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. 2015 Jul;3(7):795-805.
doi: 10.1158/2326-6066.CIR-14-0164. Epub 2015 Mar 3.

IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer

Kankana Bardhan  1 , Amy V Paschall  2 , Dafeng Yang  3 , May R Chen  1 , Priscilla S Simon  2 , Yangzom D Bhutia  1 , Pamela M Martin  1 , Muthusamy Thangaraju  4 , Darren D Browning  4 , Vadivel Ganapathy  4 , Christopher M Heaton  5 , Keni Gu  6 , Jeffrey R Lee  7 , Kebin Liu  8
Affiliations

IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer

Kankana Bardhan et al. Cancer Immunol Res. 2015 Jul.

Abstract

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development.

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Conflict of interest statement

Conflict of interest: None

Figures

Figure 1
Figure 1. The human GPR109A promoter is methylated and GPR109A expression is silenced in human colon carcinoma cells
A. GPR109A expression level in matched pairs of human normal colon and colon carcinoma tissues. Colon carcinoma tissues and adjacent normal tissues were collected from 6 patients, and analyzed for GPR109A expression by RT-PCR. GAPDH was used as normalization control. Bottom panel: the GPR109A levels were quantified using the NIH J program. The ratio of GPR109A vs GAPDH in patient #1 was arbitrarily set at 1. The GPR109A expression levels of the remaining five specimens were then normalized based on patient #1. B. Methylation status of the GPR109A gene promoter in human colon carcinoma specimens. Genomic DNA was isolated from colon carcinoma specimens of 5 colon cancer patients and modified with bisulfate. The modified DNA was then analyzed by MS-PCR (U, unmethylated; M, methylated). Numbers above the figure are patient codes. C. Inhibition of DNA methylation increases GPR109A expression. SW116 and T84 cells were treated with Aza-dC for 3 days at the indicated doses and analyzed for GPR109A expression level by semi-quantitative RT-PCR (top panel) and real-time RT-PCR (bottom panel). The GPR109A expression levels of untreated cells were arbitrarily set at 1. Column: mean, bar: SD. D. Methylation level of the human GPR109A gene promoter in human colon carcinoma cell lines. The human GPR109A gene DNA sequence was exported from the human genome database and analyzed for CpG islands using MethyPrimer computer program. Top panel: the human GPR109A gene promoter structure. Vertical bars under the line indicate location of CpG dinucleotides, and the number under the line indicates nucleotide number relative to GPR109A transcription initiation site (+1). Bottom panel: methylation level of the GPR109A gene promoter in the indicated cell lines. Genomic DNA was modified with bisulfite. The indicated DNA fragment was amplified by PCR and cloned into pCR2.1 vector. Individual clones for each cell line were sequenced and the methylation level of the cytosine in the CpGs was analyzed using QUMA computer program (open circle, unmethylated CpG; closed circle, methylated CpG).
Figure 2
Figure 2. IFNγ activates GPR109A transcription from the methylated GPR109A promoter
A. Tumor cells were treated with IFNγ and analyzed for GPR109A expression level by RT-PCR (top panel). The GPR109A expression in T84 cells were also analyzed by real-time PCR (middle panel). Column: mean, Bar: SD. Bottom panel: SW116 cells were treated with IFN-γ at 10 and 100 U/ml for 24h and analyzed by Western blotting analysis. B. Tumor cells were stained with IFN-γR mAb and analyzed for cell surface IFN-γR protein level. Grey area: IgG isotype control; solid line: IFN-γR-specific staining. C. T84 cells were treated with IFN-γ for the indicated time and analyzed for pSTAT1 protein level by Western blotting. D. The human GPR109A promoter structure. The GAS element locations and consensus sequences are shown under the bar. The ChIP PCR amplified regions are indicated above the bar. Bottom panel: SW116 and T84 cells were either untreated (−IFN-γ) or treated with IFN-γ (+IFN-γ) for 6 h and analyzed by ChIP using pSTAT1-specific mAb. Purified genomic DNA (gDNA) was used as a positive control for the PCR (left panel). The ChIP DNA was then analyzed by real time PCR (right panel). E. T84 cells were either untreated or treated with IFN-γ (+IFN-γ) for 6 h and used for nuclear extract preparation. The nuclear extracts were incubated with the GAS element-containing DNA probe in the absence or presence of IgG or pSTAT1 mAb and then analyzed for protein-DNA interaction by EMSA. A mutant GAS DNA probe (MT probe) was used as negative control.
Figure 3
Figure 3. GPR109A protein level and T-cell infiltration level in human colon carcinoma tissues
