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Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells.

Liu H, Zhang L, Niu Z, Zhou M, Peng C, Li X, Deng T, Shi L, Tan Y, Li G - BMC Cancer (2008)

Bottom Line: Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter.We found that DNA methylation suppresses BRD7 expression in NPC cells.DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan, PR China. huayingcsu@yahoo.com.cn

ABSTRACT

Background: Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription.

Methods: The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter.

Results: We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals.

Conclusion: BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.

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Related in: MedlinePlus

Bisulfite treatment and sequencing analysis identifies methylation of BRD7 promoter. (A) Representative sequencing graphs of BRD7 promoter region in 5–8F cells treated with or without 5-Aza-CdR. Modified and unmodified cytosines are indicated by arrows. (B) Schematic depiction of methylated cytosine in BRD7 promoter region. The Cytosine under the "*" in the dinucleotide CpG indicates methylated cytosines.
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Figure 4: Bisulfite treatment and sequencing analysis identifies methylation of BRD7 promoter. (A) Representative sequencing graphs of BRD7 promoter region in 5–8F cells treated with or without 5-Aza-CdR. Modified and unmodified cytosines are indicated by arrows. (B) Schematic depiction of methylated cytosine in BRD7 promoter region. The Cytosine under the "*" in the dinucleotide CpG indicates methylated cytosines.

Mentions: Sodium bisulfite deaminates unmethylated cytosine to uracil in single-stranded DNA under conditions in which the 5-methylcytosine remains nonreactive. Thus, all cytosine residues remaining at the time of sequencing represent cytosines that were methylated in the original DNA sequence. Genomic DNA from 5–8F cells treated with or without 3.75 μM of 5-Aza-CdR was analyzed. Sequencing analysis showed that the cytosines (labeled with *) at -374, -362, -352, -329, -226, -9, -5 bp were methylated in BRD7 promoter of 5–8F cells and were unmethylated in 5–8F cells treated with 3.75 μM of 5-Aza-CdR (Fig. 4B). Two "C" (labeled with *) at -260 and -170 bp appear. It could not be due to CpG methylation, possibly due to insufficient bisulfited treatment.


Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells.

Liu H, Zhang L, Niu Z, Zhou M, Peng C, Li X, Deng T, Shi L, Tan Y, Li G - BMC Cancer (2008)

Bisulfite treatment and sequencing analysis identifies methylation of BRD7 promoter. (A) Representative sequencing graphs of BRD7 promoter region in 5–8F cells treated with or without 5-Aza-CdR. Modified and unmodified cytosines are indicated by arrows. (B) Schematic depiction of methylated cytosine in BRD7 promoter region. The Cytosine under the "*" in the dinucleotide CpG indicates methylated cytosines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543047&req=5

Figure 4: Bisulfite treatment and sequencing analysis identifies methylation of BRD7 promoter. (A) Representative sequencing graphs of BRD7 promoter region in 5–8F cells treated with or without 5-Aza-CdR. Modified and unmodified cytosines are indicated by arrows. (B) Schematic depiction of methylated cytosine in BRD7 promoter region. The Cytosine under the "*" in the dinucleotide CpG indicates methylated cytosines.
Mentions: Sodium bisulfite deaminates unmethylated cytosine to uracil in single-stranded DNA under conditions in which the 5-methylcytosine remains nonreactive. Thus, all cytosine residues remaining at the time of sequencing represent cytosines that were methylated in the original DNA sequence. Genomic DNA from 5–8F cells treated with or without 3.75 μM of 5-Aza-CdR was analyzed. Sequencing analysis showed that the cytosines (labeled with *) at -374, -362, -352, -329, -226, -9, -5 bp were methylated in BRD7 promoter of 5–8F cells and were unmethylated in 5–8F cells treated with 3.75 μM of 5-Aza-CdR (Fig. 4B). Two "C" (labeled with *) at -260 and -170 bp appear. It could not be due to CpG methylation, possibly due to insufficient bisulfited treatment.

Bottom Line: Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter.We found that DNA methylation suppresses BRD7 expression in NPC cells.DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan, PR China. huayingcsu@yahoo.com.cn

ABSTRACT

Background: Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription.

Methods: The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter.

Results: We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals.

Conclusion: BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC.

Show MeSH
Related in: MedlinePlus