second site within the GC-box (Fig. 4d).
The next series of experiments investigated the effects of Sp1 and Sp3 on GCH1 promoter function. Luciferase assays were carried out in SL2 cells co-transfected with either the wild-type GCH1 proximal promoter reporter construct p0.27GCH1-GL3 or the empty pGL3 parental vector along with equal amounts of pPac-Sp1 or pPac-Sp3 expression vector DNA. Equal amounts of Sp1 or Sp3 plasmid DNA generate equal amounts of functional Sp1 and Sp3 proteins, as shown by analysis of band densities from EMSA experiments using nuclear extracts derived from different preparations of transfected SL2 cells (see Figs 4b–d, 5b and c). Preliminary experiments noted that both Sp1 and Sp3 enhanced activity from cryptic Sp-protein binding sites within the pGL3 vector itself (Fig. 4e). Indeed, Sp1 was found to have no statistically significant effect on transcription from the GCH1 promoter when luciferase activity induced by transfection with pPac-Sp1was corrected for this background activity (Fig. 4e). In contrast, the effect of Sp3 was robust, enhancing transcription by more than fivefold over the empty pGL3 vector (Fig. 4e). Co-transfection of equal amounts of Sp1 and Sp3 plasmid DNA showed that Sp1 actually inhibits Sp3-dependent activation by 50% (Fig. 4e). In agreement with the functional competition of Sp3 by Sp1, EMSA combining Sp1- and Sp3-containing nuclear extracts demonstrated that Sp1 is capable of competing away the large Sp3 complex (Fig. 4f). No doubt, Sp1 also competes with Sp3 and vice versa to form the smaller complex that is common to both proteins. Importantly, the combination of Sp1 and Sp3 proteins did not produce a large complex like that observed for Sp3.
To further characterize the GC-box and Sp1 and Sp3 binding mutagenesis was performed under the guidance of the triad model. Accordingly, GGG sequences within the GC-box were strategically replaced by TTT mutations so as to inactivate each or all of the three putative Sp-protein binding sites. Figure 5a shows that mutation M1 within Site I forces Sp-protein binding to Sites II or III while mutation M2 within Sites I and II forces binding to Site III. Similarly, mutation M3 within Sites II and III forces binding to Site I while mutation M4 within Site III forces binding to Sites I or II. Mutation M1234 in Sites I, II, and III eliminates Sp-protein binding completely.
Repeated EMSA analysis of the effect of these mutations on binding of Sp1 and Sp3 using nuclear extracts from transfected SL2 cells showed that individual mutations M1, M2, M3, or M4 had no reproducible effect on Sp1 binding whereas binding was eliminated by the complete mutation of the GC-box in M1234 (Fig. 5b). We interpret this to mean that each individual mutation leaves at least one site within the triad available for Sp1 binding. In contrast, each individual mutation prevented formation of the large Sp3 complex without affecting the small Sp3 complex (Fig. 5c). Mutation M1234 eliminated Sp3 binding (Fig. 5c). Because Sp3 does not homodimerize (Yu et al. 2003) these findings indicate that the slow migrating large Sp3 complex is composed of Sp3 bound to non-overlapping Sites I and III. Similarly, the fast migrating small complexes formed by Sp1 and Sp3 must result from occupation of any one of the three sites within the GC-box. These results serve to validate the triad model and demonstrate that the GC-box is the only cis-element located between positions −142 and −54 of the proximal promoter that is capable of binding Sp-proteins.
The functional impact of GC-box mutations on GCH1 transcription was next examined in SL2 cells transfected with the Sp3 expression vector and proximal promoter luciferase constructs containing M1, M2, M3, M4, or M1234 mutations. In these experiments, Sp3 activated transcription from the wild-type promoter by ninefold. Mutations M1, M2, and M4 each reduced by 40–50% the response of the promoter to Sp3, commensurate with their capacity to prevent formation of the large Sp3 complex. However, mutation M3 did not affect the ability of Sp3 to enhance transcription. One might conclude from this that formation of the large Sp3 complex is not required for maximum activation of transcription. An alternate interpretation is that forced occupation of Site I by Sp3 best supports activation (Fig. 5a). While mutation M4 also forces Sp3 to Site I, Site II in this mutation is left open for occupation which would reduce binding to Site I and thereby decrease activation (Fig. 5a). As predicted by EMSA, mutation M1234 decreased transcription to levels observed with the empty pGL3 vector, demonstrating that the GC-box offers the only functional binding sites for Sp3 within the entire 142 bp proximal promoter.
Although Sp1 and Sp3 in PC12 cells are not recruited to the GCH1 promoter in response to cAMP, Sp1, or Sp3 protein–protein interactions might play a role in stabilizing already bound or recently recruited C/EBPβ and NF-Y. Fortunately, SL2 cells are not only null for Sp-proteins but also for CREB, C/EBPβ, and NF-Y (Lee et al. 1997; Dooley et al. 1999). Experiments were therefore performed in SL2 cells to investigate and compare interactions between Sp1, Sp3, NF-Y, and C/EBPβ at the GCH1 promoter. Luciferase assays in SL2 cells co-transfected with the reporter p0.27GCH1-GL3 in combination with plasmid DNA encoding for Sp1 or Sp3, the three NF-Y subunits A, B, and C, or C/EBPβ showed that when expressed alone Sp3 and C/EBPβ each significantly enhanced transcription while Sp1 and NF-Y did not (Fig. 5e). NF-Y and C/EBPβ interacted at the promoter to stimulate transcription to a greater degree than did C/EBPβ acting alone. The promoter response to NF-Y or C/EBPβ was further enhanced when either of these proteins was combined with Sp3 or Sp1. Finally, the combination of Sp3 or Sp1 with NF-Y and C/EBPβ synergistically enhanced transcription. These results indicate that both Sp1 and Sp3 are capable of nucleating an enhanceosome complex at the GCH1 proximal promoter that includes C/EBPβ and NF-Y, although Sp3 is significantly more effective than is Sp1.
Based upon knowledge gained using SL2 cells we began a limited series of experiments using GC-box mutations to characterize Sp-protein binding in PC12 cells. We first questioned whether the large EMSA complexes 1 and 2 in PC12 nuclear extracts represent binding of Sp3 to two sites within the GC-box. EMSA with super-shift was used to determine the effect of the M1 mutation on Sp1 and Sp3 binding patterns. Similar to what was observed using SL2 nuclear extracts, mutation M1 had no obvious affect on formation of the major Sp1 complex 4 (Fig. 6a). M1 also did not modify formation of the major Sp3 complex 3 or the minor Sp3 complex 5. Complexes 3, 4, and 5 therefore presumably represent binding of Sp1 and Sp3 to single sites within the GC-box. However, mutation M1 eliminated large complexes 1 and 2. By analogy with data obtained using SL2 cells, these results support the conclusion that complexes 1 and 2 are produced by binding of Sp3 to two sites within the GC-box. Mutation M1234 abolished binding to the GC-box (including complex 6), demonstrating that all binding in EMSA produced by PC12 nuclear extracts is dependent upon sequences located within the GC-box (Fig. 6a).
The next series of experiments were designed to determine the effect of GC-box mutations on basal and cAMP-dependent transcription in PC12 cells using GCH1 proximal promoter constructs in which the CRE and CCAAT-box cAMP-response elements are left intact. Incubation with 8Br-cAMP for 4 h increased activity from the wild-type promoter by 4- to 5-fold (Fig. 6b). Mutations M1 and M2 which force binding of Sp-proteins to Sites II and III close to the CRE and CCAAT-box cassette had no significant effect on basal or cAMP-dependent transcription. In contrast, mutations M3 and M4 which force binding to Sites I and II furthest away from the cAMP-response elements significantly reduced basal activity (see Fig. 6b insert). Furthermore, while mutation M3 did not alter cAMP-dependent transcription, mutation M4 clearly doubled the absolute response of the promoter to cAMP. Complete mutation of the GC-box in M1234 decreased basal transcription by 60% (see Fig. 6b insert) without affecting the absolute promoter response to cAMP, resulting in a fold stimulation that was three times that of the wild-type promoter.
Experiments in PC12 cells using over-expression of Sp1 and Sp3 or dominant negative forms of Sp1 and Sp3 failed to further define distinct roles for these proteins in GCH1 transcription (data not shown). We therefore chose a strategy in which the 22 bp GC-box in the wild-type GCH1 luciferase promoter construct was replaced with a single copy of the 19 bp yeast Gal4 cis-element and then challenged by co-expression of fusion proteins composed of amino acids 1–147 of the yeast Gal4 DNA binding domain and the N-terminal activation domains of Sp1 (Gal4 Sp1; amino acids 1–603), Sp3 (Gal4-Sp3; amino acids 1–527) or C/EBPβ (Gal4- C/EBPβ; amino acids 1–83) (Fig. 7a). This paradigm eliminates differences in Sp1 and Sp3 binding as well as any preferences for binding sites within the GC-box yet the cAMP response can still be studied because the CRE and CCAAT-box cassette is intact and the assay is performed in PC12 cells.
Substitution of the GC-box with a single Gal4 DNA binding element decreased basal transcription by greater than 60% (data not shown), a value virtually identical to that produced by complete mutation of the GC-box. This decrease in basal promoter activity was significantly reversed by co-transfection with Gal4-Sp3 but not by Gal4-Sp1 or the Gal4-0 empty vector (Fig 7a and insert). In addition, although Gal4-Sp3 increased basal transcription the absolute response of the promoter to cAMP remained unchanged (Fig. 7a). Experiments with Gal4-C/EBPβ demonstrated that the Gal4-GCH1 reporter construct can respond to introduction of an activation domain with large increases in basal as well as cAMP-dependent transcription (Fig. 7a). The functionality of both Gal4-Sp1 and Gal4-Sp3 was also established by experiments in PC12 cells showing five to sixfold enhancement of transcription from a heterologus luciferase reporter containing five tandem Gal4 sites (Fig. 7b). None of the Gal4-fusion proteins affected transcription from the empty pGL3 vector (data not shown). These results support our contention that the GC-box and Sp3 are important for maintaining basal but not cAMP-dependent transcription in PC12 cells.
A comparison of Sp1 and Sp3 association with the GCH1 proximal promoter GC-box in cell types with divergent basal GCH1 mRNA levels was next undertaken to test the hypothesis that the localization of Sp3 at the proximal promoter correlates with basal GCH1 transcription. With this in mind, resting levels of GCH1 mRNA in rat adrenal chromaffin-derived PC12 cells were determined to be 10-fold higher than in rat fibroblast-derived Rat2 cells (Fig. 8a). Further comparison showed that PC12 cells respond to 4 h treatment with 8Br-cAMP with a sevenfold increase in GCH1 mRNA levels whereas the response of Rat2 cells to this stimulus was insignificant (Fig. 8a). Despite these robust differences in transcription ChIP analysis of the two cell lines demonstrated equivalent amounts of Sp1 and Sp3 at the native proximal promoter (Fig. 8b). Moreover, ChIP showed that while the quantity of Pol II at the Rat2 cell promoter was significantly lower than in PC12 cells, as would be expected based upon the lower transcription rate, Pol II was still three times higher than predicted given the 10-fold difference in basal levels of GCH1 transcription (Fig. 8b).
That presence of equivalent amounts of Sp1and Sp3 at the GCH1 proximal promoter in cells with such disparate levels of transcription suggests that these proteins might have multiple functions in GCH1 transcription. Sp-proteins are known to protect genes from repression by blocking methylation of CpG dinucleotides located within and around GC-boxes (Brandeis et al. 1994; Macleod et al. 1994). With this in mind, we performed a bioinformatics analysis of −5000 bp to + 500 bp of the rat GCH1 gene using MethPrimer (Li and Dahiya 2002). This identified a single 238 bp CpG island spanning from −162 to + 77 that encompasses the entire proximal promoter. Located strategically within or immediately adjacent to the GC-box are located four CpG dinucleotides (Fig. 8c). Cloning and sequencing of PC12 and Rat2 GCH1 methylation-specific PCR products revealed that of the four possible methyl acceptor sites only a single CpG within the GC-box is methylated and this occurred in 30% of the PC12 clones and in none of the Rat2 cell clones examined (Fig. 8b).
The region −142 through −77 of the rat GCH1 proximal promoter contains the GC-box, the CRE and the CCAAT-box, each originally identified by in vitro footprinting using PC12 cell nuclear extracts or recombinant protein (Kapatos et al. 2000). The boundaries of the GC-box were defined by these experiments as a 22 bp footprint spanning from nucleotides −132 to −111 that was competed away by the sequence GGGGCGGGGGCG, thus implicating Sp1, Sp3, and Sp4 as the likely binding partners. This earlier study also showed using heterologous promoter constructs that the GC-box reduces cAMP-dependent transcription conferred by the CRE and CCAAT-box cassette, suggesting an inhibitory role for Sp-proteins in GCH1 transcription. The present studies clearly demonstrate that the GCH1 GC-box binds Sp1 and Sp3 proteins. Sp1 is generally known to activate transcription while Sp3 is a dual role transcription factor that behaves as an activator or repressor depending on promoter context but in most cases acts as a repressor in the presence of Sp1 (Hagen et al. 1994, 1995; Majello et al. 1994; Yu et al. 2003). Our observation made in SL2 cells that Sp3 activates transcription from the GCH1 promoter while Sp1 acts as a repressor in the presence of Sp3 may therefore appear to be unique but, a similar circumstance has been reported for the mouse growth hormone receptor promoter (Yu et al. 1999). The present studies also show that although Sp1 and Sp3 are not absolutely required for the cAMP response by enhancing basal levels of transcription these proteins decrease the responsiveness of the promoter to cAMP. A number of additional conclusions can be reached from the current studies regarding Sp-proteins, the GC-box and the regulation of GCH1 transcription.
First, incubation with 8Br-cAMP for 4 h did not alter the abundance of Sp1 in PC12 cells. This is in contrast to the decline in Sp1 protein observed following treatment of PC12 cells with phorbol esters (Papanikolaou and Sabban 2000) and the increase in Sp1 protein following nerve growth factor treatment of PC12 cells (Sobue et al. 2005 but see Yan and Ziff 1997). A 95 kDa form of Sp1 is also known to result from phosphorylation of Sp1 (Jackson et al. 1990) and has been observed in some (Billon et al. 1999; Sobue et al. 2005) but not all (Papanikolaou and Sabban 2000; Liu et al. 2001; Persengiev et al. 2001) studies using PC12 cells. While Sp1 is a substrate for protein kinase A (Rohlff et al. 1997) we did not detect the 95 kDa form in nuclear extracts from control or 8Br-cAMP treated PC12 cells prepared in the presence of protein phosphatase inhibitors. The reason for this is unclear but might be related to cell passage number or culture conditions.
