Essential Promoter Region Research Articles

Variation in the Promoter Region of the MC4R Gene Elucidates the Association of Body Measurement Traits in Hu Sheep.

The melanocortin 4 receptor (MC4R) gene is expressed in the appetite-regulating areas of the brain and is engaged in the leptin signaling pathway. Although previous studies have identified variants in the coding region of the sheep MC4R gene showing significant associations with birth weight, weaning weight, and backfat thickness, no such associations have been reported for the promoter region. Besides, the essential promoter region of the sheep MC4R has not been delineated. In this study, to better understand the transcriptional regulation of MC4R and to elucidate the association between regulatory variants and haplotypes with body measurement traits in sheep, we cloned and characterized the MC4R promoter. We found that the minimal promoter of the gene is located within the region −1207/−880 bp upstream of the first exon. Real-time quantitative PCR (RT-qPCR) data revealed the mRNA expression of the MC4R gene had a significant difference between sex and age. In the association analysis, eight single nucleotide polymorphisms (SNPs) had a significant association with one or more traits (p < 0.05); of these, two SNPs were novel. Notably, individuals with haplotype H1H2 (CT-GA-GT-GA-GT-GA-GA-CG) were heavier in body weight than other haplotypes. Altogether, variations in the MC4R gene promoter, most notably haplotype H1H2, may greatly benefit marker-assisted selection in sheep.

E2F site in the essential promoter region does not confer S phase-specific transcription of the ABCC10 gene in human prostate cancer cells.

ABCC10 (MRP7) plays a role in cellular detoxification and resistance to anticancer drugs. Since ABCC10 gene transcription in human prostate cancer CWR22Rv1 cells was found dependent on E2F binding sequence motif, ABCC10 expression in G1 and S phases of the cell cycle of CWR22Rv1 cells, was analyzed. The cells were synchronized in G1 phase by double thymidine block and in S phase by thymidine/mimosine double block. ABCC10 mRNA level was found to be similar in S phase-synchronized and asynchronous cell populations. In G1 phase it decreased by 2.4- to 3-fold. It is thus inferred, that ABCC10 expression in CWR22Rv1 cells is not S phase-specific but is primarily associated with cell proliferation.

Impact ofEGFRGenetic Variants on Glioma Risk and Patient Outcome

The epidermal growth factor receptor (EGFR) regulates important cellular processes and is frequently implicated in human tumors. Three EGFR polymorphisms have been described as having a transcriptional regulatory function: two single-nucleotide polymorphisms in the essential promoter region, -216G/T and -191C/A, and a polymorphic (CA)(n) microsatellite sequence in intron 1. We aimed to elucidate the roles of these EGFR polymorphisms in glioma susceptibility and prognosis. We conducted a case-control study with 196 patients with glioma and 168 cancer-free controls. Unconditional multivariate logistic regression models were used to calculate ORs and 95% confidence intervals. A Cox regression model was used to evaluate associations with patient survival. False-positive report probabilities were also assessed. None of the EGFR -216G/T variants was significantly associated with glioma risk. The -191C/A genotype was associated with higher risk for glioma when the (CA)(n) alleles were classified as short for ≤16 or ≤17 repeats. Independently of the (CA)(n) repeat cutoff point used, shorter (CA)(n) repeat variants were significantly associated with increased risk for glioma, particularly glioblastoma and oligodendroglioma. In all tested models with different (CA)(n) cutoff points, only -191C/A genotype was consistently associated with improved survival of patients with glioblastoma. Our findings implicate EGFR -191C/A and the (CA)(n) repeat polymorphisms as risk factors for gliomas, and suggest -191C/A as a prognostic marker in glioblastoma. Our data support a role of these EGFR polymorphisms in determining glioma susceptibility, with potential relevance for molecularly based stratification of patients with glioblastoma for individualized therapies.

Transcriptional regulation of the human neutral ceramidase gene

Ceramidases play a critical role in generating sphingosine-1-phosphate by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. In order to elucidate its transcriptional regulation, we identify here a putative promoter region in the 5′-UTR of the human neutral CDase (nCDase) gene. Using human genomic DNA, we cloned a 3000 bp region upstream of the translational start site of the nCDase gene. Luciferase reporter analyses demonstrated that this 3000 bp region had promoter activity, with the strongest induction occurring within the first 200 bp. Computational analysis revealed the 200 bp essential promoter region contained several well-characterized promoter elements, lacked a conical TATA box, but did contain a reverse oriented CCAAT box, a feature common to housekeeping genes. Electrophoretic mobility shift assays demonstrated that the identified candidate transcriptional response elements (TRE) bind their respective transcription factors, including NF-Y, AP-2, Oct-1, and GATA. Mutagenic analyses of the TRE revealed that these sites regulated promoter activity and mutating an individual site decreased promoter reporter activity by up to 50%. Together, our findings suggest that regulation of nCDase expression involves coordinated TATA-less transcriptional activity.

