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

Short-chain fatty acids, metabolites produced by colonic bacteria from fermentation of dietary fiber, are the essential energy source for colonocytes. Without these short-chain fatty acids for energy, colonocytes undergo autophagy and die. In addition, short-chain fatty acids, such as butyrate, also act as a histone deacetylase (HDAC) inhibitor that mediates both normal and cancerous colonocyte proliferation (1) and anti-inflammatory response in the intestinal tract (2). GPR109A is a G-protein–coupled receptor for short-chain fatty acids (2–6). It has been shown that inhibition of DNA methylation increases GPR109A expression in human colon carcinoma cells, suggesting that GPR109A is silenced by DNA methylation (2). Although the function of GPR109A has been extensively studied in normal cells and cancer cells, the molecular mechanisms underlying GPR109A expression are unknown.

IFNγ is a proinflammatory cytokine secreted primarily by activated T cells (7). IFNγ functions through signal transducer and activator of transcription 1 (STAT1) to regulate the expression of its target genes. It has been reported that IFNγ secretion is elevated in the peripheral blood (8), and IFNγ expression level is increased in the inflamed colonic mucosa tissues in patients with ulcerative colitis (9). The expression and activation level of STAT1 is also significantly increased in colonic tissues of ulcerative colitis patients (10). Furthermore, chronic IFNγ signaling increases Cox-2 and inducible nitric oxide synthase expression to promote inflammation-dependent spontaneous colon cancer development (11). These observations thus suggest that chronic IFNγ signaling plays a key role in human ulcerative colitis pathogenesis and in inflammation-dependent spontaneous colon cancer development (8, 9, 11–13). However, it is apparent that the IFNγ signaling pathway is a two-edged sword. Although chronic IFNγ signaling promotes inflammation-dependent colon cancer development, the best known function of IFNγ in the tumor microenvironment is tumor suppression (7, 14–16).

We report here a novel mechanism underlying the regulation of GPR109A expression in colon cancer cells. Our data reveal that although the GPR109 promoter is methylated in human colon carcinoma cells, exposure of tumor cells to IFNγ reverses DNA methylation–mediated GPR109A silencing both in vitro and in vivo without altering the methylation status of the GPR109A promoter.

Human colon cancer cell lines SW480, SW620, SW116, and T84, and mouse colon carcinoma cell line CT26 were obtained from the American Type Culture Collection (ATCC). The ATCC has characterized these cells by morphology, immunology, DNA fingerprint, and cytogenetics. Deidentified human colon carcinoma specimens were obtained from the Georgia Regents University Medical Center and University Hospital with approval by the Georgia Regents University and University Hospital Human Assurance Committees.

IFNγ knockout (KO) mice (129S7(B6)-Ifngtm1Ts/J) and age-matched wild-type (WT) control mice (BALB/cJ) were obtained from The Jackson Laboratory. Mice were anesthetized under constant flow of oxygen and isofluorane. A small abdominal incision was made to expose the cecum. Tumor cells (1 × 10⁴ cells in 20 μL saline) were injected into the cecal wall on the serosal side. The wound was sealed with a wound clip. The use of mice and surgery procedures was approved by Georgia Regents University Institutional Animal Care and Use Committee.

Total RNA was isolated from cells using Trizol (Invitrogen) according to the manufacturer's instructions, and used for the first strand cDNA synthesis using the Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). The cDNA was then used as the template for PCR amplification. RT-PCR was conducted as previously described (17). The sequences of primers are listed in Supplementary Table S1.

Total RNA was isolated from freshly dissected human colon carcinoma specimens and matched adjacent normal colon tissues. RNAs from 3 patients were pooled and used for cDNA probe preparation using the RT2 First Strand Kit (Cat# 330401; Qiagen). The Human Inflammasomes PCR Arrays (Qiagen; Cat# PAHS-097Z) were used to analyze the inflammation-related gene expression using real-time RT-PCR according to the manufacturer's instructions.

