Putative Sp1 Binding Sites Research Articles

An epigenetic mechanism regulates germ cell-specific expression of the porcine Deleted in Azoospermia-Like ( DAZL) gene

The Deleted in Azoospermia-Like ( DAZL) gene is specifically expressed in fetal and adult gonads. While DAZL is known to play a role during gametogenesis, the mechanisms governing its germ cell-specific expression remain unclear. We identified the 5′ untranslated region (UTR) of the porcine DAZL gene and cloned and characterized 2 kilobase pairs of its TATA-less 5′ flanking region, identifying CpG-rich regions within the proximal promoter. Nine of 18 CpG sites in proximity to one region were largely unmethylated in germ cells but hypermethylated in somatic cells, suggesting that DNA methylation may regulate DAZL promoter activity. Furthermore, DAZL expression was induced in fibroblasts treated with a demethylating agent. Deletion analyses revealed that the minimal 149 base pair promoter region was sufficient to activate transcription. In vitro methylation of a reporter construct corresponding to these 149 base pairs resulted in complete suppression of DAZL promoter activity in primordial germ cells, further supporting a role for methylation in regulating DAZL expression. Interestingly, the differentially methylated region was shown to harbor several putative Sp1-binding sites. Mutation of only the most highly conserved site significantly reduced promoter activity in a reporter assay. Furthermore, gel shift assays revealed that Sp1 was able to specifically bind to this site, and that complex formation was inhibited when CpG dinucleotides within this region were methylated. Chromatin immunoprecipitation (ChIP) assays revealed that in vivo Sp1 binding to the core DAZL promoter region was enriched in germ cells but not in fibroblasts. Our data suggests that DNA methylation may suppress DAZL expression in somatic cells by interfering with Sp1 binding. This study provides insights into the potential mechanisms underlying the regulation of germ cell-specific gene expression.

Promoter characterization and regulation of expression of an imprinted gene SLC22A18AS

The SLC22A18/ SLC22A18AS genes are a sense–antisense pair located at human chromosome segment 11p15.5. These genes are paternally imprinted: paternal alleles are silenced and maternal alleles are expressed. Although SLC22A18 is a well-characterized gene, very little is known regarding its antisense partner SLC22A18AS. We therefore sought to identify the potential cis-regulating elements including the promoter of this gene, differentially methylated regions (DMRs) and the translation of its putative ORF. Dual promoters (P1 and P2) were identified for this gene and both are devoid of consensus TATA and CCAAT boxes. However, the P1 promoter harbors a putative Sp1 binding site. Sp1 binds to the P1 promoter in vivo and positively regulates its activity. Promoter and CpG II island regions showed heavy methylation of CpG sites, but no DMRs were observed. Treatment of a non- SLC22A18AS expressing HuH7 cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine restored expression of this gene. The histone deacetylase (HDAC) inhibitor Trichostatin-A, on the other hand, failed to induce its expression. We suggest that the expression of this gene is methylation-dependent, but histone acetylation-independent. This gene was found to be translated with a cytoplasmic localization. The present data will help to understand the regulation of this gene and its role in tumorigenesis.

Identification and Characterization of the Human Testes-Specific Protease 50 Gene Promoter

Testes-specific protease 50 (TSP50) has been identified as one of the testis-specific proteins that is expressed at high levels in approximately 92% of human breast cancer samples, making it an attractive molecular marker and a potential target for diagnosis and therapy. However, little is known about the transcriptional mechanisms controlling TSP50 gene expression. In the present study, we have characterized the 5' regulatory region of the TSP50 gene in order to understand the molecular mechanisms regulating its expression. Analysis with a series of deletions demonstrated that a 624-bp region was essential for the basal promoter activity of the TSP50 gene. Further analysis results indicated that the two fragment regions +231 to +251 and -22 to -8, especially the putative Sp1 binding site (+237 to +239) and the putative CCAAT/enhancer binding protein (C/EBP) binding site (-15 to -13), are more important for the basal transcription activity of the human TSP50 promoter. Overexpression of Sp1 and C/EBPbeta transcriptional factors upregulated the activities of the TSP50 promoter. Taken together, these results will help to better understand the role of the TSP50 gene in signal-dependent transcriptional regulation, and to develop new reagents for therapeutic downregulation of the TSP50 gene in human breast cancer.

