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Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

Vlková V, Štěpánek I, Hrušková V, Šenigl F, Mayerová V, Šrámek M, Šímová J, Bieblová J, Indrová M, Hejhal T, Dérian N, Klatzmann D, Six A, Reiniš M - Oncotarget (2014)

Bottom Line: Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells.IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes.Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumour Immunology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, v. v. i., Prague.

ABSTRACT
Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

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Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5ACTC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2, LMP-2 & LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P<0.05 determined in Student's t-test) as compared to the values from untreated cells. Biological triplicates were used for the analysis. In all experiments, error bars show standard deviations. Relative expression numbers represent the percentage of the β-actin expression levels. The levels of relative gene expression were presented as fold changes compared to the levels found in control samples. MHC class I expression (H-2Db and H-2Kb together) was determined by FACS analysis of the control tumour cells and after the treatment with IFNγ and 5AC. Representative results are presented (C). Experiments were repeated three times with similar results.
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Figure 5: Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5ACTC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2, LMP-2 & LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P<0.05 determined in Student's t-test) as compared to the values from untreated cells. Biological triplicates were used for the analysis. In all experiments, error bars show standard deviations. Relative expression numbers represent the percentage of the β-actin expression levels. The levels of relative gene expression were presented as fold changes compared to the levels found in control samples. MHC class I expression (H-2Db and H-2Kb together) was determined by FACS analysis of the control tumour cells and after the treatment with IFNγ and 5AC. Representative results are presented (C). Experiments were repeated three times with similar results.

Mentions: To examine the kinetics by which the APM promoter regions undergo IFNγ-mediated changes in DNA methylation, as compared to the effects of a DNA methyltransferase inhibitor, TC-1/A9 cells were treated with either IFNγ or 5AC for various time periods and then by sodium bisulphite conversion and MSP. In untreated cells, the core CpG island was highly methylated, and demethylation was noticed within 2 h after IFNγ treatment, while nearly maximal demethylation was evident by 6 h (Fig. 5). After 5AC treatment, strong demethylation was evident by 24 h. The kinetics of the 5AC-induced demethylation is in agreement with the fact that 5AC-induced demethylation required DNA replication. On the other hand, the kinetics of the IFNγ-mediated DNA demethylation suggests that DNA replication was not crucial.


Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

Vlková V, Štěpánek I, Hrušková V, Šenigl F, Mayerová V, Šrámek M, Šímová J, Bieblová J, Indrová M, Hejhal T, Dérian N, Klatzmann D, Six A, Reiniš M - Oncotarget (2014)

Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5ACTC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2, LMP-2 & LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P<0.05 determined in Student's t-test) as compared to the values from untreated cells. Biological triplicates were used for the analysis. In all experiments, error bars show standard deviations. Relative expression numbers represent the percentage of the β-actin expression levels. The levels of relative gene expression were presented as fold changes compared to the levels found in control samples. MHC class I expression (H-2Db and H-2Kb together) was determined by FACS analysis of the control tumour cells and after the treatment with IFNγ and 5AC. Representative results are presented (C). Experiments were repeated three times with similar results.
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Figure 5: Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5ACTC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2, LMP-2 & LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P<0.05 determined in Student's t-test) as compared to the values from untreated cells. Biological triplicates were used for the analysis. In all experiments, error bars show standard deviations. Relative expression numbers represent the percentage of the β-actin expression levels. The levels of relative gene expression were presented as fold changes compared to the levels found in control samples. MHC class I expression (H-2Db and H-2Kb together) was determined by FACS analysis of the control tumour cells and after the treatment with IFNγ and 5AC. Representative results are presented (C). Experiments were repeated three times with similar results.
Mentions: To examine the kinetics by which the APM promoter regions undergo IFNγ-mediated changes in DNA methylation, as compared to the effects of a DNA methyltransferase inhibitor, TC-1/A9 cells were treated with either IFNγ or 5AC for various time periods and then by sodium bisulphite conversion and MSP. In untreated cells, the core CpG island was highly methylated, and demethylation was noticed within 2 h after IFNγ treatment, while nearly maximal demethylation was evident by 6 h (Fig. 5). After 5AC treatment, strong demethylation was evident by 24 h. The kinetics of the 5AC-induced demethylation is in agreement with the fact that 5AC-induced demethylation required DNA replication. On the other hand, the kinetics of the IFNγ-mediated DNA demethylation suggests that DNA replication was not crucial.

Bottom Line: Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells.IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes.Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumour Immunology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, v. v. i., Prague.

ABSTRACT
Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

Show MeSH
Related in: MedlinePlus