Western blotting analysis was performed as previously described (16). The blot was probed with antibodies specific for pSTAT1 (Cat# 612133; BD Biosciences), H3K9ac (Cat# 9649; Cell Signaling), H3K18Ac (Cat# 9675; Cell Signaling), H3K27ac (Cat# 4753; Cell Signaling), H3 (Cat# 4499; Cell Signaling), p300 (Cat# sc-584; Santa Cruz Biotechnology), GPR109A (Cat# sc-134583; Santa Cruz Biotechnology), and β-actin (Cat# A1978; Sigma-Aldrich).

For demethylation of DNA, cells were treated for 3 days with 5′-aza-deoxycytidine (Sigma) at a final concentration of 1 μg/mL. For IFNγ treatment, cells were cultured in the presence of recombinant IFNγ (R & D Systems) at a final concentration of 100 U/mL.

Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions. Sodium bisulfite treatment of genomic DNA was carried out using the DNA Modification Kit (Zymo Research) according to the manufacturer's instructions. Methylation-sensitive PCR (MS-PCR) and DNA sequencing were carried out as previously described (18). For DNA sequencing, the bisulfite-modified genomic DNA was used as the template for PCR amplification of the human GPR109A promoter region. The amplified DNA fragments were cloned into pCR2.1 vector (Invitrogen), and individual clones were sequenced. DNA methylation was analyzed using QUMA program as previously described (19). All primer sequences are listed in Supplementary Table S1.

Immunohistochemical staining was performed at the Georgia Pathology Service. CD4- and CD8-specific antibodies were obtained from Dako (Cat# IS649 and IS623; DAKO). GPR109A-specific antibody has been previously described (20). Stained tissue specimens were evaluated independently by two pathologists (C.M. Heaton and J.R. Lee). For the CD4 and CD8 immunohistochemical slides, each pathologist scored the density of peritumoral lymphocytes. In addition, the percentage of tumor staining was documented.

Chromatin immunoprecipitation (ChIP) assays were carried out using anti-pSTAT1 antibody (Cat#sc-8394; Santa Cruz Biotechnology), anti-HDAC1 (Cat#sc-7872; Santa Cruz Biotechnology), and protein A-agarose beads (Millipore) as previously described (16). The GPR109A promoter DNA was detected by PCR using gene-specific primers. All PCR primer sequences are listed in Supplementary Table S1.

DNA–protein interaction was determined by electrophoresis mobility shift assay (EMSA) as previously described (19). The probe sequences are listed in Supplementary Table S1.

T84 cells were transiently transfected with scrambled siRNA (Dharmacon), p300-specific siRNA (Cat# sc-29431; Santa Cruz Biotechnology), and STAT1-specific siRNA (Qiagen; cat# SI02662324; SI02662884), respectively, using lipofectamine 2000 (Invitrogen) overnight.

Cells (5 × 10⁶) were harvested and washed in cold PBS, and then resuspended in nuclear extraction buffer (25 mmol/L Tris-HCl, pH 8.0, 50 mmol/L KCl, 5 mmol/L MgCl₂, 8% glycerol, 0.5% NP-40, and protease/phosphatase inhibitors) and incubated on ice for 10 minutes. Then the cells were homogenized in a glass homogenizer, and spun down at 1,000 g for 5 minutes. Cell pellets were washed once with DNaseI buffer, spun down at 1,000 g for 5 minutes, and then washed once with nucleus storage buffer (NSB; 50% glycerol, 50 mmol/L Tris-HCl pH 8.0, 5 mmol/L MgCl₂, 2.5 mmol/L DTT, 0.1 mmol/L EDTA, protease/phosphatase inhibitors). Endonuclease digestion was performed in a reaction mixture containing 320 μL of nuclei in NSB, 480 μL of 2× nuclease buffer (30 mmol/L NaCl, 8 mmol/L CaCl₂), 160 μL Tris buffer (50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L MgCl₂), and DNaseI (0.5 unit/mL; Roche Applied Science). Before the addition of DNaseI, an aliquot was removed from the reaction mixture for the zero time point. After addition of DNaseI, the digestion mixture was incubated at 37°C. Aliquots were taken out of the digestion mixture at 3, 6, 9, and 12 minutes after DNaseI addition, mixed immediately with 500 μL of chilled phenol:choloroform:isoamyl alcohol (25:24:1) mixture, vortexed, and spun down at 14,000 rpm for 10 minutes. DNA was collected from the top aqueous layer, purified, and analyzed by PCR. Human GPR109A promoter–specific primers were used to amplify the purified genomic DNA.