Second, in PC12 nuclear extracts we also observed what we interpret to be three isoforms of Sp3, a major protein of 100 kDa and two less abundant proteins of 77 and 73 kDa, the relative amounts of which were not altered by cAMP treatment. It is possible that the 77 and 73 kDa immunore-active proteins represent products of Sp3 proteolysis or non-specific antibody interactions. However, similar studies of Sp3 expression in PC12 cells (Billon et al. 1999) and other cell types (Wang and Bannon 2005; Ishimaru et al. 2006) report three forms of Sp3 of approximately the same molecular weight and relative abundance we show here. The Sp3 proteins we observed are thus likely to be derived from the four alternative Sp3 mRNA ribosomal entry sites, with the 100 kDa band composed of one or both of the two full-length forms and the 77 and 73 kDa bands the two N-terminal truncated forms (Suske 1999; Bouwman and Philipsen 2002; Sapetschnig et al. 2004). Truncated isoforms of Sp3 retain the C-terminal DNA binding domain but are missing the activation domain and thus serve as dominant negative regulators of full-length Sp-proteins (Kennett et al. 1997; Sapetschnig et al. 2004). These small Sp3 isoforms are likely responsible for EMSA complex 5 which comprises 10% of the total GC-box binding activity in PC12 nuclear extracts. By competing with full-length forms of Sp1 and Sp3 for binding to the native GCH1 promoter dominant negative forms of Sp3 are predicted to reduce basal transcription and thereby enhance the overall response to cAMP.
Third, Sp4 is not expressed by PC12 cells. This is not a surprise given that PC12 cells are derived from the adrenal medulla and Sp4 appears to be selectively expressed in the nervous system (Suske 1999; Bouwman and Philipsen 2002). Accordingly, Sp4 is involved in the regulation of neuronal genes, such as neurotrophin-3 (Ishimaru et al. 2006) and the β-subunit of rod cGMP-phosphodiesterase (Lerner et al. 2002) and might play a role in the control of GCH1 transcription within central monoaminergic neurons (Lentz and Kapatos 1996). Our unpublished data indicate that like Sp1, Sp4 binds to a single site within the GCH1 GC-box and is unable to activate transcription but does inhibit Sp3-dependent transcription. Sp4 has properties that are functionally distinct from Sp1 and Sp3 (Hagen et al. 1995), however, and represents another dimension in the regulation of GCH1 transcription that should be explored in the future.
Fourth, Sp1 and Sp3 bind to three sites within the 142 bp proximal promoter and these sites are all located within the GC-box. Sites I (−134 GGGCGGGGCG−125) and III (−124 GAGGGGAGGGG −114) do not overlap while Site II (−130 GGGGCGGAGGG −121) overlaps both Sites I and III. Sites I and III thus have the potential to be occupied simultaneously by Sp-proteins whereas occupation of Sites I and II or Sites II and III would be prohibited by steric hindrance. Repeated EMSA experiments using SL2 nuclear extracts and mutated probes suggest that Sp1 and Sp3 have no obvious preference for binding to any one site within the GC-box. Nonetheless, a common theme that arises from studies of both SL2 and PC12 cells is that forced binding of Sp-proteins to Sites I and II, which are furthest away from the CRE, CCAAT-box and transcription start site, has distinct effects on transcription. In the SL2 nuclear environment the transcriptionally preferred binding site for Sp3 appears to be Site I. In PC12 cells evidence for a preferred Sp-protein binding site comes from the ability of mutation M4 to enhance cAMP-dependent transcription. Inasmuch as this effect of M4 was reversed by complete mutation of the GC-box it would appear that Sp-proteins bound by Sites I or II are somehow involved.
Unlike Sp1, Sp3 binds to the GCH1 GC-box as two protein-DNA complexes, one similar in size to that produced by Sp1 and the other significantly larger. Also unlike Sp1 (Courey et al. 1989) the N-terminal domain of Sp3 does not contain a dimerization domain (Yu et al. 2003). This means that the slowly migrating large Sp3 complexes observed using nuclear extracts from transfected SL2 cells and PC12 cells is not produced by multiple Sp3 proteins tethered to a single cis-element but rather from more than one Sp3 protein simultaneously bound to the GC-box. The slow migrating Sp3 complex must therefore represent occupation of non-overlapping Sites I and III. Because Sp3 shows no obvious preference for binding to any one site it is likely that the first site bound by Sp3 can be either Site I or III. Competition experiments suggest that Sp3 binds to the second site with lower affinity, likely because of steric hindrance produced by Sp3 binding to the first site. Unlike what was observed in SL2 cells, mutations M1 and M2 did not decrease basal transcription in PC12 cells even though mutation M1 was shown by EMSA to eliminate the large Sp3 containing complexes produced by PC12 nuclear extracts. The reason for this disparity is not known but may be related to the fact that PC12 cells express additional activators of GCH1 transcription, such as CREB, C/EBPβ and NF-Y, while SL2 cells do not. The DNA binding domains of Sp1 and Sp3 are essentially identical in size, conformation and DNA recognition (Suske 1999; Bouwman and Philipsen 2002), making the distinction between Sp1 and Sp3 binding at the GCH1 GC-box a topic for further research. Our observations that Sp1 competes with Sp3 to prevent occupation of two sites by Sp3 and that Sp1 and Sp3 cannot occupy the GC-box at the same time strongly suggest, however, that steric hindrance arising from the N-terminal of Sp1 restricts the binding of a second molecule of Sp1 to the promoter.
Fifth, Sp1 and Sp3 are each associated with the native GCH1 proximal promoter but are not recruited or required during cAMP-dependent transcription. Despite the results of these ChIP experiments it is likely that Sp1 and Sp3 proteins are involved in some subtle aspects of the cAMP response. For example, as shown here Sp1 and Sp3 might assist in the recruitment of C/EBPβ and NF-Y to the promoter (Kapatos et al. 2007). As reported for other genes (Krikun et al. 2000; Xu et al. 2000; Wang and Bannon 2005), competition between Sp1 and Sp3 proteins for binding to the GC-box might also determine the basal rate of GCH1 transcription and thereby the relative magnitude of the cAMP response. Our EMSA results using SL2 cells indicate that Sp1 and Sp3 cannot co-occupy the GC-box yet ChIP experiments in PC12 cells show that Sp1 and Sp3 are both associated with the native promoter. One interpretation of this apparent contradiction is that Sp1 and Sp3 are bound to different copies of the native GCH1 promoter. A GCH1 promoter with Sp3 bound has greater basal activity and therefore a smaller fold response to cAMP than does a promoter which has bound Sp1, even when that promoter is occupied by C/EBPβ and NF-Y.
Sixth, association of Sp1 and Sp3 with the native proximal promoter does not predict levels of GCH1 transcription but does correlate with hypo-methylation of four CpG dinucleotides located within or around the GC-box. Cytosine methylation within consensus Sp-binding sites does not affect binding of Sp1 and Sp3 (Harrington et al. 1988; Zhu et al. 2003) whereas methylation of CpG immediately adjacent to a consensus site can reduce Sp1 and Sp3 binding (Zhu et al. 2003). The single cytosine found methylated in 30% of the PC12 cell GCH1 promoters is located immediately adjacent to Site III and in the native promoter would be predicted to drive Sp-protein binding to Sites I or II. Mutation M4 also forces Sp-protein binding to Sites I or II and increases in the response of the promoter to cAMP. The select methylation of a single CpG dinucleotide located with the GCH1 proximal promoter of PC12 cells might therefore be involved in the cell type-specific response to cAMP.
In cells that express very low levels of GCH1 transcription the native proximal promoter is characterized by levels of bound Pol II and acetylated nucleosomes that are normally associated with active chromatin (Kapatos et al. 2007). As shown here, cells such as these typically do not respond to cAMP but they do respond to inflammatory cytokines with a rapid induction of GCH1 transcription that is part of the program required for the BH4-dependent production of nitric oxide (NO) by the inducible form of nitric oxide synthase (Kwon et al. 1989; Werner et al. 1990; D'Sa et al. 1996). In order to retain this cytokine response these cells must maintain the GCH1 promoter in a paused but transcriptionally competent state. Methylation of cytosines in CpG dinucleotides is widely accepted as a gene-silencing signal (Herman and Baylin 2003; Jaenisch and Bird 2003; Klose and Bird, 2003) and the binding of Sp-proteins is known to interfere with promoter DNA methylation both within and surrounding Sp-binding sites (Brandeis et al. 1994; Macleod et al. 1994; Clark et al. 1997; Siegfried et al. 1999; Chan et al. 2004; Pang et al. 2004). We hypothesize that in cell types that express low levels of GCH1 the association of Sp1 and Sp3 proteins with the GCH1 proximal promoter serves to protect promoter bound nucleosomes from repressive histone marks and the subsequent recruitment of DNA methyltransferases. In this way, even naïve cells would be able to respond to cytokine stimulation with an increase in GCH1 gene expression and BH4 biosynthesis.
This study was supported by NINDS grant NS26081.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Merseylea TAS 7305, AustraliaEmail to
Lions Park
Railton Road, Railton
Open: Open daylight hours
Railton Recreation Ground
Native Rock Road, Railton
Open: Open variable hours
Goliath Park
Ennis Avenue, Railton
Open: Open daylight hours
Rest Area
Bass Highway, Sassafras
Open: Open 24 hours
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text:
Thursday, June 6, 2013 - 8:00 a.m.
Jimmy Barton is Elizabeth Barton's son and also attended the Marshall family reunion on May 25, 2013. Detectives Murphy and Parker interviewed him at the Yoknapatawpha County Sheriff's Department. The interview was recorded with the witness's knowledge and consent.
- Detective S. Murphy
- Detective E. Parker
- Jimmy Barton
Detective Parker: For the record, what is your name and address?
Jimmy Barton: My name is Jimmy Barton, and I live at 1710 Anderson Road.
Detective Parker: All right, now that we have that out of the way, have you seen these posters around town or on the news?
Jimmy Barton: Sure.
Detective Parker: Good. So what can you tell us about Oscar Knight?
Jimmy Barton: Nothing. I wish I could help, but I didn't know the man.
Detective Parker: Really? OK. Where were you on Saturday, May 25th?
Jimmy Barton: I was at the Marshall family reunion at Wall Doxey State Park. I go every year from as far back as I can remember. Mother and the Marshalls are close friends.
Detective Murphy: Who's your mother?
Jimmy Barton: Elizabeth Barton.
Detective Parker: Did you meet Oscar Knight at the reunion?
Jimmy Barton: No. I wasn't introduced to him, but I did see him and hear all the buzz about him being a jerk. I didn't really speak with him, so I know nothing concerning him.
Detective Parker: Does your mother know Mr. Knight?
Jimmy Barton: Yeah, she did once, but I didn't. As a matter of fact, I never even heard his name until that day at the reunion.
Detective Murphy: What did you hear about him?
Jimmy Barton: Only that he was a rotten excuse for a man. No one liked him, it seems. He must have had a very nasty disposition. I never heard one good word about him. That's pretty bad, wouldn't you agree?
Detective Murphy: Who told you that Mr. Knight was a rotten excuse for a man?
Jimmy Barton: Mother, for one. And Steve. And Pauline. And Albert, just to mention a few. They couldn't all be wrong.
Detective Murphy: What made them say that?
Jimmy Barton: I don't know. Because he was acting like a jerk to everyone at the reunion, I guess.
Detective Murphy: To you?
Jimmy Barton: No. I told you, I didn't even meet him. But he wasn't very nice to my mother.
Detective Parker: Really? Tell us about that.
Jimmy Barton: I told you. He was just being a real jerk, saying rude things. Mother didn't do anything to deserve that.
Detective Parker: What did Mr. Knight say to her?
Jimmy Barton: I don't think it was so much what he said as how he behaved.
Detective Parker: And how did he behave?
Jimmy Barton: Mother said that Oscar smelled of alcohol and was just being obnoxious. She said he said something about her looking old. Believe me, that isn't something anyone should say to my mother. She prides herself on the fact that she doesn't look her age, so when he said that, she got riled up a little.
Detective Murphy: Is that what caused the fight?
Jimmy Barton: Fight? There was no fight. Are you kidding? I admit Mother is a little vain, but she isn't stupid. She tried to just walk away, and Oscar wouldn't leave it alone.
Detective Parker: What happened when she walked away?
Jimmy Barton: Nothing. She kept walking; he kept talking. I think someone else caught his attention, and that's when she got away from him.
Detective Parker: So Mr. Knight was drunk?
Jimmy Barton: I don't know if he was or not, but he reeked, according to what Mother said.
Detective Parker: How does your mother know Mr. Knight? Did she tell you?
Jimmy Barton: Sure. They were in the same grade in school. She said she thought he was a nice guy back then, but she saw through him one day and just stayed away from him after that.
Detective Murphy: You know a lot about what happened for not being there. When did you learn all this, Jimmy?
Jimmy Barton: I hadn't heard any of this until later in the day. Mother left the reunion early and she usually stays until the end, so I stopped by the house to see if she was sick. Plus, I heard a little bit about Oscar insulting just about everyone that day, so I wanted to see if she was OK.
Detective Parker: And was she?
Jimmy Barton: Oh, sure. She just decided she didn't feel comfortable at the park any longer with Oscar running around being such a creep. Hopefully, he won't be back next year. Anyway, we both just laughed a little about it, and then we sat down and had a big piece of her double chocolate cake. That's one of her specialties.
Detective Parker: Sounds delicious. Is that something your mother typically does? Leave when she's uncomfortable in a situation?
Jimmy Barton: What was she supposed to do? Stay there and put up with that?
Detective Murphy: You mentioned Pauline was one of the people who told you Oscar was a rotten excuse for a man. Who is that?
Jimmy Barton: She's my mother's best friend, Pauline Jones.
Detective Murphy: And she was at the reunion too?
Jimmy Barton: Yeah, and she had a little run-in with Oscar too. I think quite a few people at the reunion had a problem with him. I'm glad I wasn't around him. Steve told me that he never did like Oscar, but he didn't want to go so far as to ask him to leave. He said that would probably just make matters worse.
Detective Murphy: Who is Steve?
Jimmy Barton: Steve Marshall. He's Mother's other best friend. He sets up the reunion every year. Does a real good job too.
Detective Murphy: You mentioned one other name of someone who told you about Oscar being a jerk. Who was that again?
Jimmy Barton: Albert Plum. He's my boss. And my friend too. He was in that same class with Mother and Steve.
Detective Murphy: What did he tell you about Oscar?
Jimmy Barton: Not much really. He just told me the best thing I could do would be to stay as far away from Oscar as possible.
Detective Parker: How did you miss out on meeting Oscar when so many other people ran into him. Where were you? Did you leave the park?
Jimmy Barton: Oh no. I had walked over to the lake with Todd and Steve to do some fishing.
Detective Parker: Todd?
Jimmy Barton: Todd is a good friend of mine, and he's Steve's son.
Detective Murphy: All right, let's go back to Pauline. What did she tell you?
Jimmy Barton: Basically, the same thing I heard from everyone else. Oscar was a jerk, didn't smell good, and seemed to be on a mission.
Detective Murphy: A mission?
Jimmy Barton: Yes. I guess it was a mission to cause trouble because that's exactly what he did. Do you need anything else? I really need to get to work.
Detective Parker: We're almost finished. Do you know of anyone who would want Mr. Knight dead?
Jimmy Barton: Probably anyone who knew him from what I hear. But if you mean do I know anyone personally who actually said they'd like to kill him, no.
Detective Parker: OK, you can go. If we need to talk to you again, we'll contact you.
Jimmy Barton: Sure thing. Good luck.
Interview ended: 8:24 a.m.