Functional Analysis of Neurosteroidal Oestrogen Using Gene‐Disrupted and Transgenic Mice

The brain aromatase (oestrogen synthase) hypothesis predicts that oestrogen plays important roles in both sexual behaviours and brain sexual differentiation. To elucidate the functions of oestrogen in the brain, we generated aromatase knockout (ArKO) mice, which showed undetectable oestrogen and enhanced androgen levels in blood. These ArKO mice exhibited an enhanced appetite and disorders in sexual motivation, sexual partnership preference, sexual performance, aggressive behaviour, parental behaviour, infanticide behaviour and exploratory (anxiety) behaviour. We characterised the brain-specific promoter of the mouse aromatase gene, and identified several crucial cis-acting elements and the minimal essential promoter region for brain-specific expression. Next, we introduced a transgene of human aromatase, controlled by the minimal promoter region, into the ArKO mouse. The resulting mouse (ArKO/hArom), whose preoptic area, hypothalamus and amygdala were exposed to oestrogens only in the perinatal period, and then to enhanced androgens and no oestrogens in adulthood, showed near recovery from behavioural disorders. These results suggest that local oestrogens acting in specific brain regions are involved in the organisation of sex-specific neural networks during the perinatal period. Finally, we examined effects of oestrogens on gene expression within specific brain regions in mice during the perinatal period using DNA microarray analysis. This assay revealed both up-regulated and down-regulated brain-specific genes, including those related to neuronal function. Specifically, genes involved in energy metabolism, cell proliferation/apoptosis and secretory/transport system were altered in ArKO mice compared to wild mice. These results suggest that brain oestrogens participate in the sexual differentiation of the brain by influencing gene expression.

Transcriptional regulation of the human neutral ceramidase promoter

Promitogenic and proinflammatory metabolites of ceramide, including sphingosine‐1‐phosphate (S‐1‐P), contribute to atherogenic plaque formation. Ceramidases play a critical role in generating S‐1‐P by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. The objective of the present study is to elucidate the transcriptional regulation of human neutral ceramidases (hnCDase) in vascular smooth muscle (VSM) cells. Using human genomic DNA, we cloned a 3000 base pair (bp) region upstream of the translational start site of the hnCDase gene. Luciferase reporter analyses identified a 200 bp essential promoter region with multiple candidate transcriptional response elements (TRE). Electrophoretic mobility shift assays identified several growth factor regulated transcription factors, including AP‐1, AP‐2, and NF‐Y. Mutagenic analyses of the TRE revealed that these sites regulated promoter activity. We demonstrated that PDGFββ, TGFβ, as well as serum induced hnCDase expression in human coronary artery smooth muscle cells, which correlated with elevation of the mass of hnCDase‐dependent metabolites, sphingosine and S‐1‐P. Taken together, our studies indicate that hnCDase is transcriptionally regulated by growth factors that contribute to VSM cell proliferation or inflammation, both hallmarks of atherosclerotic arterial injury. These studies were funded by NIH RO1 HL076789 to MK.

Identification of seven novel germline mutations in the human E-cadherin (CDH1) Gene

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.

Mutation analysis of the DBC2 gene in sporadic and familial breast cancer

The expression of the recently identified tumor suppressor gene, DBC2 (Deleted in Breast Cancer 2), is frequently extinguished in breast cancer cells or tissues. Mutation analysis of the essential promoter region, all exons and exon/intron boundaries of the DBC2 gene was performed in 100 sporadic breast cancer cases by PCR-SSCP, and DHPLC, followed by direct sequencing. An additional 17 breast cancer families, who were negative for the BRCA1/2 mutations, were analyzed by direct sequencing. Three novel mutations were observed in the promoter and 5′-untranslated region (UTR) of the gene; a germ-line G>A transition in the promoter at nt −238 from the transcription start site, and two tumor-specific mutations at nt −121C>T and nt +48G>A. No deleterious mutations were detected in the coding sequence of the gene in familial and sporadic breast cancer cases. The sequence variations found within the promoter and 5′-UTR region of the gene warrant expression analysis and screening more tumor samples at this region.