Statistical analysis was performed using the Student t test. A P value of <0.05 was taken as statistically significant.

Analysis of GPR109A mRNA levels from matched normal colon tissues and colon carcinoma tissues indicates that GPR109A is highly expressed in normal human colon epithelial cells but is silenced in human colon carcinoma cells. GPR109A expression is approximately 1.4-fold to 150-fold higher in the normal colon tissues as compared with that in the matched colon carcinoma tissues among the six matched pairs of tissues analyzed (Fig. 1A). To determine whether the GPR109A promoter is methylated in vivo in colon carcinoma, genomic DNA was isolated from human colon carcinoma tissues of 5 colon cancer patients. The MethPrimer program was used to design MS-PCR primers, and MS-PCR analysis of the bisulfite-modified genomic DNA indicated that the GPR109A promoter is methylated in all 5 colon cancer specimens examined (Fig. 1B). To determine whether GPR109A expression can be reactivated by inhibition of DNA methylation, tumor cells were treated with 5′-aza-deoxylcytidine (Aza-dC) and analyzed for GPR109A expression. Consistent with the heavy DNA methylation level, Aza-dC treatment dramatically increased GPR109A expression in human colon carcinoma cells in a dose-dependent manner (Fig. 1C).

Inhibition of DNA methylation upregulates GPR109A expression in human colon carcinoma cells (2). However, DNA sequence analysis indicates that there are no classical CpG islands in the human GPR109A gene promoter region (Fig. 1D). To determine whether DNA methylation directly regulates GPR109A expression, we analyzed the methylation level of the GPR109A promoter. Genomic DNA was isolated from human colon carcinoma SW116 and T84 cells and modified with bisulfite. Bisulfite sequencing PCR primers were designed using the MethPrimer program and were used to amplify one region of the GPR109A promoter. The amplified region was cloned. Single clones were then sequenced and examined for DNA methylation level. It is clear that the majority of cytosines of the CpG dinucleotides in this region are methylated in SW116 and T84 cells (Fig. 1D). Taken together, these data demonstrate that GPR109A is silenced in human colon carcinoma cells by methylation of its promoter DNA.

While analyzing the inflammation-related gene expression in colon carcinoma cells, we observed, surprisingly, that GPR109A was upregulated by IFNγ, a proinflammatory cytokine secreted by activated T cells. RT-PCR analysis of four human colon carcinoma cell lines revealed that GPR109A was dramatically upregulated by IFNγ despite its promoter DNA being methylated (Fig. 2A). Analysis of GPR109A expression kinetics revealed that GPR109A mRNA starts to increase 3 to 6 hours after IFNγ treatment and continued to increase even 24 hours after the treatment (Fig. 2A). Furthermore, IFNγ at a concentration as low as 10 μ/mL can reregulate GPR109A expression (Fig. 2A). Flow cytometry analysis revealed that IFNγR is expressed on the cell surface of all 4 cancer cell lines examined (Fig. 2B). Western blotting analysis indicated that pSTAT1, the key mediator of the IFNγ signaling pathway, is rapidly activated by IFNγ through phosphorylation in human colon carcinoma cells. pSTAT1 was detected as early as 10 minutes after IFNγ treatment (Fig. 2C). Analysis of the human GPR109A gene promoter revealed the presence of two gamma activation sequence (GAS) elements in the region (Fig. 2D). ChIP analysis with pSTAT1-specific mAb showed that IFNγ-activated pSTAT1 is associated with both GAS sites in the GPR109A promoter chromatin (Fig. 2D). Furthermore, EMSA indicated that pSTAT1 binds to the GAS DNA element directly (Fig. 2E). IFNγ functions through both STAT1-dependent and STAT1-independentl signaling pathways (21). To determine whether IFNγ-induced GPR109A expression depends on STAT1, STAT1 was silenced using specific siRNAs in human colon carcinoma T84 cells. Analysis of GPR109A expression revealed that silencing STAT1 diminished IFNγ-induced GPR109A expression (Supplementary Fig. S1). These observations thus indicate that IFNγ upregulates GPR109A expression through direct binding of pSTAT1 to the methylated GPR109A promoter DNA to activate its transcription.