Transcriptional auto-regulation of the dopamine receptor regulating factor (DRRF) gene

The murine dopamine receptor regulating factor (DRRF) gene is transcribed from a TATA-less promoter that has several putative Sp1 binding sites. The present investigation identifies functional transcription factors that modulate the expression of this gene. In the D2-dopamine receptor expressing NB41A3 cells, Sp1 potently activates transcription from the DRRF promoter in pCAT-DRRF-1153/+17, while DRRF itself effectively inhibits it. Sp1 also activates this promoter in pCAT-DRRF-310/+17. In competitive co-transfection experiments, DRRF represses the transcriptional activation induced by Sp1, while Sp1 de-represses the inhibitory effect of DRRF. Deletion of the 31 bp fragment between -1153 and -1122 decreased basal transcription down to about 60%. This fragment contains a functional AP1 binding site. In addition, deletion of the 129 bp region between -901 and -772 further decreased transcription. The latter region has a functional AP2 binding site. The present observations suggest that DRRF auto-regulates its own promoter by competing with Sp1 and that both AP1 and AP2 modulate expression of this gene.

Functional polymorphisms in carboxylesterase1A2 ( CES1A2) gene involves specific protein 1 (Sp1) binding sites

Carboxylesterase 1 (CES1) is involved in metabolic activation of a variety of prodrugs into active derivatives and plays an important role in pharmacokinetics. We previously reported that a single nucleotide polymorphism (SNP), −816A/C of the CES1A2 gene associates with the responsiveness to an angiotensin-converting enzyme (ACE) inhibitor, imidapril, whose activity is achieved by CES1. To identify relevant functional polymorphisms, we re-sequenced the CES1A2 promoter region (∼1 kb) in 100 Japanese hypertensive patients. Altogether 10 SNPs and one insertion/deletion (I/D) were identified, among which seven SNPs and one I/D residing between −62 and −32 were in almost complete linkage disequilibrium ( D′ = 1.00, r2 = 0.97). They consisted a minor and a major haplotype, the allele frequencies of which were 22% and 74%, respectively. The minor haplotype possessed two putative Sp1 binding sites while the major haplotype did not have any Sp1 binding site. The minor haplotype had a higher transcription and Sp1 binding activities than the major haplotype, in vitro. The original −816A/C was in high linkage disequilibrium with these haplotypes ( D′ = 0.92, r2 = 0.85), and well agreed with the efficacy of imidapril medication. These results suggest that the Sp1 binding site variation in the CES1A2 promoter is functional, and are good candidates for the pharmacogenetic studies of CES1-activated drugs.

Critical roles of Sp1 in gene expression of human and rat H+/organic cation antiporter MATE1

A H+/organic cation antiporter (multidrug and toxin extrusion 1: MATE1/SLC47A1) plays important roles in the tubular secretion of various clinically important cationic drugs such as cimetidine. We have recently found that the regulation of this transporter greatly affects the pharmacokinetic properties of cationic drugs in vivo. No information is available about the regulatory mechanisms for the MATE1 gene. In the present study, therefore, we examined the gene regulation of human (h) and rat (r) MATE1, focusing on basal expression. A deletion analysis suggested that the regions spanning -65/-25 and -146/-38 were essential for the basal transcriptional activity of the hMATE1 and rMATE1 promoter, respectively, and that both regions contained putative Sp1-binding sites. Functional involvement of Sp1 was confirmed by Sp1 overexpression, a mutational analysis of Sp1-binding sites, mithramycin A treatment, and an electrophoretic mobility shift assay. Furthermore, we found a single nucleotide polymorphism (SNP) in the promoter region of hMATE1 (G-32A), which belongs to a Sp1-binding site. The allelic frequency of this rSNP was 3.7%, and Sp1-binding and promoter activity were significantly decreased. This is the first study to clarify the transcriptional mechanisms of the MATE1 gene and to identify a SNP affecting the promoter activity of hMATE1.

TNF-α inducesMUC1gene transcription in lung epithelial cells: its signaling pathway and biological implication

The current study was conducted to elucidate the mechanism through which TNF-alpha stimulates expression of MUC1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-alpha exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-alpha-treated cells compared with controls. However, TNF-alpha did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-alpha increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-alpha-induced MUC1 promoter activation, and anti-TNFR1 antibody blocked TNF-alpha-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-alpha also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Sp1 binding site in the MUC1 promoter located between nucleotides -99 and -90. TNF-alpha-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-alpha induces MUC1 gene transcription through a TNFR1 --> MEK1/2 --> ERK1 --> Sp1 pathway.

Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer

Oligoarray analysis of a matched pair of prostate cancer and normal cell lines derived from the same radical prostatectomy specimen identified 113 candidate hypomethylated genes that were overexpressed in the cancer cells and contained CpG islands. Hypomethylation of wingless-related MMTV integration site 5A (WNT5A), S100 calcium-binding protein P (S100P) and cysteine-rich protein 1(CRIP1) was confirmed in the cancer cells by bisulfite sequencing. Treatment of the corresponding normal prostate epithelial cells 1542-NPTX with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-CdR) induced higher levels of mRNA expression and partial loss of methylation on these genes. Primary prostate cancers were tested using methylation-specific polymerase chain reaction. WNT5A was hypomethylated in 11/17 (65%) tumors, S100P in 8/16 (50%) and CRIP1 in 13/20 (65%). Bisulfite sequencing of a section of the 5' untranslated region (UTR) of WNT5A revealed that three CpG sites (15, 24 and 35) were consistently methylated (93%) in the normal cell line and normal tissues, but not in the prostate cancer cell line and eight primary prostate cancers. Multiple putative binding sites for the transcription factors SP1 and AP-2 were found adjacent to CpG sites 15 and 24. A putative c-Myb binding site was located within the CpG site 35. Anti-c-Myb antibody co-precipitation with WNT5A was methylation-sensitive in 1542-NPTX cells. It is likely that an epigenetic mechanism regulates WNT5A expression in prostate cancer.

생쥐 신경교세포 유래 신경영양인자 유도성 전사인자 (mGIF) 유전자의 유전체 구조 및 프로모터 특성 분석

To study the transcriptional mechanisms by which expression of the murine glial cell-derived neurotrophic factor inducible transcription factor (mGIF) gene is regulated, a murine genomic clone was isolated using a mGIF cDNA as probe. A 13-kb genomic fragment, which comprises 4-kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Sp1. The mGIF gene also has consensus sequences for AP2 binding sites. The transcriptional activity of five deletion mutants of a 2.1-kb fragment was analyzed by modulating transcription of the heterologous luciferase gene in the promoterless plasmid pGL2-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3. Transient expression assays suggested the presence of a positive regulator between -213 and -129 while a negative regulator was found in the region between -806 and -214. Relatively strong transcriptional activity was observed in neuronal NB41A3, glial C6 cells and hepatic HepG2, but very weak activity in skeletal muscle C2C12 cells. These findings confirm the tissue-specific activity of the mGIF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

DNA Methylation-dependent Epigenetic Regulation of Dimethylarginine Dimethylaminohydrolase 2 Gene in Trophoblast Cell Lineage

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

β1,4-Galactosyltransferase V Functions as a Positive Growth Regulator in Glioma

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1-->6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.

Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3

We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.

Functional analysis of two Sp1/Sp3 binding sites in murine Nanog gene promoter

Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5' flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity of Nanog gene. In this report, we identified the role of two putative Sp1 binding sites located in the Nanog gene 5'-flanking region in regulation of murine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Sp1 and Sp3. Furthermore, overexpression of dominant-negative Sp1 or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Sp1 and Sp3 are important for Murine Nanog gene expression.

Cross-species comparison of hepatic sex differences identified in transcript profiles

Screening of a foetal brain genomic DNA library allowed to isolate a 10-kb fragment of the gene encoding the human α2B-adrenergic receptor, that contained 5.5 kb of the 5′-flanking region, the open reading frame and 2.9 kb of the 3′-flanking region. The 1-kb fragment upstream from the start codon was rich in GC, lacked consensus TATA or CAAT box, but contained several Sp1-binding sites. Other potential cis-regulatory elements found in the 5′-flanking region included AP2, USF, Stat-6, NFκB and Olf-1. A single canonical polyadenylation signal (AATAAA) was found at position +3252/+3257 and the polyadenylation site was 3274 nucleotides downstream from ATG. Transfection experiments with chimeric luciferase contructs containing various truncated fragments of the 5′-region showed that the fragment −3160/+3 exhibited promoter activity in all tested cell lines and permitted the definition of a minimal 200-bp promoter (−603/−411) containing three putative Sp1-binding sites and two initiator elements. Transcriptional activity of this region was inhibited by the addition of mithramycin, a specific inhibitor of Sp1 binding to GC-rich sequences. The search for sequence variants within a fragment covering 1.7 kb of 5′-flanking region and the coding region allowed us to identify five novel single nucleotide polymorphisms. Interestingly, the G/C substitution at position −98 relative to the start codon was common and in complete linkage with a previously identified insertion/deletion polymorphism in the coding region which was showed to affect α2B-adrenergic receptor function. Based on transfection data and computer-assisted sequence analysis, the −98 G/C single nucleotide polymorphism was located within a portion of the 5′-UTR (−127/+3) affecting luciferase activity and it created additional putative binding site for Sp1. However, G/C substitution had no significant incidence on promoter activity in BHK-21 or HeLa cells.

Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors

The phosphate carrier (PiC) catalyses the import of phosphate into mitochondria where it is needed for ATP synthesis. We have analysed the 5'-flanking region of the human PiC gene and found that it has a single transcriptional initiation site and lacks a TATA box. Through deletion analysis of the -1213/-25 nt region, we identified an activation domain (-223/-25) and an inhibition domain (-1017/-814). The most effective promoter activity in transfected HeLa cells corresponded to the region containing putative binding sites for Sp1 (-163/-142; where Sp1 stands for stimulating protein-1) and CREB (-138/-116; where CREB stands for cAMP-response-element-binding protein). These DNA sequences were active in gel-shift assays in the presence of HeLa cell nuclear extracts or recombinant Sp1 and CREB respectively. Forskolin increased PiC promoter activity via the CREB site. Both footprinting and transfection of deletion constructs of the inhibition region (-1017/-814) showed that PiC silencer activity extends over 25 nt (-943/-919), which specifically binds two proteins present in HeLa cell nuclear extracts. These transcription factors were purified by DNA affinity, analysed by MS and identified as p54(nrb)/NonO (nuclear RNA binding protein) and PSF (protein-associated splicing factor). The PiC silencer region cloned in front of the ferritin promoter conferred a strong inhibition to the heterologous promoter. These findings may provide insight into control of PiC gene expression in different cell types and under different growth conditions. To our knowledge, this is the first study to analyse the regulation of the PiC gene expression in any cell.

Sp1 and Sp3 regulate the basal transcription of receptor activator of nuclear factor kappa B ligand gene in osteoblasts and bone marrow stromal cells

Receptor activator of nuclear factor kappa B ligand (RANKL), a potent regulator of osteoclast formation and function, is expressed by osteoblasts and bone marrow stromal cells. However, the molecular mechanism underlying RANKL expression in osteoblast/stromal cells remains largely unclear. Here, we characterized the molecular mechanism controlling RANKL basal transcription in osteoblast/stromal cells. We cloned a 1,103-bp murine RANKL promoter (from -953 to +150, relative to the transcription start site). Using a series of deletion mutants of the 1,103-bp promoter, we identified a 100-bp region (-154 to -54) mediating RANKL basal transcription in both osteoblasts and bone marrow stromal cells. Electrophoretic mobility shift assay (EMSA) using five overlapping oligonucleotides (Probes 1-5) spanning the 100-bp region showed that Probes 1 and 2 specifically bound nuclear proteins with high affinity from both cell types. Computer analysis revealed that Probes 1 and 2 contain a putative Sp1-binding site. Supershift assays with Sp1 and Sp3 antibodies confirmed that the nuclear proteins binding to Probes 1 and 2 are Sp1 and Sp3. Functionally, the mutation of the Sp1/Sp3 site in Probe 1 profoundly reduced the basal promoter activity while the mutation of the one in Probe 2 resulted in moderate reduction in the basal promoter activity. Moreover, the mutation of both sites abrogated the RANKL basal promoter activity, indicating that Sp1 and Sp3 play a key role in the RANKL basal transcription in osteoblasts and bone marrow stromal cells.

