Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells
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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.
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.
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.
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.
KeywordsEnhance Green Fluorescence Protein Normal Nasopharyngeal Epithelium BRD7 Gene BRD7 Expression BRD7 Promoter
NPC is a head and neck malignancy with high occurrence in South-East Asia and Southern China [1, 2]. The development of this EBV-associated cancer may involve cumulative genetic and epigenetic changes in a background of predisposed genetic and environmental factors [3, 4]. Genome-wide studies have unraveled multiple chromosomal abnormalities with involvement of specific oncogenes and tumor suppressor genes [5, 6]. BRD7 has been recently identified as a bromodomain gene in NPC cells by cDNA Representational Difference Analysis (cDNA RDA) . As a member of the bromodomain genes family, BRD7 may be considered as a component of chromatin remodeling complexes which possess histone acetyltransferase activity [8, 9]. Together with E1B-AP5, BRD7 functions as an inhibitor of basic transcription in several viral and cellular promoters in the nucleus . An alternative role of BRD7 arises from the evidence that BRD7 exhibits a much higher level of mRNA expression in normal nasopharyngeal epithelia than in NPC biopsies and cell lines [11, 12]. Indeed, over-expression of BRD7 in NPC cells can effectively inhibit cell growth and cell cycle progression from G1 to S phase by transcriptional regulation of some key cell cycle related genes [13, 14, 15]. Our previous studies revealed the full-length promoter -404/+46 of BRD7 gene, and showed that Sp1 specifically bound to BRD7 promoter . However, little is known about the down-expression of BRD7 in NPC cells. In this report, we reveal that DNA methylation results in the suppression of BRD7 expression in NPC cells. BRD7 promoter activity is regulated by methylation of CpG sites with the (G+C)-rich promoter region. DNA methylation inhibitor, 5-Aza-CdR, up-regulates BRD7 expression in NPC 5–8F cells. More importantly, the methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than that in the blood samples from normal individuals. These results will be helpful in further understanding the transcription-repression mechanism of the BRD7 gene in NPC cells and the establishment of noninvasive approach in the early detection and surveillance of NPC.
Cell culture and antibodies
Most of the cell lines used in this study was from the American Type Culture Collection (ATCC). NPC CNE1, 5–8F (high tumorigenic and metastatic ability) and 6–10B (tumorigenic, but lacking metastatic ability) cell lines were provided by the Cancer Center of Sun Yet-Sen University, (Guangzhou, China). NPC HNE1 cells were provided by Cancer Research Institute of Central South University (Hunan, China). COS7 and BHK-21 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C, 5% CO2. HNE1, CNE1, 6–10B, 5–8F, SW480 and Hella cells were cultured in RPMI1640 medium containing 10% FBS.
The presence of CpG islands within the upstream region spanning from -1 to -2000 bp of BRD7 gene was analyzed with EMBOSS (European Molecular Biology open software Suite http://www.ebi.ac.uk/emboss/cpgplot program CpGplot and Softberry CpGFinder program http://www.softberry.com/berry.phtml?topic=CpGisland, respectively.
Construction of plasmids
pGL3-404/+46 was generated as previously described. pGL3-404/+46/GFP was generated by replacing the luciferase gene of pGL3-enhancer with enhanced green fluorescence protein (EGFP) as follows: EGFP coding region was amplified by PCR using primers 5'-GACTTTCCAAAATGTCGTAACAACTCC-3' (forward) and 5'-GGCTCTAGATTACTTGTACAGCTCGTC-3' (reverse) with pEGFP-C2as template, cut with double restriction enzyme NcoI and XbaI, then cloned into vector fragment of pGL3-404/+46 which was cut with the same restriction enzymes NcoI and XbaI to release the luciferase coding region.
Luciferase assay was performed as previously described . Briefly, 4 × 105 cells were seeded in each well of 12-well plates 24 h prior to transfection, then transfected with 0.5 μg of various BRD7 promoter constructs and 0.25 μg pSV40 β-galactosidase per well by Lipofectamine 2000 Reagent (Invitrogen) according to manufacturer's instruction. Luciferase activity was measured in cell lysates 38 h after transfection using Luciferase Assay kit (Promega). β-galactosidase activity was measured in cell lysates by β-galactosidase Enzyme Assay System (Promega). Experiments were repeated at least three times with three replicates per sample. Results were normalized against β-galactosidase activity.
Direct GFP fluorescence assay
4 × 105 cells were seeded in each well of 12-well plates 24 h prior to transfection. Next day, every well was transfected with 0.5 μg of pGL3/-404,+46/EGFP or CH3-pGL3/-404,+46/EGFP in by using Lipofectamine 2000 Reagent according to manufacturer's instruction. EGFP fluorescence was observed 38 h after transfection using an AX-80 analytical microscope system (Olympus, Tokyo, Japan).