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AREMA Railway Careers Network
Due to an internal promotion, Union Sanitary District (USD) is now recruiting for an Organizational Performance Program Manager (OPPM). The OPPM conducts and coordinates strategic planning efforts, performance measurement, continuous improvement and organizational development processes to maintain and improve the District's overall effectiveness and performance; facilitates collaborative problem-solving among and between teams and workgroups; and acts as an internal strategic and organizational consultant to management and staff. This position works extensively with the six-member Executive Team and performs special projects as assigned by Management.
Applicants must apply online at http://www.unionsanitary.ca.gov by 4:00 p.m., Friday, June 9, 2017.
You will need the following education and experience. A typical way to acquire the knowledge, skills and abilities for this position include:
Additional Salary Information: •CalPERS retirement 2.5% at 55 formula. (If new to CalPERS or not from an agency with reciprocity with CalPERS, the formula is 2% at 62 by pension reform law.) •Medical, dental and vision insurance substantially paid for by the District for the employee and family. •Two weeks'' vacation per year (increasing with longevity); and 13 paid holidays per year. •Management Administrative Leave. •STD, LTD and Life Insurance paid by the District. •Alternative Work Week Schedule. •Deferred Compensation Plan with District Match. •Educational Reimbursement, Student and Computer Loan programs for non-probationary employees.