Human colon carcinoma specimens were stained with GPR109A-, CD4-, and CD8-specific antibodies. Shown are images from two patients with high (A, patient 3 as in Fig. S2–4) and low (B, patient 9 as in Fig. S2–4) GPR109A, CD4 and CD8 staining, respectively.
Figure 4
Figure 4. IFNγ activates gpr109a transcription from their methylated promoters in vivo
A. Genomic DNA was isolated from mouse colon carcinoma cell line CT26 (left bottom panel), and analyzed for gpr109a promoter DNA methylation by MS-PCR. U: unmethylated, M: methylated. CT26 cells were also treated with IFN-γ (100U/ml) and analyzed for GPR109A expression level by real-time RT-PCR (right panel). Column: mean, Bar: SD. B. CT26 cells (1x104/mouse) were surgically implanted into the cecal wall of WT (n=9) and IFN-γ KO (n=7) mice. Tumor growth on the colon tissues were analyzed 21 days after tumor transplant. Left panel: representative image of WT and IFN-γKO mouse colon tissues showing colon tumor development (red arrows). The tumor volumes were quantified and presented in the right panel. C. Colon carcinoma tissues as shown in B were dissected from the colon tissues of three WT and three IFN-γKO mice, respectively, and analyzed by real-time RT-PCR for GPR109A expression levels. Each column represents relative GPR109A expression level in one mouse.
Figure 5
Figure 5. IFNγ does not alter the methylation level of the GPR109 Apromoter DNAs in human colon carcinoma cells
T84 cells were cultured in the absence or presence of IFN-γ for 24 h. Genomic DNA was isolated from the cells and analyzed for the GPR109A promoter DNA methylation level in the 2 GAS-containing GPR109A promoter regions as shown. Open circle: Unmethylated CpG; Closed circle: Methylated CpG.
Figure 6
Figure 6. IFNγ regulates GPR109A expression through direct regulation of p300 expression
A. T84 cells were treated with TSA (200 nM) or IFN-γ (100U/ml) for 24 h and analyzed for the expression of the indicated genes by RT-PCR (left panel) and real-time RT-PCR (middle panel). The untreated (−IFN-γ) or treated cells (+IFN-γ) were also analyzed by ChIP using HDAC1-specific antibody for HDAC1 association with the GPR109A promoter region (right panel). B. T84 cells were treated with IFN-γ for the indicated time and analyzed by RT-PCR for p300 expression level by semi-quantitative PCR (left panel) and real-time PCR (middle panel). p300 protein level in T84 cells was analyzed by Western blotting (right panel). C. Top panel: The human p300 promoter structure. The ChIP PCR-amplified regions are indicated above the bar. The GAS element locations and consensus sequences are shown under the bar. Bottom panel: T84 cells were either untreated (−IFN-γ) or treated with IFN-γ (+IFN-γ) for 6 h and analyzed by ChIP using pSTAT1-specific mAb for pSTAT1 association with the p300 promoter region. Left panel: semi-quantitative PCR. Right panel: real-time PCR. The value of untreated cells was set at 1. D: EMSA of pSTAT1 binding to the GAS element-containing p300 promoter DNA. T84 cells were either untreated or treated with IFNγ (+IFN-γ) for 6 h and used for nuclear extract preparation. The nuclear extracts were incubated with the GAS element-containing DNA probe in the absence or presence of IgG or pSTAT1 mAb and then analyzed for protein-DNA interactions. E. T84 cells were cultured in the absence (−IFN-γ) or presence (+IFN-γ) of IFNγ for 6 h and then analyzed by ChIP using p300-specific antibody to detect p300 association with the GPR109A promoter chromatin. Left panel: semi-quantitative PCR. Right panel: real-time PCR. The value of IgG of untreated cells was set at 1. F. T84 cells were transiently transfected with scrambled or p300-specific siRNAs overnight, and then treated with IFNγ for 8 h. p300 and GPR109A expression levels were then analyzed by real-time RT-PCR.
Figure 7
Figure 7. IFNγ up-regulates p300 expression to mediate H3K18 acetylation in the GPR109A promoter region
A. IFN-γ induces global acetylation of H3K9, H3K18 and H3K27. SW116 and T84 cells were treated with IFN-γ. Histone acidic extracts were prepared at the indicated time points from the cells and analyzed for the indicated acetylated lysine residues of H3 by Western blotting. B. H3K27 is not acetylated in the GPR109A promoter regions. T84 cells were treated with IFN-γ overnight and analyzed for acetylated H3K27 in the GPR109A promoter regions by ChIP. C. p300 mediates H3K18 acetylation in the GPR109A promoter region. T84 cells were transiently transfected with either scramble siRNA or p300-specific siRNA overnight, followed by treatment with IFN-γ for 8h. Cells were then analyzed by ChIP for acetylated H3K9 and H3K18 levels in the GPR109A promoter region. Top panel: semi-quantitative PCR. Bottom panel: real-time PCR. The value of IgG of untreated cells was set at 1.
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