RT-PCR was performed as previously described . The single-stranded cDNA was amplified by using primers as follows: BRD7 primer (forward) 5'-CAAGCTCTTTAGCCAAACAAGAA-3', (reverse) 5'-TCATTCCTGAGTGCAACAGC-3'; GAPDH primer (forward) 5'-TCTAGACGGCAGGTCAGGTCCACC-3', (reverse) 5'-CCACCCATGGCAAATTCCATGGCA-3'. PCR was carried out for 28 cycles using a step cycle of 94°C for 40 s, 58°C for 40 s, 72°C for 1 min, followed by 72°C for 10 min. GAPDH primer was added to the reactions at the end of the fifth cycle.
Nested methylation-specific PCR analyses
The DNA methylation status was established by PCR analysis of bisulfite-modified genomic DNA, which induces chemical conversion of unmethylated, but not methylated, cytosine to uracil, using two procedures. First, methylation status was analyzed by bisulfite genomic sequencing of both strands of the corresponding CpG islands. The second analysis used methylation-specific PCR using primers specific for either the methylated or modified unmethylated DNA. Methylation-specific primer (forward): 5'-AGTTTGAGCGGTGGATTTCGTTTC-3', (reverse) 5'-GGTTCGGTCGGATATGGGTAAGAAG-3'; Unmethylation-specific primer (forward): 5'-AAAGATGAGAGTTTGAGTGGTGGATTTT-3', (reverse) 5'-GGGGTTTGGTTGGATATGGGTAAGAAG-3'.
Sodium bisulfite modification and genomic sequencing
Genomic DNA was extracted from blood or cultured cells with or without a 72 h pretreatment with 5-Aza-CdR, using the DNA-easy kit (Qiagen) according to the manufacturer's instructions. Two μg of DNA was denatured in 50 μl of 0.3 M NaOH for 15 min at 37°C. For the chemical modification of DNA, 520 μl of 3 M sodium bisulfite (Sigma) and 30 μl of 10 mM hydroquinone (Sigma) were added to the DNA solution and the samples were mixed, overlaid with mineral oil, and incubated at 50°C overnight. Modified DNA was purified with the Wizard DNA Clean-up system (Promega) and eluted in water. As a final step, NaOH was added to a final concentration of 0.3 M, and the samples were incubated for 5 min at room temperature. DNA was precipitated by ethanol and resuspended in water. The sequence of interest in the bisulfite-reacted DNA was PCR-amplified in a reaction mixture containing dNTPs, PCR buffer, Taq enzyme, and primers. For each reaction, 1 μl (~50 ng) of bisulfited DNA was used in 25 μl reaction volume. DNA fragments were gel-purified with the QIAquick Gel Extraction kit (Qiagen) cloned into pGEM/T-easy vector (Invitrogen). Clones with appropriate sized inserts were sequenced.
In vitro DNA methylation and transient transfection
The methylated plasmids (Met-pGL3/-404,+46 and Met-pGL3/-404,+46/GFP) were generated by incubating 40 μg of plasmid DNA (pGL3/-404,+46 and pGL3/-404,+46/GFP) with 100 units SssI methylase in reaction buffer consisting of 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9, and 160 μM S-adenosylmethionine according to the manufacturer's instructions (New England Biolabs, Inc.). Reactions were carried out at 37°C overnight. Complete methylation was verified by digestion with the methylation-sensitive restriction enzyme HpaII. Only plasmids that showed a complete protection from HpaII digestion were used in the transfection experiments. The methylated plasmid DNA was purified by the Wizard DNA Clean-up system (Promega) and transfected into COS7 and 5–8F cells in parallel with the unmethylated pGL3/-404,+46 and pGL3/-404,+46/GFP, respectively. Luciferase activity was analyzed at 38 h after transfection.
Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were prepared, quantified, and used for EMSA with double strand probes or competitors as described previously . The methylated -353/-337 and -330/-317 oligonucleotide were prepared by incubating 20 μg of unmethylated -353/-337 (Sense: 5'-GATCCCGCCCCGGCCCCGCCCTCGG-3', anti-sense:5'-CCGAGGGCGGGGCCGGGGCGGGATC-3') and -330/-317 (Sense: 5'-CGGCCCCGCCCCCGGCCCGCGAGCT-3', anti-sense: 5'-AGCTCGCGGGCCGGGGGCGGGGCCG-3') with 80 units of SssI CpG methylase at 37°C overnight. The reaction mixture was then heated at 65°C to inactivate the methylase, purified by polyacrylamide gel electrophoresis, and concentrated with Centricon 3 microconcentrators. Nuclear extracts were incubated for 20 min on ice in the presence or absence of unlabeled competitor oligonucleotides followed by the addition of the end-labeled probe and 15 min incubation on ice.
5-Aza-CdR and TSA treatment
For the 5-Aza-CdR treatment, DNA methyltransferase inhibitor, 5-Aza-CdR, was added to 2 × 106 cells at final concentrations from 1.875 to 15 μM for 72 h. For trichostatin A (TSA) treatment alone, deacetylase inhibitor TSA was added to 2 × 106 cells at final concentrations from 150 to 5000 nM for 48 h. For the treatment of 5-Aza-CdR combined with TSA, 1000 nM of TSA was added to 2 × 106 cells for 48 h at the end of the treatment of 3.75 μM 5-Aza-CdR.