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Baymont Inn & Suites
Select your stay dates to check availability at Baymont Inn & Suites
Baymont Inn & Suites
About the Hotel
Property Location
Located in Indianapolis (East Indianapolis), Baymont Inn & Suites is close to Mid-America Motorcycle Speedway and Pleasant Run Golf Course. This hotel is within the vicinity of Great Times Fun Park and Greatimes Familiy Fun Park.
Make yourself at home in one of the 75 air-conditioned rooms featuring refrigerators and flat-screen televisions. Complimentary wired and wireless Internet access keeps you connected, and cable programming provides entertainment. Bathrooms feature shower/tub combinations, complimentary toiletries, and hair dryers. Conveniences include safes and desks, as well as phones with free local calls.
Enjoy a range of recreational amenities, including an indoor pool, a spa tub, and a fitness center. Additional amenities at this hotel include complimentary wireless Internet access and a television in a common area.
Mingle with other guests at a complimentary reception, held on select days. A complimentary hot/cold buffet breakfast is served daily.
Business, Other Amenities
Featured amenities include complimentary high-speed (wired) Internet access, a 24-hour business center, and express check-out. Free self parking is available onsite.
Hotel Facilities
- Television in common areas
- Fitness facilities
- Number of buildings/towers - 1
- Free self parking
- 24-hour business center
- Free WiFi
- Total number of rooms - 75
- Express check-out
- Free breakfast
- 24-hour front desk
- Indoor pool
- Free newspapers in lobby
- Spa tub
- Accessible bathroom
- In-room accessibility
- Smoke-free property
- Free wired high-speed Internet
- Coffee/tea in common areas
- Elevator/lift
- Laundry facilities
- Number of floors - 3
- RV, bus, truck parking
- Free reception
Room Facilities
- Free cribs/infant beds
- Premium bedding
- Refrigerator
- Coffee/tea maker
- Hair dryer
- Television
- Daily housekeeping
- Free wired high-speed Internet
- Microwave
- Free toiletries
- Cable TV service
- Free WiFi
- Connecting/adjoining rooms available
- Free weekday newspaper
- Flat-panel TV
- In-room safe
- Shower/tub combination
- Desk
- Phone
- Iron/ironing board
- Free local calls
- Air conditioning
Hotel Policy
Know Before You Go
- The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
The following fees and deposits are charged by the property at time of service, check-in, or check-out.
- Pet fee: USD 15 per accommodation, per night plus USD 50 one-time cleaning fee
The above list may not be comprehensive. Fees and deposits may not include tax and are subject to change.Show all hotel policy Show less hotel policy


