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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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IFNγ stimulates DNA demethylation of the APM gene promoter regionsDNA from tumour cell lines cultured in the absence or presence of IFNγ were bisulphite treated and subjected to MSP analysis of the TAP-1/LMP-2, TAP-2 and LMP-7 promoter sequences. Higher proportion of DNA methylation, as compared to TC-1 cells and DNA demethylation induced by IFNγ, is documented in TC-1/A9 cells (A). Similar results were obtained in TRAMP-C2 cells (B), while no effects were noticed in IFNγ-insensitive RVP-3 tumour cells (C). U = unmethylated primer, M = methylated. Experiments were repeated three times with similar results.
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Figure 2: IFNγ stimulates DNA demethylation of the APM gene promoter regionsDNA from tumour cell lines cultured in the absence or presence of IFNγ were bisulphite treated and subjected to MSP analysis of the TAP-1/LMP-2, TAP-2 and LMP-7 promoter sequences. Higher proportion of DNA methylation, as compared to TC-1 cells and DNA demethylation induced by IFNγ, is documented in TC-1/A9 cells (A). Similar results were obtained in TRAMP-C2 cells (B), while no effects were noticed in IFNγ-insensitive RVP-3 tumour cells (C). U = unmethylated primer, M = methylated. Experiments were repeated three times with similar results.

Mentions: Enhanced APM gene expression in the MHC class I-deficient tumour cells was associated with DNA demethylation of the corresponding gene promoter regions determined by MSP (Fig. 2). We demonstrated DNA demethylation of the promoter sequences of selected antigen-presenting machinery genes (TAP-1/LMP-2, TAP-2) upon IFNγ treatment both in the MHC class I-deficient tumour cell line TC-1/A9 (Fig. 2a) and in the prostate cancer cell line TRAMP-C2 (Fig. 2b). For LMP-7, we did not see any dramatic changes in the MSP analysis targeting cytosines located at positions -186, -190 and -335 upstream from the LMP-7 transcription start (proximal primers). We therefore analysed CpGs in a more distant region covering CpGs at the positions -1219, -1233 and -1238 and -1087. In this region, we indeed noticed massive demethylation upon IFNγ treatment (distant primers). As a positive control, we used DNA from the MHC class I-positive TC-1 cell line (Fig. 2a), and as a negative control, we used MHC class I-deficient RVP-3 cell line, which did not respond to the IFNγ treatment (Fig. 2c). Comparative analysis of the TC-1 and TC-1/A9 cell lines demonstrated association of the cell surface MHC class I expression levels with DNA demethylation of the APM genes. No demethylation of the APM genes upon IFNγ treatment was seen in the RVP-3 cells.


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)

IFNγ stimulates DNA demethylation of the APM gene promoter regionsDNA from tumour cell lines cultured in the absence or presence of IFNγ were bisulphite treated and subjected to MSP analysis of the TAP-1/LMP-2, TAP-2 and LMP-7 promoter sequences. Higher proportion of DNA methylation, as compared to TC-1 cells and DNA demethylation induced by IFNγ, is documented in TC-1/A9 cells (A). Similar results were obtained in TRAMP-C2 cells (B), while no effects were noticed in IFNγ-insensitive RVP-3 tumour cells (C). U = unmethylated primer, M = methylated. Experiments were repeated three times with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196173&req=5

Figure 2: IFNγ stimulates DNA demethylation of the APM gene promoter regionsDNA from tumour cell lines cultured in the absence or presence of IFNγ were bisulphite treated and subjected to MSP analysis of the TAP-1/LMP-2, TAP-2 and LMP-7 promoter sequences. Higher proportion of DNA methylation, as compared to TC-1 cells and DNA demethylation induced by IFNγ, is documented in TC-1/A9 cells (A). Similar results were obtained in TRAMP-C2 cells (B), while no effects were noticed in IFNγ-insensitive RVP-3 tumour cells (C). U = unmethylated primer, M = methylated. Experiments were repeated three times with similar results.
Mentions: Enhanced APM gene expression in the MHC class I-deficient tumour cells was associated with DNA demethylation of the corresponding gene promoter regions determined by MSP (Fig. 2). We demonstrated DNA demethylation of the promoter sequences of selected antigen-presenting machinery genes (TAP-1/LMP-2, TAP-2) upon IFNγ treatment both in the MHC class I-deficient tumour cell line TC-1/A9 (Fig. 2a) and in the prostate cancer cell line TRAMP-C2 (Fig. 2b). For LMP-7, we did not see any dramatic changes in the MSP analysis targeting cytosines located at positions -186, -190 and -335 upstream from the LMP-7 transcription start (proximal primers). We therefore analysed CpGs in a more distant region covering CpGs at the positions -1219, -1233 and -1238 and -1087. In this region, we indeed noticed massive demethylation upon IFNγ treatment (distant primers). As a positive control, we used DNA from the MHC class I-positive TC-1 cell line (Fig. 2a), and as a negative control, we used MHC class I-deficient RVP-3 cell line, which did not respond to the IFNγ treatment (Fig. 2c). Comparative analysis of the TC-1 and TC-1/A9 cell lines demonstrated association of the cell surface MHC class I expression levels with DNA demethylation of the APM genes. No demethylation of the APM genes upon IFNγ treatment was seen in the RVP-3 cells.

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