STAT4 Expression in Human T Cells Is Regulated by DNA Methylation but Not by Promoter Polymorphism

STAT4, which plays a pivotal role in Th1 immune responses, enhances IFN-gamma transcription in response to the interaction of IL-12 with the IL-12R. Mice deficient in STAT4 lack IL-12-induced IFN-gamma production and Th1 differentiation and display a predominantly Th2 phenotype. Although these findings indicate that STAT4 expression levels are important for the development of cytokine-producing Th1 cells, the transcriptional and posttranscriptional mechanisms regulating STAT4 expression are unknown. We sought to identify and characterize the transcriptional regulatory elements in the promoter region of the human STAT4 gene. We found that disruption of multiple transcriptional regions covering the CREB, OCT1, and SP1 motifs significantly reduced STAT4 promoter activity. However, genomic DNA isolated from 91 patients with asthma or rheumatoid arthritis showed no evidence of mutations in the defined STAT4 essential promoter region. The 5' flanking region of the promoter was found to contain a -149A/G change in approximately 20-35% of patients, but this polymorphism had no effect on promoter activity. Interestingly, STAT4 expression was drastically increased in human T cells following treatment with a DNA methyltransferase inhibitor, and truncation of methylation sites in the proximal regulatory elements of the STAT4 promoter markedly enhanced transcriptional activity. Thus, our findings provide molecular insight into STAT4 expression and suggest that, in human T cells, STAT4 expressional regulation is associated with DNA hypermethylation, but not promoter polymorphisms.

Alterations of the RB1 gene in dedifferentiated liposarcoma

Dedifferentiated liposarcoma is a malignant adipocytic neoplasm containing a nonlipogenic sarcoma of variable histological grade that arises against the background of a pre-existing well-differentiated liposarcoma. The phenomenon of dedifferentiation is considered to be time-dependent, but the mechanism is not well known. The retinoblastoma protein, encoded by the RB1 gene located at 13q14, is a key regulator of proliferation, development, and differentiation of certain cell types, including adipocytes. In the current study, we investigated the genetic alterations of the RB1 gene, such as mutation (the essential promoter region and the protein-binding pocket domain; exons 20–24) and methylation of the promoter region, in addition to pRB expression and loss of heterozygosity (LOH) status, in two morphologically distinct areas (nonlipogenic dedifferentiated and well-differentiated components) in 27 patients. As a control, 11 undifferentiated high-grade pleomorphic sarcoma/pleomorphic malignant fibrous histiocytoma samples and 11 well-differentiated liposarcoma samples were also evaluated. Dedifferentiated components showed LOH (15/25; 60%) and abnormal retinoblastoma protein expression (18/27; 66.7%) more frequently than noted in the well-differentiated components (3/24; 12.5% and 9/27; 33.3%, respectively). Five and four out of the 27 dedifferentiated components harbored mutations and promoter methylation, respectively, whereas none of these alterations were seen in the well-differentiated components. These results suggest that retinoblastoma protein has a major role to play in dedifferentiation and that a ‘two-hit' mechanism is involved in the altered retinoblastoma protein expression in dedifferentiated liposarcoma.

Introduction of an initiator element in the mouse thymidylate synthase promoter alters S phase regulation but has no effect on promoter bidirectionality

The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element, is bidirectional and initiates transcription at multiple start sites across broad initiation windows upstream and downstream of the 30 nt essential promoter region. The TS promoter also plays an essential role in the post-transcription regulation of TS gene expression during the G(1)-S phase transition. The goal of this study was to determine if the addition of a TATAA box or an initiator element would have a significant effect on start-site pattern, promoter bidirectionality and S phase regulation of the TS gene. A TATAA box and/or an initiator element were inserted downstream of the TS essential promoter region, and the modified promoters were used to drive expression of indicator genes. The engineered genes were transfected into cultured mammalian cells, and the effects of the mutations were determined. Addition of the TATAA box and especially the initiator element had a significant effect on the transcription start site pattern, indicating that the elements were functional. Unexpectedly, addition of one or both of these elements had no effect on promoter bidirectionality. However, inclusion of the initiator element led to a significant reduction in S phase regulation of TS mRNA levels, indicating that changes in promoter architecture can perturb normal S phase regulation of TS gene expression.