text:
Sagittarius Love Match – Valentine's Day Special
A fun, mutually stimulating relationship that will need constant novelty to sustain itself. The initial phase of this relationship is bound to be certainly rocking, given each person's love and need for amorous, exciting times, but their independent views and ways are sure to strew this path with challenges, at the later stages.
This can be one of the good matches of the Zodiac, for the wandering soul of the Sagittarius finds a perfectly stable foundation in the solid Taurus. This relationship is likely to work out better, if the woman in this pair is Sagittarius. The sexy and modest Taurus will always be there for the she-Archer, and she will love it! So, it will be all sweets, until the Bull gets a tad too clingy.
They are similar and yet naturally opposite to each other. What happens when they come together? Fireworks! The chemistry is magnetic, and often lasting, provided each respects and honours the other's need for space and freedom. They can beautifully delegate the tasks, where Gemini takes care of the bigger picture, letting the Archer care for the details.
Let's accept that these two are essentially very different people – Sagittarius loves to wander, while the Cancer loves to cling to his home; the Archer is a free bird, while the Crab feels the happiest when tied in a loving bond. So, there's not much in common, you see! The difference may be intriguing for starters, but for the long-term, not much can be said.
Although the Leo too is an independent person and indulges harmless flirting, he can't handle the Sagittarius's tendency to take the matters of the heart lightly beyond a limit. And hence, while there may be scintillating physical chemistry in this relationship, it may lack emotional connection as the eternal wander of the Zodiac – Sagittarius – would not make a long-term commitment easily.
On the face of it, this may be quite a stable relationship. But go little deeper and you will find the problem areas! To say the least, the punctual Virgo cannot ignore the Sagittarius unreliability and the Sagittarius simply can't understand why the Virgo wants everything to be perfect. The relationship will succeed only if both of them go an extra mile to nurture it.
Do these Zodiac Sign based guidelines satisfy your curiosity about your Love Match? You can get our Free Love Horoscope Reading to know even more!
When these two first get together, the chemistry is stunning! The relationship is amorous and intense, as each loves the wild abandon of the other. The love story continues happily till the Archer's lover-of-dreams Libra starts getting clingy. As the 'couple' perspective seeps in, the Archer may start feeling claustrophobic, and so shall begin the trouble.
This one can be a physically breathtaking bond! There may be too many 'ifs' and 'buts', yet this relationship has the potential to work. Why? Because, each is vastly different from the other, and thus can act as a perfect complement to the other, if a workable, practical arrangement is to be made. Yes, there will be adjustments to be made, but aren't they always there! The irresistible Scorpio may, however, not approve of the free, errant ways of the Archer.
The Sagittarius loves himself to the core, and above anyone else! And so, it's very easy for them to fall hook, line and slinker for their mirror image! No wonder, there exists a long-term potential in this fiery relationship, but there are strings attached. They both are capable of feeling very bound and limited in a committed bond, which may take them their separate ways, at least temporarily. Plus, each will have to honour the other's need to come first!
The Sagittarius is turned on by power, money and status, and the Capricorn often manages to achieve all that and even more. But, here's a difference! The Capricorn believes in earning it all, while the Sagittarius may not have that much patience – the patience to wait on the sidelines, as the Goat goes about conquering self-constructed milestones. Also, the stable and modest Capricorn may not find much allure in the Archer's over-the-top, wavering tendencies.
Both the Signs are attuned well to each other's need for freedom and space. Hence, the scope for jealousies, possessiveness and insecurities is rare. But, the Sagittarius may be more egocentric than the Aquarius, which will greatly affront the Aquarius. Yet, the eternal humanitarian Aquarius will not say it loud – but, he will find his separate way! Physically, this will be a happy and exciting relationship, though.
This one pair has a great possibility of a fantastical romance and an utterly satisfying physical union. The clandestine Pisces meets the in-your-face Sagittarius, and the fireworks ensue! But, both are very different people, and the journey ahead would not be struggle or hurdle free. Each needs his/ her own space, but in a very different way. The dreamy Pisces, however, may not resent much the Archer's errant ways, for they (the Pisces) may not even notice, when lost in their dreams.
Happy with the aforementioned information? Does it satisfy all your confusions regarding your love equation this Valentine's Day? If no, then we suggest you get a personalised, Birth Chart based reading that will brighten your life and love – Love Ask 3 Questions – 100% tailor-made, Horoscope-based answers and remedies to put right your love equation on this Valentine's Day.
Read here about other Signs and their Love Equation this Valentine's Day!
25 Jan 2017


