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Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins.

Li L, Su X, Choi GC, Cao Y, Ambinder RF, Tao Q - BMC Cancer (2012)

Bottom Line: Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle.Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection.Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer, Hong Kong, China.

ABSTRACT

Background: Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples.

Methods: We evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.

Results: We found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.

Conclusions: Hypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.

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(A-D) Pharmacologic demethylation with 5-aza-dC at indicated concentrations and time points analyzed by MSP in Rael, NK-YS and C666-1 cell lines. Each bottom panel is the plotted densitometry ratios as % of methylation (U/U + M) of Zp and Rp. M, methylated; U, unmethylated. (E, F). Detailed BGS analysis of demethylation after 5-aza-dC treatment in Rael and C666-1 cell lines. "x" circle, CpG site abolished by sequence variation or undetermined methylation status due to an unconverted C in nearby CpN site.
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Figure 5: (A-D) Pharmacologic demethylation with 5-aza-dC at indicated concentrations and time points analyzed by MSP in Rael, NK-YS and C666-1 cell lines. Each bottom panel is the plotted densitometry ratios as % of methylation (U/U + M) of Zp and Rp. M, methylated; U, unmethylated. (E, F). Detailed BGS analysis of demethylation after 5-aza-dC treatment in Rael and C666-1 cell lines. "x" circle, CpG site abolished by sequence variation or undetermined methylation status due to an unconverted C in nearby CpN site.

Mentions: To determine whether methylation directly mediates the transcriptional repression of BZLF1 and BRLF1, Rael, NK-YS and C666-1 were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor. Rael treated with 5-aza-dC at different concentrations (0.005, 0.015, 0.03, 0.06, 0.5 and 1 μM) for 72 h. MSP analysis showed that unmethylated Rp and Zp alleles were increased in a dose-dependent manner, with significant demethylation of Rp and Zp observed by treatment with 1 μM 5-aza-dC. Subsequently, 1 μM of 5-aza-dC was chosen to evaluate the demethylation of Zp and Rp for different indicated times (5 h, 24 h, 30 h, 48 h) in Rael cell line. It was found that both Zp and Rp were demethylated in a time-dependent manner (Figure 5A, B). Similarly, NK-YS and C666-1 cells treated with 5-aza-dC at different concentrations showed obvious demethylation of Zp and Rp, compared with untreated cells (Figure 5C, D). Concomitantly, Zp and Rp alleles were partially demethylated after 5-aza-dC treatment by high-resolution BGS analysis (Figure 5E, F, Table 2),


Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins.

Li L, Su X, Choi GC, Cao Y, Ambinder RF, Tao Q - BMC Cancer (2012)

(A-D) Pharmacologic demethylation with 5-aza-dC at indicated concentrations and time points analyzed by MSP in Rael, NK-YS and C666-1 cell lines. Each bottom panel is the plotted densitometry ratios as % of methylation (U/U + M) of Zp and Rp. M, methylated; U, unmethylated. (E, F). Detailed BGS analysis of demethylation after 5-aza-dC treatment in Rael and C666-1 cell lines. "x" circle, CpG site abolished by sequence variation or undetermined methylation status due to an unconverted C in nearby CpN site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3362778&req=5

Figure 5: (A-D) Pharmacologic demethylation with 5-aza-dC at indicated concentrations and time points analyzed by MSP in Rael, NK-YS and C666-1 cell lines. Each bottom panel is the plotted densitometry ratios as % of methylation (U/U + M) of Zp and Rp. M, methylated; U, unmethylated. (E, F). Detailed BGS analysis of demethylation after 5-aza-dC treatment in Rael and C666-1 cell lines. "x" circle, CpG site abolished by sequence variation or undetermined methylation status due to an unconverted C in nearby CpN site.
Mentions: To determine whether methylation directly mediates the transcriptional repression of BZLF1 and BRLF1, Rael, NK-YS and C666-1 were treated with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor. Rael treated with 5-aza-dC at different concentrations (0.005, 0.015, 0.03, 0.06, 0.5 and 1 μM) for 72 h. MSP analysis showed that unmethylated Rp and Zp alleles were increased in a dose-dependent manner, with significant demethylation of Rp and Zp observed by treatment with 1 μM 5-aza-dC. Subsequently, 1 μM of 5-aza-dC was chosen to evaluate the demethylation of Zp and Rp for different indicated times (5 h, 24 h, 30 h, 48 h) in Rael cell line. It was found that both Zp and Rp were demethylated in a time-dependent manner (Figure 5A, B). Similarly, NK-YS and C666-1 cells treated with 5-aza-dC at different concentrations showed obvious demethylation of Zp and Rp, compared with untreated cells (Figure 5C, D). Concomitantly, Zp and Rp alleles were partially demethylated after 5-aza-dC treatment by high-resolution BGS analysis (Figure 5E, F, Table 2),

Bottom Line: Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle.Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection.Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer, Hong Kong, China.

ABSTRACT

Background: Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of BZLF1 and BRLF1 promoters (Zp and Rp) in tumor samples.

Methods: We evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as BZLF1 and BRLF1 expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.

Results: We found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing BZLF1 and BRLF1. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of BZLF1 and BRLF1 was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene BHRF1 and late lytic gene BLLF1.

Conclusions: Hypermethylation of Zp and Rp mediates the frequent silencing of BZLF1 and BRLF1 in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.

Show MeSH
Related in: MedlinePlus