Cloning and biological characterization of buffalo ( Bubalus bubalis) interferon-γ

Interferon-γ, a major immunomodulatory cytokine, of Indian water buffalo ( Bubalus bubalis) was characterized at molecular level. Complementary DNA and essential promoter region were cloned and sequenced, and functional recombinant protein was expressed in bacterial system. The cDNA has 97.8% nucleotide identity with 11-nucleotide and four-amino acid variations, and the essential promoter region has 98.4% identity with five-nucleotide variations and a four-nucleotide deletion in comparison with the corresponding bovine sequences. All the major promoter elements such as NF IL-2 like motif, cyclosporin sensitive binding element and GATA motif are strictly conserved. Recombinant buffalo-IFN-γ expressed in bacterial system reacted with an anti-bovine-IFN-γ monoclonal antibody in Western blot and showed antiviral activity against buffalo pox virus in cultured Madin-Darby bovine kidney (MDBK) cells by inhibiting virus induced cytopathic effect. The study shows high level sequence similarity of IFN-γ among ruminants. In view of the immunomodulatory and antiviral activities of IFN-γ, this molecule will be useful in better understanding of the immune system of water buffaloes.

Induction of Disease-associated Keratin 16 Gene Expression by Epidermal Growth Factor Is Regulated through Cooperation of Transcription Factors Sp1 and c-Jun

Overexpression of keratin 16 has been observed in keratinocytes in those skin diseases characterized by hyperproliferation such as psoriasis. Therefore, keratin 16 is usually referred to as a disease-associated keratin. In the present study, we found that epidermal growth factor (EGF) increased the expression of keratin 16 mRNA and protein synthesis in a time-dependent manner in HaCaT cells. Reporter assays revealed that the EGF response region was in the range of -162 to -114 bp. Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the keratin 16 promoter by site-directed mutagenesis significantly inhibited keratin 16 promoter activity induced by EGF. Furthermore, keratin 16 gene expression induced by Ras activation was also regulated in the same manner as the EGF response. By using the DNA affinity precipitation assay in HaCaT and SL2 cells, Sp1 directly interacted with the Sp1 site of the promoter, and c-Jun and c-Fos precipitated with the Sp1 oligonucleotide was attributable to the interaction between the Sp1 and AP1 proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activate keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of keratin 16 promoter, and EGF-induced promoter activation was blocked by the viral oncoprotein E1A. Taken together, these results suggest that Sp1 and AP1 sites in the essential promoter region are critical for EGF response, and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of EGF-induced keratin 16 gene expression.

CpG island methylation status and mutation analysis of the RB1 gene essential promoter region and protein-binding pocket domain in nervous system tumours.

A series of 136 nervous system tumours were studied to determine the methylation status of the CpG island contained within the promoter region of the RB1 gene, as well as mutation analysis of the essential promoter region and exons 20–24 (and surrounding intronic regions) coding for the protein-binding pocket domain. Methylation of the RB1 CpG island was detected in 26 samples corresponding to nine glioblastomas, three anaplastic astrocytomas, one mixed oligo-astrocytoma, one ependymoma, two medulloblastomas, two primary central nervous system lymphomas, two neurofibrosarcomas, and six brain metastasis from solid tumours. No inactivating mutations were found within the RB1 promoter region, whereas one glioblastoma and one oligodendroglioma displayed similar sequence variations consisting of 12 and 8 base pair deletions at intron 21. These results suggest that RB1 CpG island hypermethylation is a common epigenetic event that is associated with the development of malignant nervous system tumours.

Human pituitary adenomas infrequently contain inactivation of retinoblastoma 1 gene and activation of cyclin dependent kinase 4 gene.

Components of cyclinD1/cyclin-dependent kinase 4 (CDK4)/p16INK4a/pRb pathway are the frequent target of many tumor types. We examined the role of retinoblastoma susceptibility gene (RB1) and the CDK4 gene in human pituitary tumorigenesis. For the RB1 gene, pRb expression and loss of heterozygosity (LOH) on 13q in pituitary adenomas were analysed. Immunostaining of pRb revealed lack of expression in 1 of 29 pituitary adenomas. In 4 of 31 pituitary adenomas, allelic imbalances including LOH of RB1 on 13q14 were detected. Three of 4 pituitary adenomas, in which one adenoma lacked pRb expression, had a common LOH region at least from D13S219 on 13q12.3-q13 to D13S265 on 13q31-32. Interphase fluorescence in situ hybridization with a probe of RB1 showed 2 copies of RB1 gene suggesting that mitotic recombination events, not deletion or chromosome loss, led to LOH in the 3 pituitary adenomas analyzed. All 27 exons, intron-exon boundaries, and essential promoter region of RB1 gene were then sequenced in genomic DNA from 4 pituitary adenomas with allelic imbalance on 13q14 including one adenoma without pRb expression and 3 adenomas with pRb expression. Any somatic mutations, insertions, or microdeletions in the RB1 gene were not detected in 4 pituitary adenomas. Methylation sensitive (MS)-polymerase chain reaction (PCR) and bisulfite sequencing analysis revealed hypomethylated status of CpG islands in the promoter region of the RB1 genes of 4 pituitary adenomas. In addition, activating mutations of CDK4 gene, which is a component of cyclinD1/CDK4/p16INK4a/pRb pathway, were not detected in 31 pituitary adenomas. Based on these results, it is concluded that somatic mutations of the RB1 gene or CDK4 gene do not appear to play a major role in pituitary tumorigenesis. This supports the presence of potential tumor suppressor gene(s) on 13q12.3-q13 to 13q31-32 in pituitary adenomas.