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The effect of family structure on parents' child care time in the United States and the United Kingdom
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s11150-007-9017-y
- Cite this article as:
- Kalenkoski, C.M., Ribar, D.C. & Stratton, L.S. Rev Econ Household (2007) 5: 353. doi:10.1007/s11150-007-9017-y
- Citations
- 302 Downloads
We use time-diary data from the 2003 and 2004 American Time Use Surveys and the 2000 United Kingdom Time Use Study to estimate the effect of family structure on the time mothers and fathers spend on primary and passive child care and on market work, using a system of correlated Tobit equations. Our results indicate that estimates are sensitive to the inclusion of a common household factor that controls for selection into family type. Estimates from the selection-controlled models indicate that single parents in both countries spend more time in child care than married or cohabiting parents, perhaps in part to compensate for the missing parent, but that there is no difference in the time allocation of married and cohabiting parents. There are substantial cross-country differences, however, as single parents in the U.S. work more than other parents and single parents in the U.K. work less.


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Not quite what you're looking for? View similar properties or start a new property search.
Lovely 7-bedroom family house for sale in the desirable Puerto Santa Maria area with gardens, pool and stables as well as excellent access to Puerto Sherry and the beaches
- 7 Bedrooms
- 5 Bathrooms
- Size 400m² (4,305ft²)
Comfortable 7-bed family house in the desirable area of Puerto Sta Maria close to Puerto Sherry and beaches. Stables, gardens, guest or staff house and a pool. Easy access to town centre and local amenities
This spacious family house near Puerto Santa Maria in Cadiz is built on one level consisting of 5 bedrooms and has a 2-bedroom staff or guest house. The main house consists of a large reception room, dining room and TV room leading to the kitchen area with a utility room, pantry and staff area. Lovely lawned garden with mature trees and private pool area and barbeque. What's more, there are 3 stables behind house with a paddock area covering a total of 6,200m². The property benefits from mains electricity and central heating but has its own private well for the water supply. There are terraced areas all around the house and a garage onsite.
- Swimming pool
- Terrace
- Garden
- New build
- Parking
All on one floor:
- Gardens, pool, garage and terraces
- Large living room
- Dining room
- TV room
- Equipped kitchen
- Utility room and service area
- Pantry
- 5 bedroom
- Guest or staff house with a further 2 bedrooms
- Air conditioning
- 3 Stables
- Paddocks
- Pool
- Secluded position but in prime location of Puerto Sta Maria
- Staff/guest Cottage
- Own well
- No IVA (VAT)
- Land available to develop
Property located in a sought-after areaof Puerto Santa Maria, Cadiz.
- 3 minutes to the heart of Puerto Santa Maria
- 3 minutes to Puerto Sherry
- 25 minutes to Jerez and the airport
- 3 minutes to the beach
- 1 hour 10 minutes to Sevilla and its airport
- 1 hour 10 minutes to Gibraltar
View more properties for sale in Cadiz
Contact us about this propertyLucas Fox Marbella
+34 951 319 994
- €1,500,000
- Equestrian Property For sale
- 7 Bedrooms, 5 Bathrooms
- Size 400m² (4,305ft²)
- Plot size 6,200m² (66,736ft²)
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A 12.03-carat blue diamond has been auctioned at Sotheby's in Geneva for $48.4m (£31.7m) to a Hong Kong tycoon.
Joseph Lau confirmed he bought the ring-mounted, cushion-shaped stone for his daughter, renaming it "Blue Moon of Josephine" after her.
He is also confirmed as the buyer of a 16.08-carat pink diamond, which Christie's sold for $28.5m on Tuesday.
Lau, a convicted property billionaire, has a history of buying precious stones for his daughter.
In 2009, he bought another blue diamond for her - the 7.03-carat "Star of Josephine" - paying what was then a record $9.5m.
The pink diamond purchased earlier this week has been renamed "Sweet Josephine", a spokeswoman for Mr Lau told BBC News.
Last year he was sentenced to five years in jail in Macau for corruption, but has avoided prison by not visiting the territory. There is no extradition treaty between Macau and Hong Kong


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pp 69-100
Introducing Imperative Programming
- Don Syme
- , Adam Granicz
- , Antonio Cisternino
* Final gross prices may vary according to local VAT.Get Access
In Chapter 3 you saw some of the simple but powerful data types and language constructs that make up F# functional programming. The functional programming paradigm is strongly associated with "programming without side effects," called pure functional programming. In this paradigm, programs compute the result of a mathematical expression and do not cause any side effects, except perhaps reporting the result of the computation. The formulae used in spreadsheets are often pure, as is the core of functional programming languages such as Haskell. F# is not, however, a "pure" functional language; for example, you can write programs that mutate data, perform I/O communications, start threads, and raise exceptions. Furthermore, the F# type system doesn't enforce a strict distinction between expressions that perform these actions and expressions that don't.
Chapter Metrics
Reference tools
Other actions
- Title
- Introducing Imperative Programming
- Book Title
- Expert F#
- Pages
- pp 69-100
- Copyright
- 2007
- 10.1007/978-1-4302-0285-1_4
- Print ISBN
- 978-1-59059-850-4
- Online ISBN
- 978-1-4302-0285-1
- Publisher
- Apress
- Copyright Holder
- Don Syme, Adam Granicz, and Antonio Cisternino
- Additional Links
- Topics
- Industry Sectors
- eBook Packages
- Authors
Continue reading...
To view the rest of this content please follow the download PDF link above.


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I need to now what size is two twin size beds when put together?
**its a king
What size are two twin beds?
Two twin mattresses laid side by side are 78 inches wide and 75 inches long. If the two mattresses are extra long twins, then they are 78 inches wide and 80 inches long.Source:Better Homes and Gard...
Read More »
What is the length of twin size beds?
The standard size of a twin size bed is 75 inches. However, according to Dimensions Guide, there is an extra-long twin size bed, which is 80 inches in length, and the super single bed (twin size) h...
Read More »
How much are twin size adjustable beds?
The cost of a twin size adjustable bed depends on the manufacturer and store. For example, an Elite ECTB-set costs $1,049 at TotallyFurniture.com while a luxury model costs $1,491 at UcanHealth.com...
Read More »
How much are twin-size adjustable beds?
Twin-sized adjustable beds with mattress, steel frame, stainless steel chromed mattress guard and fabric color cost approximately $1,100 for a standard model. A luxury twin size adjustable bed with...
Read More »


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ImpressArt doesn't stop at offering high-quality stamps. It also offers quality metal stamping helpmates to make your custom metal stamped jewelry the very best.
- Stamp Straight Tape provides a thick, strong ridge to line metal stamps against, allowing precision and accuracy when using letter stamps. It is non-residual and leaves a clean surface on metal after use.
- The stubby brass hammer is designed especially for metal stamping. The short, ergonomic handle makes this hammer easy to handle and helps prevent double impressions. The soft brass head is easy on your stamps, ensuring they'll last longer.


text: Hello, I have read through all of what is written on this topic. I still cannot figure out how to have my banner ads start automatically after they are uploaded. The banner shows as "loading" and never starts. If I right-click the banner and select "play" the ad begins, no problem. What am I doing wrong? How can I get the ads to begin automatically? http://www.andydahl.net/My_Ad.swf for a sample of what is happening! I'm using RapidWeaver for my sites, modifiying the code to accept the swf file, and then ftping the file and assets folder to my web host (1&1). And help will be much appreciated! Thanks, Andy email@example.com Mac OS X