To determine whether other cytokines induce GPR109A expression, T84 cells were treated with IFNα, IFNβ, TNFα, IL23, and GM-CSF for 24 hours, and analyzed for GPR109A expression. None of these cytokines upregulate GPR109A in human colon carcinoma cells (Supplementary Fig. S2).

IFNγ is primarily produced by activated T cells in vivo. To determine the expression levels of GPR109A and T-cell infiltration in human colon carcinoma, we analyzed 18 human colon carcinoma specimens using immunohistochemical staining with GPR109A-, CD4-, and CD8-specific antibodies. The stained tissues were then analyzed by two pathologists (C.M. Heaton and J.R. Lee). The images are presented in Fig. 3 and Supplementary Figs. S3 to S5. The staining scores are presented in Supplementary Table S2. Four colon carcinoma specimens (patients 7, 9, 10, and 13) showed no detectable GPR109A protein level. Tumor tissues from 2 of these 4 patients (patients 7 and 9) also had minimal to no CD4⁺ and CD8⁺ T-cell infiltrations. However, tumor specimen from patient 10 had moderate to abundant T-cell filtration, but GPR109 protein levels were low to nondetectable in those tumor cells. In the tumor tissues from the remaining 14 patients, either one or both CD4⁺ and CD8⁺ T cells were present inside the tumors, and GPR109A protein was detectable in the tumor cells. These observations indicate that although the GPR109A promoter DNAs are methylated in human colon carcinoma cells (Fig. 1), GPR109A is expressed in most of the human colon carcinoma cells (with the exception of samples from patients 10 and 13) if CD4⁺ or CD8⁺ T cells are present in the tumor microenvironment (Supplementary Table S2).

To determine the inflammation gene expression profiles, we analyzed the expression levels of inflammation-related genes in human colon carcinomas and matched adjacent normal colon tissues using PCR arrays. Real-time RT-PCR analysis identified 41 inflammation-related genes that are differentially expressed in normal colon tissues and in colon carcinoma tissues. Among these 41 genes, the expression level of IFNγ is about 4 times higher in carcinoma tissues than in the adjacent normal colon tissues (Supplementary Table S3).

To determine functionally whether IFNγ activates GPR109A transcription from the methylated promoter under pathophysiologic conditions, we established mouse colon carcinoma CT26 cell orthotopic transplant models in WT and IFNγ KO mice. The gpr109a promoter is methylated in CT26 cells (Fig. 4A); however, IFNγ treatment dramatically increased gpr109a mRNA levels in CT26 cells in vitro (Fig. 4A). A low-dose of CT26 (1 × 10⁴ cells/mouse) and a short tumor growth time (21 days) were used in this study to unmask the difference between WT and IFNγ KO mice. When surgically implanted into mouse cecum, CT26 tumors grew much faster in IFNγ KO mice than in WT mice (Fig. 4B). Analysis of the dissected tumor tissues revealed that mRNA levels of gpr109a are significantly higher in tumors grown in WT mice than that in IFNγ KO mice (Fig. 4C). Our data thus demonstrate that IFNγ activates gpr109a transcription from their methylated promoters in vivo.

IFNγ has been shown to induce DNA demethylation (22). To determine whether IFNγ upregulates GPR109A expression through inhibition of DNA methylation, human colon carcinoma cells were cultured in the absence or presence of IFNγ for 24 hours, and the DNA methylation level in the pSTAT1-binding consensus regions (Fig. 2D) of the GPR109A promoter was analyzed. DNA sequencing analysis indicated that the GPR109A promoter DNA is still methylated after IFNγ treatment in human colon carcinoma cells (Fig. 5).