Neutrophil elastase stimulatesMUC1gene expression through increased Sp1 binding to theMUC1promoter

We previously reported MUC1 was a cell surface receptor for Pseudomonas aeruginosa, and binding of bacteria to cells was significantly reduced by pretreatment with neutrophil elastase (NE) (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181-L187, 2001). The current study was conducted to ascertain NE effects on MUC1 gene transcription, and MUC1 protein synthesis and degradation. A549 human lung carcinoma cells treated with NE exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Also, MUC1 protein shed into cell-conditioned medium was rapidly and completely degraded by NE. Actinomycin D blocked NE-stimulated increase in MUC1 protein expression, suggesting a mechanism of increased gene transcription that was confirmed by measurement of quantitatively greater MUC1 mRNA levels in NE-treated cells compared with controls. However, NE did not alter MUC1 mRNA stability, implying increased de novo transcription induced by the protease. NE increased promoter activity in A549 cells transfected with MUC1 gene promoter-luciferase reporter plasmid. This effect of NE was completely blocked by mithramycin A, an inhibitor of Sp1, as well as mutation of one of the putative Sp1 binding sites in MUC1 promoter located at -99/-90 relative to transcription initiation site. EMSA revealed NE enhanced binding of Sp1 to this 10-bp segment in a time-dependent manner. These results indicate the increase in MUC1 gene transcription by NE is mediated through increase in Sp1 binding to -99/-90 segment of MUC1 promoter.

Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family

The melanocortin-4 receptor (MC4-R) plays a key role in the hypothalamic control of food intake, lending importance to the understanding of the mechanisms that regulate its expression. To identify factors controlling the expression of the human (h) MC4-R gene, a fragment containing 1253 bp of the 5'-flanking region of the hMC4-R gene was isolated. A series of hMC4-R luciferase constructs were developed and used to transiently transfect HEK293 and GT1-7 cell lines, both expressing endogenous MC4-R mRNA. Deletion analysis of the 1253 bp fragment showed that the basal promoter activity is mainly restricted to the 179 bp upstream of the transcription start site in both cell types. Mutation of a putative Sp1-binding site located at position -76 bp resulted in a dramatic reduction of the luciferase activity in HEK293 and GT1-7 cells by 87 and 80% respectively. Both in vitro and in vivo studies (gel shift and chromatin immunoprecipitation analyses) revealed binding of both Sp1 and Sp3 to this site in HEK293 cells. Cotransfection with an Sp1 expression vector in Drosophila cells that do not express Sp1, in conjunction with treatment of HEK293 cells with mithramycin A, a specific inhibitor of Sp1, confirmed the role of Sp1. For the first time, we have demonstrated that the constitutive activity of the hMC4-R promoter is dependent upon Sp transcription factors.

PTGS2 (COX-2) −765G > C Promoter Variant Reduces Risk of Colorectal Adenoma among Nonusers of Nonsteroidal Anti-inflammatory Drugs

Prostaglandin H synthase 2 (PTGS2) or cyclooxygenase-2 (COX-2) has been shown to play a key role in the regulation of inflammation, and its inhibition is associated with a reduced risk of colon cancer. The PTGS2 (COX-2) -765G > C promoter variant is located in a putative SP1 binding site and reduces PTGS2 expression. In a Minnesota-based case-control study of cases with adenomatous (n = 494) or hyperplastic polyps (n = 186) versus polyp-free controls (n = 584), we investigated the role of the PTGS2 -765G > C promoter polymorphism. Multiple logistic regression analysis was used, adjusting for age, body mass index, caloric intake, alcohol, fiber, sex, hormone use, and smoking. For colorectal adenoma, odds ratios (OR) compared with PTGS2 -765GG as reference were GC 1.00 [95% confidence interval (95% CI), 0.74-1.35] and CC 0.53 (95% CI, 0.22-1.28). For hyperplastic polyps, the comparable adjusted odds ratios were GC 0.97 (95% CI, 0.65-1.46) and CC 0.24 (95% CI, 0.05-1.11). Risk associated with the -765G > C variant differed by aspirin or other nonsteroidal anti-inflammatory drug (NSAID) use. Among nonusers of aspirin or other NSAIDs, the CC genotype conferred a significant decrease in risk of adenoma (OR, 0.26; 95% CI, 0.07-0.89). Use of aspirin or other NSAIDs reduced risk of adenoma only among those with the -765GG (wild type) and possibly -765CG genotypes (OR, 0.66; 95% CI, 0.48-0.92 and OR, 0.64; 95% CI, 0.40-1.02, respectively). These data suggest that COX-2 expression or activity may be beneficially suppressed, and risk of colorectal polyps reduced, by aspirin or other NSAIDs in PTGS2 -765GG (wild type) individuals and by the -765 CC variant genotype in nonusers of NSAIDs.