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Posted by Mc Timothy Associates- VERIFIED EMPLOYER VERIFIED27 Jan 3:53 pmIkeja, Lagos
Mc Timothy is a full service Management consulting, Recruitment and Business Training firm that enables business greatness and effectiveness through customer-centric innovative solutions. We provide leading edge management strategy and HR infrastructure support with exceptional customer service leading to improved performance of our client organisations.
Our client required the services of an experienced person to fill the post of a Marketing Manager urgently
To develop and execute a fully integrated brand marketing communications plan supporting the overall brand strategy.
To promote and implant the company's brand nation-wide to enhance our culture and essence.
Make decisions relating to marketing aspects of brand internally and externally and deliver promotional activities.
Develop a strategy that will build a strong brand that is fit for purpose and will support our business objectives.
Develop and implement a brand research, tracking programme that will monitor the brand and build an understanding of brand perception.
Make informed decisions on brand development, support new brand activities, ensure the brand values and culture are reflected accurately.
Liaising with legal and compliance personnel, to ensure the brand collateral is registered for trademark and intellectual property rights where necessary and that this value is understood
Carry out marketing strategy development by analyzing market trends & consumer insights and developing business opportunities for brands.
Ensure marketing communications plan for the brandincludes internal and external customers through strong stakeholder planning.
Monitor customer and supporter reactions to brand through focus groups and market research ensuring this feeds into the brand communication planning.
Implement the brand guidelines – making sure they are followed and adapted appropriately.
Oversee the production of marketing products through a variety of channels which may include TV and radio adverts, newspaper and magazine advertisements, email or on-line campaigns, and road shows.
Use all necessary channels to ensure new staff and volunteers understand their roles in replicating and promoting the brand such that the organisation breathes the brand
Develop and communicate strong brand messages reinforced through media stories, award success, and internal story telling
Work with HR to ensure that living the brand is evident in throughout our HR practice
Co-ordinate and deliver a range of activities inline with our company's goal and objectives
Responsible for ensuring staff compliance /adherence to established internal control procedures for financial and other transactions;
Meet marketing and sales financial objectives by forecasting requirements; preparing an annual budget; scheduling expenditures; analyzing variances; initiating corrective actions.
Accomplishes marketing and sales objectives by planning, developing, implementing, and evaluating advertising, merchandising, and trade promotion programs; developing field sales action plans.
Identifies marketing opportunities by identifying consumer requirements; defining market, competitor's share, and competitor's strengths and weaknesses; forecasting projected business; establishing targeted market share.
Protects organization's value by keeping information confidential.
Accomplishes marketing and organization mission by completing related results as needed.
Engage with cross functional resources (leadership, Quality, Supply Chain) in Head Office and use their support to successfully implement market activation activities on ground
Gain Management approval and successfully launch new products.
The post holder will be working in a developing environment and he/she will therefore be expected to undertake other appropriate duties as required for the effective operation of the company
Respect the unique contribution of every individual and work positively in an equal opportunity and diverse environment
Researching consumer markets, monitoring market trends and identifying potential areas in which to invest, based upon consumer needs and spending habits
Generating names for new and existing products and services, coming up with ideas for new packaging designs, including shape, size, colours, fonts and imagery
Supervising the sign off of marketing literature and campaigns, liaising with legal and compliance personnel, ensuring the designs and messages meet the company brand and regulatory guidelines
• Minimum of HND/BSc in any relevant field with appreciable knowledge in fast food industry.
• Experience in the application of digital marketing and social media as part of integrated marketing campaigns.
• Minimum of three (3) years' experience
Interested Applicant should submit their CV or Click the "Apply Button"
Application Deadline 20-Feburary-2016
Stay safe!
Watch out for:


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Sea Breeze, a delightful, very comfortable, terraced cottage in a spectacular waterfront position in Noss Mayo, has its own waterside terrace and summerhouse to enjoy the glorious views of the Yealm estuary, whatever the weather. The estuary, sheltered by wooded hillsides, filled with all manner of boating craft, is home to a wonderful variety of bird life and is one of the prettiest in Devon. A short walk takes you into the centre of the village with its excellent waterfront restaurant inns. Sea Breeze is perfect for exploring this area of outstanding natural beauty, close to the scenic South West coastal path, the sandy beaches of Mothecombe and Wonwell and just a short drive from the Cornish border.
The front door opens from the roadside into a spacious, attractive kitchen/diner. Kitchen with dresser and dining table to seat 5, gas hob, electric built-under oven, dishwasher and microwave. The room features an original slate floor and beams that originate from Napoleonic times! Four steps lead down to the sitting room with pretty, decorative Victorian fireplace, two comfortable sofas, TV with DVD, stunning views onto the estuary and French doors out to the terrace. Carpeted stairs lead up from the kitchen to a pretty double bedroom (4ft 6in bed) with dreamy, far reaching water views and a small decorative fireplace. Further stairs up to a spacious twin bedroom with TV/DVD and 3ft single beds, one with a 3ft pull-out beneath. Adjacent bathroom with bath, shower over, basin and WC.
The cottage has two terrace areas overlooking the water, one with table, chairs and built-in seating around the enclosing walls – a stunning spot to enjoy the views and summer sunshine. The delightful summer house, accessed from the first terrace, is comfortably furnished with a sofa and enclosed with large patio doors, so even on inclement days the scenic views can be appreciated. This room extends on into a utility area housing a washing machine, a small table top freezer and shower room with basin and WC. There is gated access from the lower terrace, via steps, down to a jetty on the estuary and the property's private running mooring. Please Note: Children will need supervision on the terraces at all times.
Parking: On road parking is usually available close to Sea Breeze on The Hard or on nearby Pillory Hill. There is also a free car park in the village centre. It is possible to unload across the road for a short space of time.


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Lolita Sandals High Platform Shoes PU Leather with Buckles
( 3 Reviews )
I love this cute shoes .You never feel tired even the tall heel .The design is unique .I wanna say thanks .The customer service representative Selena gave me good suggestions on size choosing .
By joyce Aug 29, 2012View All Reviews
Fantastic Buckle PU Leather White Lolita Sandals
I was so excited that I have bought this pair of shoes. It is just what I thought. I don't like high heels, I like wedge heels. It is very comfortable.
By Renata Aug 31, 2012View All Reviews
White Chunky Heel PU Platform Bow Ankle Strap Lolita Sandals
I love the design of this pair of shoes. It is comfortable too. This is my third lolita shoes from Milanoo. I will come again.
By Esther Sep 07, 2012View All Reviews
Black Lolita Sandals Chunky Heels Platform Ankle Straps Bow
( 2 Reviews )
The shipping is faster than planed .I am quite surprised the quality .The shoes is nice and very comfortable to wear .I love it!
By Vicky Aug 27, 2012View All Reviews
PU Leather Bow White High( 3-3.9") Open Toe Lolita Sandals
I like the bow and the ties. It is cuter than I thought. Really amazing. I have told my friends where to get a great pair of lolita shoes.
By Fay Sep 07, 2012View All Reviews
Wedge Heel PU Platform Lolita Sandals
The product was just as pictured. They're really cute and fairly comfortable. Though I'm not quite used to them and can't wear them for prolonged periods of time yet as they are a little tight on my toes and theres not much breathing room. Also, maybe don't wear these in warm or hot weather, they are a leather type material and they'll make your feet sweat after a while outside in warm weather.
By Becca May 14, 2015View All Reviews
Gothic Black Lolita Sandals High Chunky Heels High Plarform Ankle Straps Buckles
prodotto di ottimo materiale, non vedo l'ora di indossare! attenzione alle spese doganali!
By Vanessa Puggioni Aug 23, 2016View All Reviews


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Are you required to log in to use Wikianswers?
1,074,936questions onAdd New Page
No, you can use Wikianswers without being logged in, just as with every Wikia wiki. However, creating and using a free account has several benefits, such as your IP address not being publicly displayed, following pages, email notifications, and many more.