Promoter DNA methylation of CpG islands often causes chromatin condensation to block transcription factor binding to the DNA, thereby silencing gene expression (23). However, histone acetylation may mediate the switch between repressive and permissive chromatin and thus dictate the functional state of genes (24–26). Our above observations that IFNγ upregulates GPR109A expression from the methylated GPR109A promoter without altering the promoter DNA methylation level suggest that histone acetylation might play a role in IFNγ-mediated GPR109A expression. Therefore, we hypothesized that inhibition of HDAC activity may enhance GPR109A transcription activation by IFNγ. To test this hypothesis, human colon carcinoma cells were treated with either TSA or IFNγ, or both TSA and IFNγ together, and analyzed for GPR109A expression. RT-PCR analysis revealed that either Trichostatin A (TSA) or IFNγ treatment dramatically increased GPR109A expression (Fig. 6A). However, combined TSA and IFNγ treatment resulted in an even greater upregulation of GPR109A than either agent alone (Fig. 6A).

It is known that HDAC1 mediates histone acetylation level in many gene promoters (27). Our observation that inhibition of HDAC activity dramatically increased IFNγ-induced GPR109A expression in human colon carcinoma cells led us to reason that IFNγ might regulate GPR109A expression by repressing HDAC1 expression. To test this hypothesis, we analyzed HDAC1 association with the GPR109A promoter. ChIP analysis showed that HDAC1 is associated with the chromatin at one of the two regions analyzed at the GPR109A promoter region (Fig. 6A). Consistent with the observation that HDAC1 expression is not regulated by IFNγ, IFNγ did not alter HDAC1 association with the GPR109A promoter chromatin (Fig. 6A).

Histone acetyltransferases (HAT) and HDACs antagonize each other through modification of the lysine side chains (27, 28). p300 is a HAT that often acts as a transcription cofactor (29–33). For example, p300 can mediate H3K18/27 acetylation (34–37). Therefore, we sought to determine whether IFNγ mediates p300 expression to regulate GPR109A expression. RT-PCR analysis revealed that IFNγ treatment indeed rapidly upregulates p300 expression level in human colon carcinoma cells. The increase in p300 mRNA and protein levels occurred just 10 minutes after IFNγ treatment and reached a plateau at 6 hours (Fig. 6B). Analysis of the human p300 gene promoter region identified two GAS sites (Fig. 6C), and ChIP analysis indicated that IFNγ-activated pSTAT1 is associated with one of the two GAS sites at the p300 promoter region in human colon carcinoma cells (Fig. 6C). Consistent with the ChIP results, EMSA indicated that pSTAT1 directly and specifically binds to the GAS DNA of the p300 promoter (Fig. 6D). Taken together, our data indicated that p300 is an immediate early target of the IFNγ signaling pathway in human colon cancer cells.

To determine whether p300 directly mediates IFNγ regulation of GPR109A transcription, we first analyzed p300 association with the GPR109A promoter chromatin. ChIP analysis revealed that IFNγ treatment dramatically increased p300 association with the GPR109A promoter chromatin (Fig. 6E). Next, we sought to determine whether p300 mediates GPR109 expression. p300 was silenced with p300-specific siRNA in human colon carcinoma cells, and GPR109A expression levels were then analyzed. Real-time RT-PCR analysis revealed that silencing p300 diminished IFNγ-induced GPR109A (Fig. 6F). Thus, we conclude that p300 directly mediates pSTAT1-activated GPR109A transcription in human colon carcinoma cells.