Tumor and blood samples
All samples were collected from the Xiangya Hospital of Central South University and the Hunan Tumor Hospital, Changsha, Hunan, China. All patients were diagnosed by pathological examination. Totally 18 NPC patients and 16 normal individuals were used in this study. Written informed consent was obtained from all studied participants. The study was approved by the ethical review committees of the appropriate institutions. Five-to-10 ml peripheral blood samples were taken from each individual.
A CpG island is overlapped with BRD7 promoter
Down-regulation of BRD7 gene expression in NPC cells is due to partly methylation of BRD7 promoter
DNA methylation inhibitors 5-Aza-CdR augments endogenous mRNA and reverses the methylation status of BRD7 promoter in NPC cells
Bisulfite treatment and sequencing analysis identifies methylated cytosines in BRD7 promoter
Cytosine methylation inhibits nuclear protein binding to BRD7 promoter
In vitro cytosine methylation of BRD7 promoter silence its activity
Frequent aberrant methylation of BRD7 promoter in NPC patients
BRD7 is a recently identified bromodomain gene. It exhibits much higher-level of mRNA expression in normal nasopharyngeal epithelia than in NPC biopsies and cell lines [11, 12]. Over-expression of BRD7 in NPC cells is effective in inhibiting cell growth and cell cycle progression of NPC cells [13, 14, 15], but little is known about its down-expression in NPC cells. In this study, we found that BRD7 promoter is hemimethylated in a number of NPC cell lines including HNE1, CNE1, 6–10B and 5–8F cell lines, and that the methylation status of BRD7 promoter is inversely proportional with BRD7 mRNA expression in NPC cells. Thus, pharmacological inhibition of DNA methylation by 5-Aza-CdR enhanced BRD7 mRNA expression in NPC cells. This is in agreement with previous studies that, indeed, hemimethylation is sufficient to inhibit the expression of p16ink4A  and hMLH1 gene  in HCT116 and HT29 cell lines, respectively. Numerous studies have suggested that DNA methylation can suppress gene transcription either by directly inhibiting the interaction of transcription factors with their regulatory sequences or by attracting methylated DNA binding proteins that, in turn, recruit histone deacetylases and histone methyltransferases, resulting in an inactive chromatin structure [19, 20]. Our study indicates that DNA methylation represses BRD7 gene transcription by directly inhibiting the interaction of transcription factors with their regulatory elements, as judged by the inability of TSA to potentiate 5-Aza-CdR-mediated expression of BRD7 gene. Sp1 is a well-investigated factor that regulates transcription through specific sequences in G/C-rich promoter regions and is often critical for transcription initiation of TATA-less promoters . We identified several Sp1 binding sites in BRD7 promoter. Sp1 has high affinity to BRD7 promoter . Sequence analysis of the bisulfite-modified BRD7 promoter demonstrated that cytosine residues flanking functional Sp1 elements at -353/-337 and -330/-317 are methylated. It is known that methylation of specific cytosine residues in or near transcription regulatory motifs can block accessibility of the transcription factor [22, 23, 24]. Indeed, we found that methylation of cytosines flanking the -353/-337 and -330/-317 element impaired the ability of nuclear protein to bind the Sp1 binding sites in BRD7 promoter. Moreover, in vitro methylation of BRD7 promoter construct with SssI methylase leads to an almost complete loss of the activity of BRD7 promoter in NPC cell lines. NPC is highly radiosensitive and chemosensitive, but treatment of patients with locoregionally advanced disease remains problematic [25, 26]. New biomarkers for NPC, including DNA copy number of EBV or methylation of multiple tumour suppressor genes, which can be detected in serum and nasopharyngeal brushings, have been developed for the molecular diagnosis of this tumor. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC, such as aberrant methylation of the 5-CpG island of Ras association domain family 1A (RASSF1A), RARβ2, death-associated protein kinase (DAP-kinase), p16 (CDKN2A), p15 (CDKN2B), p14 (ARF) and O6-methylguanine DNA methyltransferase (MGMT), DLC1, TSLC1, TIG1 in NPC [27, 28, 29, 30, 31, 32, 33]. In the present study, among the 18 NPC patients, aberrant promoter methylation of BRD7 gene was detected in 100% of tumor biopsies and matched blood samples of NPC patients. In contrast, weak promoter methylation of BRD7 gene was observed in half of the blood samples from normal, healthy, age-matched individuals, indicating that epigenetic inactivation of BRD7 gene plays an important role in the tumorigenesis of NPC. This is a provocative observation, suggesting that the methylation status of BRD7 promoter may serve as a clinical biomarker for early detection and prescreening patients with clinical symptoms or individuals at high risk as well as in monitoring patients for recurrence. Further studies are necessary to confirm this.
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.
This work was supported by Natural Science Foundation of China 30772481, 30400528, 30470367, 30400238 and 973 key program (2006CB910500).
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