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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-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencingDNA isolated from treated and control untreated TC-1/A9 cells was subjected to bisulphite conversion and cloned. Sequences from 11 clones from each sample are presented. After treatment with IFNγ, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed. For LMP-7, we did not see any dramatic changes in bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. White and black circles indicate unmethylated and methylated CpGs, respectively. Rhombuses indicate the CpG islands that were investigated with bisulphite sequencing. White colour marks the CpG islands investigated with MSP. TS: transcription start.
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Figure 3: IFNγ-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencingDNA isolated from treated and control untreated TC-1/A9 cells was subjected to bisulphite conversion and cloned. Sequences from 11 clones from each sample are presented. After treatment with IFNγ, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed. For LMP-7, we did not see any dramatic changes in bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. White and black circles indicate unmethylated and methylated CpGs, respectively. Rhombuses indicate the CpG islands that were investigated with bisulphite sequencing. White colour marks the CpG islands investigated with MSP. TS: transcription start.

Mentions: Results from the MSP were confirmed by bisulphite sequencing using the TC-1/A9 cell line (Fig. 3). Again, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed after the treatment with IFNγ. For LMP-7, we did not see any dramatic changes in a bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. This corresponds with the result from MSP analysis with LMP-7 proximal primers. Based on these results, we can suggest that the methylation status of the distant rather than proximal regulatory sites in the LMP-7 region is crucial for their expression.


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-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencingDNA isolated from treated and control untreated TC-1/A9 cells was subjected to bisulphite conversion and cloned. Sequences from 11 clones from each sample are presented. After treatment with IFNγ, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed. For LMP-7, we did not see any dramatic changes in bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. White and black circles indicate unmethylated and methylated CpGs, respectively. Rhombuses indicate the CpG islands that were investigated with bisulphite sequencing. White colour marks the CpG islands investigated with MSP. TS: transcription start.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: IFNγ-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencingDNA isolated from treated and control untreated TC-1/A9 cells was subjected to bisulphite conversion and cloned. Sequences from 11 clones from each sample are presented. After treatment with IFNγ, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed. For LMP-7, we did not see any dramatic changes in bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. White and black circles indicate unmethylated and methylated CpGs, respectively. Rhombuses indicate the CpG islands that were investigated with bisulphite sequencing. White colour marks the CpG islands investigated with MSP. TS: transcription start.
Mentions: Results from the MSP were confirmed by bisulphite sequencing using the TC-1/A9 cell line (Fig. 3). Again, strong DNA demethylation of both the TAP-2 and TAP-1/LMP-2 gene promoter regions was observed after the treatment with IFNγ. For LMP-7, we did not see any dramatic changes in a bisulphite sequencing analysis targeting cytosines located at the positions -502 upstream to +130 downstream from the LMP-7 transcription start site. This corresponds with the result from MSP analysis with LMP-7 proximal primers. Based on these results, we can suggest that the methylation status of the distant rather than proximal regulatory sites in the LMP-7 region is crucial for their expression.

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