Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2014 Aug 30;5(16):6923-35.
doi: 10.18632/oncotarget.2222.

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

Affiliations
Free PMC article

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

Veronika Vlková et al. Oncotarget. .
Free PMC article

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.

Figures

Figure 1
Figure 1. IFNγ upregulation of the cell-surface MHC class I expression cells is associated with APM gene expression in experimental tumour cells
MHC class I expression (H-2Db and H-2Kb together) was determined by the FACS analysis of control tumour cells and after the treatment with IFNγ. Representative results are presented. (A) Upregulation of APM genes in TC-1/A9 and TRAMP-C2 tumours after treatment with IFNγ. (B) Expression levels of selected APM genes in TC-1/A9 and TRAMP-C2 control tumour cells and after the treatment with IFNγ. As a negative control, MHC class I-deficient RVP-3 cell line that did not respond to the IFNγ treatment was used. TC-1 cells, a MHC class I-positive parental cell line to the TC-1/A9 cells, that also displayed higher APM expression levels compared to TC-1/A9 cells, served as a MHC class I-positive control. *denote significant changes (P<0.05 determined in Student's t-test) as compared to the values for 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. Experiments were repeated three times with similar results.
Figure 2
Figure 2. IFNγ stimulates DNA demethylation of the APM gene promoter regions
DNA 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.
Figure 3
Figure 3. IFNγ-induced DNA demethylation of the TAP-2 and TAP-1/LMP-2 promoters in TC-1/A9 cells analysed by bisulphite sequencing
DNA 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.
Figure 4
Figure 4. Histone H3 acetylation levels in the APM regulatory gene sequences in TC-1/A9 cells are lower than those in TC-1 cells, but can be increased by IFNγ
ChIP analysis of chromatin from the TAP-1/LMP-2, TAP-2, and LMP-7 promoter sequences isolated from control and treated TC-1/A9 cells demonstrates an increase in acetylated histone H3 (H3K18) after IFNγ treatment. Results were normalized to the levels of the relative input in TC-1 cells. Experiments were repeated five times with similar results. * denotes significant changes (P<0.05 determined in Student's t-test) as compared to the values from TC-1 cells. ** denotes significant changes (P<0.05 determined in Student's t-test) as compared to the values from TC-1/A9 cells.
Figure 5
Figure 5. Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5AC
TC-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.
Figure 6
Figure 6. JAK/STAT inhibitors abrogated IFNγ-induced DNA demethylation of the APM gene promoters in TC-1/A9 cells
Inhibitor of Janus kinases (JAK inhibitor 1), as well as STAT1 phosphorylation inhibitor fludarabine, blocked the IFNγ induction of the MHC class I cell-surface expression (A), as well as APM gene activation (B), and caused impaired demethylation of the corresponding gene promoter regions (C). Both inhibitors blocked IFNγ-induced STAT1 phosphorylation (D). The effect of fludarabine was much weaker, as compared to Janus kinase inhibitor 1. * denotes significant changes (P<0.05 determined in Student's t-test) as compared to the values from untreated control cells. ** denotes significant changes (P<0.05 determined in Student's t-test) as compared to the values from TC-1/A9 cells after IFNγ treatment. 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. U = unmethylated primer, M = methylated primer. Experiments with JAK inhibitor 1 and fludarabine were repeated five and two times, respectively, with similar results.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

References

    1. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–692. - PMC - PubMed
    1. Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358:1148–1159. - PubMed
    1. Sigalotti L, Coral S, Fratta E, Lamaj E, Danielli R, Di Giacomo AM, Altomonte M, Maio M. Epigenetic modulation of solid tumors as a novel approach for cancer immunotherapy. Semin Oncol. 2005;32:473–478. - PubMed
    1. Tomasi TB, Magner WJ, Khan AN. Epigenetic regulation of immune escape genes in cancer. Cancer Immunol Immunother. 2006;55:1159–1184. - PubMed
    1. Garrido F, Ruiz-Cabello F, Cabrera T, Perez-Villar JJ, Lopez-Botet M, Duggan-Keen M, Stern PL. Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol Today. 1997;18:89–95. - PubMed

Publication types

Substances

LinkOut - more resources

Feedback