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Definition of outlet
1a : a place or opening through which something is let out : exit, ventb : a means of release or satisfaction for an emotion or impulse sexual outletsc : a medium of expression or publicationd : a publication or broadcast organization media outlets
2 : a stream flowing out of a lake or pond
3a : a market for a commodityb : an agency (such as a store) through which a product is marketed retail outlets
4 : a receptacle for the plug of an electrical device
Examples of outlet in a Sentence
this road is the only outlet for traffic coming from the racetrack
technology has provided many new outlets for amateur writers and artists to share their work with a global audience
Origin and Etymology of outlet
Middle English ut-lete, from ut out + -lete watercourse, from Old English gelǣt, from lǣtan to let
First Known Use: 13th century
Other Business Terms
OUTLET Defined for English Language Learners
Definition of outlet for English Language Learners
: something that people use to express their emotions or talents
: a store that sells products made usually by one company and often at reduced prices
: a television, radio, or publishing company
OUTLET Defined for Kids
Definition of outlet for Students
1 : a place or opening for letting something out The lake has several outlets.
2 : a way of releasing or expressing a feeling or impulse I needed an outlet for my anger.
3 : a device (as in a wall) into which the prongs of an electrical plug are inserted for making connection with an electrical circuit
Medical Dictionary
Medical Definition of outlet
1: an opening or a place through which something is let out the pelvic outlet
2: a means of release or satisfaction for an emotion or impulse counseling provides an outlet for expressing and evaluating their feelings—Vicki Glaser
Learn More about outlet
See words that rhyme with outlet Thesaurus: All synonyms and antonyms for outlet Spanish Central: Translation of outlet Nglish: Translation of outlet for Spanish speakers Britannica English: Translation of outlet for Arabic speakers
Seen and Heard
What made you want to look up outlet? Please tell us where you read or heard it (including the quote, if possible).


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Promotional Sewing Kits
Promotional sewing kits are an affordable giveaway your recipients will really appreciate. These compact kits easily fit in a purse or suitcase and contain all the essentials for making simple clothing repairs at home or on the go.
Personalized sewing kits are the perfect giveaway for dry cleaners, travel agencies, hotels, or any business that wants to align itself with a handy product that offers real convenience.
|Copyright © 1996 - 2017 Motivators Inc. Promotional Products All Rights Reserved.


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Yes, I know. Every time I talk about the innovative electronic features that auto and truck makers are putting into their vehicles, you tell me that you are already smartest, safest drivers... Read more
We haven't driven GMC's new mid-size Canyon yet. Its introduction isn't scheduled until fall, but I suspect there will be a range of new features designed to set this truck... Read more
I know. I know. You think a minivan is so uncool. But, if you have a gang of kids that you are taking out to one of the neighborhood lakes stocked by the Texas Department of Parks and Wildli... Read more
If you want to look dangerous when you are behind the wheel, the people at Ram are making it easy for you. The Black Package, which was successfully brought to market on the 1500-level pick... Read more
What is happening with General Motors? I hate to do negative articles about any single vehicle or supplier, but a thinking person has to question the number of serious recalls that GM has be... Read more
Imagine, if you will, a sportsman's world without Ford Super Duty trucks. Since they account for more than half of the big, bad truck category, there would be an equally big hole in t... Read more
While I am working on a major truck feature for the print version of the April issue of the magazine, this was important enough to require a quick piece to let you know about two recalls tha... Read more
While there is no formal NHTSA recall as yet, Toyota last week stopped sales and delivery of about 36,000 vehicles already in dealerships. The affected models include 2013 and 2014 Tun... Read more
Earlier this week in Dallas I had a chance to climb into and around Ford's 2015 F-150. My first impression is that this is a worthy successor to the iconic trucks that have prece... Read more
It is extremely rare for one vehicle manufacturer to win both the Car of the Year and Truck of the Year honors at the North American International Auto Show in Detroit, but Chevy proved... Read more


text:
Driver Magician 4.7 inclu Keygen
Driver Magician 4.7 inclu Keygen
Driver Magician offers a professional solution for device drivers backup, restoration, update and removal in Windows operating system. It identifies all the hardware in the system, extracts their associated drivers from the hard disk and backs them up to a location of your choice. Then when you format and reinstall/upgrade your operating system, you can restore all the "saved" drivers just as if you had the original driver diskettes in your hands. After one system reboot, your PC will be loaded and running with the required hardware drivers.
What's more, Driver Magician has a built in database of the latest drivers with the ability to go to the Internet to receive the driver updates. It saves lots of time to find the correct drivers and mature drivers will obviously increase the performance of hardware. If there are unknown devices in your PC, Driver Magician helps you detect them easily and quickly with its built in hardware identifier database.
- Back up device drivers of your computer in four modes.
- Restore device drivers from backup in one mouse click.
- Update device drivers of your PC to improve system performance and stability.
- Uninstall device drivers
- Live Update device identifier database and driver update database.
- Detect unknown devices.
- Back up more items such as My Documents and Desktop.
- Restore more items from backup.
- Get detailed information of the hardware drivers.
- Clone all drivers to an auto-setup package (.exe), so you can restore drivers without installing Driver Magician.
v4.7 (2015-08-02):
- Support Windows 10
- Some minor tweaks
1. Disconnect Internet connection
2. Run app's setup
3.After installation,Copy Patch & Keygen it into install directory & apply it.Use Keygen for registration
4. Block its IneterNet connection with firewall
5. Disable anti virus but not virus 100% Secure
Release Date: 2015-08-02
OS: XP/2003/Vista/7/8/10
Language: Multilingual


text:
Yonge Eglinton Laser Eye + Cosmetic CentreLASIK - Laser Eye Surgery, Ophthalmology, Minimally Invasive Surgery (BOTOX®), CataractsOther Doctors in this Practice
Contact Information
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Arnold Kravetz, MD, FRCSC
What Sets Us Apart
At Yonge Eglinton Laser Eye & Cosmetic Centre you will only find the best of the best willing to assist you and make you feel as comfortable as possible through every step of your procedure.
University of Toronto Medical School - Medical Degree and Residency in Family Practice/Medical and Surgical Ophthalmology
Other Affiliations
Diplomate of the American Board of Ophthalmology
Fellow of the Royal College of Physicians and Surgeons of Canada
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ED Magedson – Founder
Ripoff Report
Quality Paperback Book ClubInternet USA
Quality Paperback Book Club Book club scam Internet
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Fix it the right way.
Corporate Advocacy Program™
I bought books from these people and they automatically enrolled me in their book club. I am getting books every month from them and they are charging me $45-$55 for each delivery. I am sending the boxes back unopened. Still waiting on a refund to show up in my account. Cannot "talk" to a real person. To go forward I HAVE to open an account. I do not want to open an account. Argh
This report was posted on Ripoff Report on 05/03/2013 06:04 AM and is a permanent record located here: http://www.ripoffreport.com/reports/quality-paperback-book-club/internet/quality-paperback-book-club-book-club-scam-internet-1048134. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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Part of the contract
SUBMITTED: Tuesday, November 05, 2013POSTED: Tuesday, November 05, 2013
I've been a QPB.com member twice. I have successfully stopped my membership twice when my life no longer matched well with QPB's service.
Under the terms of the club buying any books from them is signing up for the club. Since you bought books you already have an account.
Under the terms of the club if you do not log in and opt out of the monthly book you have agreed to purchase it and have it sent to you. Returning the package is only you mailing a book you own to someone else. *You must refuse the book in advance in order to not be charged for it.*
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