Although p300 acetylates multiple lysine residues in both H3 and H4 (37), p300 has been shown to specifically mediate H3K18/27 acetylation (34, 36). We therefore analyzed the effects of IFNγ on H3K18/27 acetylation. H3K9ac was also included as a positive control. IFNγ treatment increased the global acetylation levels of H3K9, H3K18, and H3K27 in human colon cancer cells (Fig. 7A). However, repeated ChIP analysis revealed that H3K27 is not acetylated in the GPR109A promoter regions and that IFNγ treatment does not induce acetylation of H3K27 in the GPR109A promoter region in human colon cancer cells (Fig. 7B). H3K9 is constitutively acetylated in the GPR109A promoter region in human colon cancer cells, and IFNγ increases H3K9 acetylation and induces H3K18 acetylation in the GPR109A promoter region in human colon cancer cells. To determine whether IFNγ induces H3K18 hyperacetylation specifically through p300, p300 was silenced with p300-specific siRNA. Silencing p300 diminished the IFNγ-induced acetylation in both H3K9 and H3K18 in the GPR109A promoter region in human colon cancer cells (Fig. 7C). Therefore, our data indicated that H3K9 is constitutively acetylated in the GPR109A promoter region, and IFNγ signaling activates p300 to induce hyperacetylation of H3K18 in the GPR109A promoter region in human colon cancer cells.

Our above findings raise the possibility that IFNγ induces chromatin remodeling at the GPR109A promoter region to convert a methylated DNA-mediated repressive chromatin conformation to a hyperacetylated and transcriptionally permissive one to facilitate pSTAT1 binding to activate GPR109A transcription. To test this hypothesis, we used limited DNaseI digestion to detect chromatin remodeling at the GPR109A promoter region. Human colon carcinoma cells were treated with IFNγ. Nuclei were then isolated from the cells and incubated with DNase I for various time periods. Genomic DNA was purified from the nuclei and used as the template for PCR amplification using the GPR109A promoter DNA-specific primers. Comparison of the PCR-amplified DNA fragment levels showed that the GPR109A promoter chromatin regions in IFNγ-treated cells are significantly more accessible to DNaseI than their counterparts in the untreated cells (Supplementary Fig. S6). Thus, our data indicate that IFNγ induces p300-mediated H3K18 hyperacetylation and resultant chromatin remodeling to create a transcriptionally permissible chromatin conformation at the methylated GPR109A promoter regions, which facilitates IFNγ-activated pSTAT1 binding to the GAS sites to activate GPR109A transcription.

In colorectal cancer, genome-wide analysis showed that many of the methylated genes have known or predicted function in the suppression of tumorigenesis (38). Therefore, colon cancer cells use DNA methylation as a mechanism to silence tumor suppressor genes to advance the disease (39). Although there are no classical CpG islands in the human GPR109A gene promoter region, we identified several CpG diculeotides in the region and observed that cytosines in these dinucleotides are methylated in human colon carcinoma cells. Analysis of six matched pairs of normal human colon tissues and colon carcinoma tissues revealed that GPR109A expression is indeed dramatically lower in the tumor tissues as compared with the adjacent normal colon tissues, which is consistent with the GPR109A promoter DNA methylation status. These observations thus indicate that GPR109A is silenced by its promoter DNA methylation in human colon carcinoma cells in vitro and in colon carcinoma tissues in vivo.

GPR109A is the receptor in colonocytes for the gut commensal bacterial metabolite butyrate (2) and mediates the butyrate-dependent anti-inflammatory effects to suppress colonic inflammation and inflammation-dependent colon cancer (2, 13). Therefore, it is not surprising that the GPR109A gene is silenced in human colon carcinoma cells. However, although the GPR109A promoter is methylated, GPR109A is expressed to some degree in a majority of colon carcinoma specimens examined, suggesting the presence of a novel mechanism that activates GPR109A transcription in the tumor microenvironment in spite of the methylation of its promoter. IFNγ is a proinflammatory cytokine secreted by activated T cells that functions as a key component of the host cancer immune surveillance system (40). IFNγ exerts its function through activation of STAT1 that acts as a master transcription factor to regulate IFNγ target gene transcription. Our observations that T cells extensively infiltrate colon cancer tissues and that high expression level of GPR109A is present in colon carcinoma cells in WT but not in IFNγ KO mice suggest that IFNγ is responsible, at least in part, for GPR109A expression in colon carcinoma in vivo. Because IFNγ is a key component of the host cancer immune surveillance system (40–42), our data thus suggest that the immune system might counteract tumor cell–induced and DNA methylation–mediated silencing of GPR109A expression through IFNγ-mediated reactivation of GPR109A transcription in tumor cells.

