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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

Cytosine methylation inhibits transcription factor binding to its corresponding binding sites in BRD7 promoter.Left panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -353/-337 probes by EMSA. Results are representative of three independent experiments. Excess amounts of unlabeled unmethylated -353/-337, methylated -353/-337 were added as competitors. Right panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -330/-317 probes by EMSA. Excess amounts of cold unmethylated -330/-317, methylated -330/-317 were added as competitors. NP: nuclear protein; Wt: wild type probe; Met: methylated probe; CH3-P: SssI methylase treated probe.
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Figure 5: Cytosine methylation inhibits transcription factor binding to its corresponding binding sites in BRD7 promoter.Left panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -353/-337 probes by EMSA. Results are representative of three independent experiments. Excess amounts of unlabeled unmethylated -353/-337, methylated -353/-337 were added as competitors. Right panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -330/-317 probes by EMSA. Excess amounts of cold unmethylated -330/-317, methylated -330/-317 were added as competitors. NP: nuclear protein; Wt: wild type probe; Met: methylated probe; CH3-P: SssI methylase treated probe.

Mentions: Cytosine methylation in the promoter region, when present within regulatory elements, could potentially interfere with binding of specific transcription factors to these motifs. Our previous studies confirmed that the MYC-MAX binding site at -260/-246 was non-specific [16]. To investigate whether cytosine methylation within Sp1 binding sites at -353/-337 and -330/-317 interfere with nuclear factor binding, we compared the binding abilities in EMSA reactions of a 20-bp oligomer (nucleotides -353/-337 and -330/-317), which contained the two different Sp1 elements and neighboring cytosines, in unmethylated and methylated forms. First we examined the abilities of unmethylated and methylated -353/-337 to compete with the unmethylated -353/-337 probe in binding to nuclear proteins from 5–8F cells. As seen in Fig. 5A, two sequence-specific gel shift complexes were observed with labeled unmethylated -353/-337 as a probe (lane 1 of Fig. 5), but no complex was formed with labeled methylated -353/-337 as a probe (lane 2 of Fig. 5). In competition EMSA reactions, 50-fold excess of unlabeled unmethylated -353/-337 oligomers were sufficient to completely inhibit complex formation (lane 3, 4 of Fig. 5), but none of the DNA-protein bands were inhibited by the addition of a 100-fold excess of unlabeled methylated -353/-337 oligomers (lane 5, 6 of Fig. 5), suggesting that the unmethylated -353/-337 oligomer binds activated protein. Then we compared the abilities of unmethylated and methylated -330/-317 oligomers to bind nuclear proteins from 5–8F cells in EMSA reactions using labeled unmethylated -330/-317 or methylated -330/-317 as probes. A strong and a weak DNA-protein complexes were formed with labeled unmethylated -330/-317 as probes (lane 7 of Fig. 5), whereas no DNA-protein complexes were formed with methylated -330/-317 as probes (lane 8 of Fig. 5). In the competition assay, both of them were completely inhibited with cold unmethylated -330/-317 (lane 9 of Fig. 5), but none of them were inhibited by cold methylated -330/-337 (lane 11, 12 of Fig. 5). These results suggested that methylation of cytosines at -353/-337 and -330/-317 significantly inhibited nuclear protein (possibly Sp1) binding to BRD7 promoter.


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)

Cytosine methylation inhibits transcription factor binding to its corresponding binding sites in BRD7 promoter.Left panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -353/-337 probes by EMSA. Results are representative of three independent experiments. Excess amounts of unlabeled unmethylated -353/-337, methylated -353/-337 were added as competitors. Right panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -330/-317 probes by EMSA. Excess amounts of cold unmethylated -330/-317, methylated -330/-317 were added as competitors. NP: nuclear protein; Wt: wild type probe; Met: methylated probe; CH3-P: SssI methylase treated probe.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cytosine methylation inhibits transcription factor binding to its corresponding binding sites in BRD7 promoter.Left panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -353/-337 probes by EMSA. Results are representative of three independent experiments. Excess amounts of unlabeled unmethylated -353/-337, methylated -353/-337 were added as competitors. Right panel: Comparison of the nuclear binding capabilities of unmethylated and methylated -330/-317 probes by EMSA. Excess amounts of cold unmethylated -330/-317, methylated -330/-317 were added as competitors. NP: nuclear protein; Wt: wild type probe; Met: methylated probe; CH3-P: SssI methylase treated probe.
Mentions: Cytosine methylation in the promoter region, when present within regulatory elements, could potentially interfere with binding of specific transcription factors to these motifs. Our previous studies confirmed that the MYC-MAX binding site at -260/-246 was non-specific [16]. To investigate whether cytosine methylation within Sp1 binding sites at -353/-337 and -330/-317 interfere with nuclear factor binding, we compared the binding abilities in EMSA reactions of a 20-bp oligomer (nucleotides -353/-337 and -330/-317), which contained the two different Sp1 elements and neighboring cytosines, in unmethylated and methylated forms. First we examined the abilities of unmethylated and methylated -353/-337 to compete with the unmethylated -353/-337 probe in binding to nuclear proteins from 5–8F cells. As seen in Fig. 5A, two sequence-specific gel shift complexes were observed with labeled unmethylated -353/-337 as a probe (lane 1 of Fig. 5), but no complex was formed with labeled methylated -353/-337 as a probe (lane 2 of Fig. 5). In competition EMSA reactions, 50-fold excess of unlabeled unmethylated -353/-337 oligomers were sufficient to completely inhibit complex formation (lane 3, 4 of Fig. 5), but none of the DNA-protein bands were inhibited by the addition of a 100-fold excess of unlabeled methylated -353/-337 oligomers (lane 5, 6 of Fig. 5), suggesting that the unmethylated -353/-337 oligomer binds activated protein. Then we compared the abilities of unmethylated and methylated -330/-317 oligomers to bind nuclear proteins from 5–8F cells in EMSA reactions using labeled unmethylated -330/-317 or methylated -330/-317 as probes. A strong and a weak DNA-protein complexes were formed with labeled unmethylated -330/-317 as probes (lane 7 of Fig. 5), whereas no DNA-protein complexes were formed with methylated -330/-317 as probes (lane 8 of Fig. 5). In the competition assay, both of them were completely inhibited with cold unmethylated -330/-317 (lane 9 of Fig. 5), but none of them were inhibited by cold methylated -330/-337 (lane 11, 12 of Fig. 5). These results suggested that methylation of cytosines at -353/-337 and -330/-317 significantly inhibited nuclear protein (possibly Sp1) binding to BRD7 promoter.

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