Synergistic Activation of the TATA-less Mouse Thymidylate Synthase Promoter by the Ets Transcription Factor GABP and Sp1

The mouse thymidylate synthase (TS) promoter lacks a TATA box and an initiator element and directs transcriptional initiation at multiple sites over a 90-nucleotide region. The minimum sequence required for wild-type promoter activity has been mapped to a 30-nucleotide essential promoter region that partially overlaps the 5′ end of the transcriptional initiation window. The essential promoter region contains two potential binding sites for members of the Ets family of transcription factors as well as a binding site for Sp1. Promoter mutation analyses revealed that all three of these sites are important for promoter activity. Transient cotransfection assays showed that GABP, a heterodimeric Ets factor, is able to stimulate expression of reporter genes driven by the wild-type mouse TS promoter whereas several other Ets factors have no effect. Electrophoretic mobility shift assays revealed that recombinant GABP binds to both Ets elements in the essential promoter region. Stimulation of promoter activity by GABP is diminished when either Ets element is inactivated and is prevented when both Ets elements are inactivated. Transient cotransfection assays revealed that Sp1 and GABP stimulate TS promoter activity in a highly synergistic manner.

Transcriptional Control Elements of the Rat Thymidylate Synthase Promoter: Evolutionary Conservation of Regulatory Features

The sequence elements that are important for the transcription and regulation of the rat thymidylate synthase (TS) gene were analyzed. The rat TS promoter lacks a TATA box and directs transcriptional initiation at multiple sites between 60 and 20 nt upstream of the AUG translational start codon. Promoter deletion analyses showed that the region between −100 and −42 nt relative to the AUG codon was both necessary and sufficient for high level promoter activity and was designated the essential promoter region. The essential region also had bidirectional promoter activity. Site-directed mutagenesis revealed that four elements were especially important for promoter activity. These include Ets motifs at −85 and −50, an Sp1 motif at −80, and an LSF motif that overlapped the upstream Ets and Sp1 motifs. Inactivation of E2F motifs that are upstream and downstream of the essential promoter region had no measurable effect on promoter activity in transient transfection assays. The rat TS promoter region directed S-phase-specific expression of a stably transfected minigene if a spliceable intron was included in the transcribed region. When the intron was deleted or the E2F motifs were inactivated, expression of the minigene changed very little during the G1 to S transition.

Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication.

Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells.

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter.

The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

Inhibition of Mouse Thymidylate Synthase Promoter Activity by the Wild-Type p53 Tumor Suppressor Protein

The p53 tumor suppressor protein is an important negative regulator of the G1to S transition in mammalian cells. We have investigated the effect of p53 on the expression of the mouse thymidylate synthase (TS) gene, which normally increases as cells enter S phase. A luciferase indicator gene that was driven by the wild-type or various modified forms of the TATA-less mouse TS promoter was transiently cotransfected with a p53 expression plasmid into TS-deficient hamster V79 cells and the level of luciferase activity was determined. We found that wild-type p53 inhibited TS promoter activity by greater than 95% but had a strong stimulatory effect on an artificial promoter that contained multiple p53-binding sites. In contrast, an expression plasmid that encodes a mutant form of p53 or a wild-type retinoblastoma tumor suppressor protein had little effect on TS promoter activity. Deletion of sequences upstream or downstream of the TS essential promoter region, or inactivation of each of the known elements within the essential promoter region, had no effect on the ability of wild-type p53 to inhibit TS promoter activity. Our observations indicate that the inhibition of TS promoter activity by p53 is not due to the presence of a specific p53 negative response element in the TS promoter. Rather, it appears that p53 inhibits the TS promoter by sequestering (“squelching”) one or more general transcription factors.