It has been shown that DNA methylation may render tumor cells resistant to IFN-induced apoptosis and that IFN-stimulated gene expression determines tumor cell sensitivity to DNA demethylation–induced apoptosis (43, 44). Strikingly, in the present study we observed that IFNγ can override the silencing effects of DNA methylation to activate GPR109A transcription without obvious demethylation of the GPR109A promoter DNA in human colon carcinoma cells. Current approach to reactivate DNA methylation–silenced tumor suppressor genes primarily relies on chemical inhibitors, mainly 5′-aza-dC that inhibits DNA methyltransferase (DNMT) activity (43, 45). This type of DNA methylation inhibitor is highly cytotoxic, and its use as chemotherapeutic agents in cancer therapy is associated with extensive toxicity (45). Our data suggest that immunotherapeutic approach, such as adoptive or active CTL immunotherapy (46), might be an alternative and yet effective and less toxic approach to activate DNA methylation–silenced expression of genes in cancer tissues. In addition, 5′-aza-dC is a general DNA methylation inhibitor that causes global DNA demethylation. In contrast, IFNγ-activated pSTAT1 only binds to specific DNA sequences (GAS element) to activate specific gene transcription. Therefore, IFNγ-activated expression of genes from DNA methylation–silenced promoters is gene specific; this approach is thus likely to be associated with low toxicity.

p300 is a histone acetyltransferase that also functions as a master transcriptional mediator in mammalian cells (27, 34, 47–49). In this study, we demonstrate that p300 is an immediate early target of the IFNγ signaling pathway. p300 transcription activation was detected 10 minutes after IFNγ treatment. STAT1 was also activated within 10 minutes after IFNγ treatment. Although it has been shown that pSTAT1 and p300 directly interact with each other (50), we observed that pSTAT1 physically binds to the p300 promoter region. Therefore, the likely signaling transduction cascade is that pSTAT1 is activated first, and it then binds to the p300 promoter to activate p300 transcription. Elevation of p300 enhances its association with the GPR109A promoters to induce H3K18 hyperacetylation, and chromatin remodeling, resulting in a permissive chromatin conformation at the GPR109A promoter to facilitate pSTAT1 binding to activate GPR109A transcription.

Based on the activation and expression kinetics of pSTAT1, p300, and GPR109A, we propose a model to illustrate IFNγ-induced GPR109A transcription activation from their methylated promoters. We propose that the GPR109A promoter is methylated and thus transcriptionally inactive in human colon carcinoma cells. Exposure of human colon carcinoma cells to IFNγ induces rapid STAT1 activation. Activated STAT1 directly binds to the p300 promoter region to rapidly activate p300 transcription. P300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation and resultant chromatin remodeling to create a transcriptionally permissive chromatin at the GPR109A promoter regions without an obvious change in the methylation status of the promoter DNA. A transcriptionally permissive chromatin structure allows pSTAT1 binding to activate GPR109A transcription despite DNA methylation (Supplementary Fig. S7).

No potential conflicts of interest were disclosed.

Conception and design: K. Bardhan, M. Thangaraju, V. Ganapathy, K. Liu

Development of methodology: K. Bardhan, A.V. Paschall, M. Thangaraju

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Bardhan, A.V. Paschall, M.R. Chen, P.S. Simon, Y.D. Bhutia, K. Gu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Bardhan, A.V. Paschall, C.M. Heaton, K. Liu

Writing, review, and/or revision of the manuscript: K. Bardhan, D.D. Browning, V. Ganapathy, K. Liu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Yang, M.R. Chen, P.M. Martin, J.R. Lee, K. Liu

Study supervision: D.D. Browning, K. Liu

The authors thank Kimberly Smith and Kimya Jones for their excellent technical assistance in immunohistochemical staining of tumor tissues.

This work was supported by NIH grants CA133085 and CA182518 and the VA merit review award I01BX001962 (to K. Liu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.