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Egr-1 regulates the transcription of NGX6 gene through a Sp1/Egr-1 overlapping site in the promoter.

ABSTRACT: BACKGROUND: As a novel candidate metastasis suppressor gene, Nasopharyngeal carcinoma-associated gene 6 (NGX6) is involved in cellular growth, cell cycle progression and tumor angiogenesis. Previous studies have shown that NGX6 gene is down-regulated in colorectal cancer (CRC). However, little is known about its transcriptional regulation. RESULTS: We defined the minimal promoter of NGX6 gene in a 186-bp region (from-86 to +100) through mutation construct methods and luciferase assays. Results from Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) revealed that Early growth response gene 1 (Egr-1) binds to the Sp1/Egr-1 overlapping site of NGX6 minimal promoter. Overexpression of Egr-1 increased the promoter activity and mRNA level of NGX6 gene; while knock-down of endogenous Egr-1 decreased mRNA expression of NGX6 gene. CONCLUSION: These results demonstrate that Egr-1 regulates NGX6 gene transcription through an overlapping Sp1/Egr-1 binding site as a positive regulator of NGX6 gene.

SUBMITTER: Liu M

PROVIDER: S-EPMC4134679 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Egr-1 regulates the transcription of NGX6 gene through a Sp1/Egr-1 overlapping site in the promoter.

Liu Minji M Wang Xiaoyan X Peng Ya Y Shen Shourong S Li Guiyuan G

BMC molecular biology 20140716

<h4>Background</h4>As a novel candidate metastasis suppressor gene, Nasopharyngeal carcinoma-associated gene 6 (NGX6) is involved in cellular growth, cell cycle progression and tumor angiogenesis. Previous studies have shown that NGX6 gene is down-regulated in colorectal cancer (CRC). However, little is known about its transcriptional regulation.<h4>Results</h4>We defined the minimal promoter of NGX6 gene in a 186-bp region (from-86 to +100) through mutation construct methods and luciferase assa ...[more]

PMID: 25029911

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Project description:BackgroundNasopharyngeal carcinoma associated gene 6 (NGX6) is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC). In this study, we explored the role of DNA methylation in regulation of NGX6 transcription.MethodsIn the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation.ResultsThe sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P < 0.05). An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the NGX6 promoter was associated with colorectal cancer patients age (P < 0.05). Moreover, a trend was shown toward metastasis status and primary site in colorectal carcinomas with NGX6 promoter methylation (p = 0.056 and P = 0.067, respectively). In addition, 5-Aza-dC could induce NGX6 mRNA expression and NGX6 promoter demethylation in HT-29 cells.ConclusionsDown-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might be a potential molecular marker for diagnosis or prognosis, or serve as a therapeutic target.

Project description:Pigs are commonly used as an animal model to evaluate the toxic effects of exogenous compounds. Cytochrome P450 1A1 (CYP1A1) metabolizes numerous exogenous compounds and is abundantly expressed in the liver, kidneys, and intestines. The high amino acid similarity between human and porcine CYP1A1 indicates that they probably have the same metabolic characteristics. Therefore, understanding the regulatory mechanism of CYP1A1 expression in pigs is particularly important for predicting the toxicology and metabolic kinetics of exogenous chemicals. Currently, the transcriptional regulation of porcine CYP1A1 has rarely been studied, especially regarding basal transcription. In this study, we first confirmed that the key regulatory elements of porcine CYP1A1 basal transactivation are in the proximal promoter region using promoter truncation analysis via a dual luciferase assay in a porcine kidney cell line LLC-PK1. Two overlapping cis-elements, the xenobiotic response element (XRE) and GC box, in this proximal region potentially play key roles in the basal transactivation of porcine CYP1A1. Furthermore, using electrophoretic mobility shift assay and chromatin immunoprecipitation, the GC box binding protein Sp1 was confirmed to bind to the proximal promoter of porcine CYP1A1, instead of AhR, the XRE binding protein. In LLC-PK1 cells, by knocking down either Sp1 or AhR, the expression of porcine CYP1A1 at the mRNA level and protein level was significantly downregulated, suggesting both proteins are important for porcine CYP1A1 expression. However, promoter activity analysis in LLC-PK1 cells treated with an AhR agonist and antagonist confirmed that AhR does not participate in the basal regulation of porcine CYP1A1 at the proximal promoter. In conclusion, our study revealed that the proximal promoter is the key regulatory region for porcine CYP1A1 basal expression. Although AhR plays an important role in the transactivation of porcine CYP1A1 expression, the key determinant transcription factor for its basal transactivation is Sp1 at the proximal promoter of porcine CYP1A1.

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Egr-1 regulates the transcription of NGX6 gene through a Sp1/Egr-1 overlapping site in the promoter.

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Egr-1 regulates the transcription of NGX6 gene through a Sp1/Egr-1 overlapping site in the promoter.

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