1. Volume 138, Issue 5, Pages 1898-1908.e12 (May 2010)
Functional Switching of TGF-β1 Signaling in Liver Cancer via Epigenetic Modulation of a Single CpG Site in TTP Promoter 
Bo Hwa Sohn, In Young Park, Jung Ju Lee, Suk–Jin Yang, Ye Jin Jang, Kyung Chan Park, Dong Joon Kim, Dong Chul Lee, Hyun Ahm Sohn, Tae Woo Kim, Hyang–Sook Yoo, Jong Young Choi, Yun Soo Bae, Young Il Yeom 
Gastroenterology 
Volume 138, Issue 5, Pages e12 (May 2010)
DOI: /j.gastro
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2. Figure 1
TTP is down-regulated via DNA methylation in HCC cell lines. (A) TTP mRNA levels in HCC cell lines were measured by quantitative real-time RT-PCR using β-Actin as the control for normalization. Error bars; mean ± standard error. (B) TTP induction by a DNMT inhibitor. HCC cell lines were treated with AzadC (72 hours) and TTP mRNA levels measured by semiquantitative RT-PCR. (C) Dose-dependent induction of TTP by AzadC. HCC cell lines were treated with indicated concentrations of AzadC and TTP mRNA levels measured by quantitative real-time RT-PCR.
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3. Figure 2
Single CpG site methylation at the TTP promoter in HCC cell lines. (A) CpG islands of the TTP gene locus predicted by “CpG island plot” ( CpG sites between −600 and +878 bp from transcription start site are presented (vertical bars) and numbered from −600 bp. Nucleotide sequences for TRR (−531 to −471 bp) are shown. (B) DNA methylation status of the TTP promoter and enhancer regions in HCC cell lines, determined by bisulfite sequencing. Circles symbolize CpG sites residing in 3 subregions (R1, R2, and R3). Open circle, unmethylated; solid circle, methylated.
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4. Figure 3
TTP down-regulation associated with single CpG site methylation at the TTP promoter in HCC patient tissues. (A) DNA methylation pattern of the TTP promoter and enhancer regions in 2 sets of paired tumor and adjacent, normal-appearing nontumor tissues. Bisulfite sequencing results for normal liver genomic DNA are also shown. (B) Methylation quantities at the cytosine residue (“the −500C”) of the CpG No. 2, located at −500 bp from transcription start site of TTP, were determined by pyrosequencing from the tissues of 24 HCC patients. N.L, normal liver; N.T, non-tumor; T, tumor. Methylation levels of paired tumor and nontumor tissues from the same individuals are linked with a line. (C) TTP mRNA levels in the tissues of 24 HCC patients were determined by quantitative real-time RT-PCR using β-Actin as the normalization control. (D) Inverse correlation between the TTP mRNA levels and the methylation quantities at the −500C in HCC patient tissues. Data for all the examined tissues (normal liver, nontumor, and tumor) were plotted (γ = − , P < .001).
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5. Figure 4
Regulation of TTP expression via the specific DNA methylation at the −500C. (A) TTP induction by TGF-β1 in SK-Hep1 and PLC/PRF/5 cells. Quantitative real-time RT-PCR experiments were performed after treating cells with TGF-β1 (5 μg/L) for the indicated time period with or without AzadC (2 μmol/L) pretreatment. (B–D) Luciferase reporter assays for various TTP promoter constructs. Cells were transfected with plasmid DNAs and, after 48 hours, treated with TGF-β1 for an additional 2 hours. (B) Luciferase activities measured from SK-Hep1 cells transfected with TTP promoter constructs carrying wild-type sequences (WT), a single base substitution at the −500C (C→T), or TRR deletion (Del). AzadC pretreatment was performed 12 hours prior to the transfection. (C) Luciferase activities measured in PRC/PRF/5 cells transfected with in vitro methylated promoter constructs. In vitro methylated TTP promoter sequences and TRR sequences were ligated with linearized pGL3-basic vector. (D) In vitro methylated TRR sequences were ligated with pGL3 vector containing SV40 promoter and directly transfected into PLC/PRF/5 cells. TRR contains only 1 CpG site, ie, the −500C.
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6. Figure 5
Differential epigenetic status of the TTP locus in SK-Hep1 and PLC/PRF/5 cells. (A) ChIP analysis of the TTP locus in response to TGF-β1. Cells were treated with/without AzadC (2 μmol/L) and/or TGF-β1 (5 μg/L) as indicated and their chromatins immunoprecipitated using antibodies against unphosphorylated RNA polymerase II (RNAPII; unP), RNAPII phosphorylated at C-terminal domain serine residues 5 and 2 (pS5 and pS2, respectively), or acetylated histones H3 and H4 (Ac-H3 and Ac-H4, respectively). PCR-amplified regions are designated as “a” (−561 to −352 bp from transcription start site of TTP genomic sequence), “b” (−17 to +162 bp), “c” (+2029 to bp), and “d” (+3041 to bp) under the genomic diagram of the TTP locus. Region “a” harbors TRR and −500C. (B and C) Graphic representation of the ChIP results in Figure 5A. Error bars: mean ± SE. *Significantly different from control (P < .05). (D) ChIP analysis of region “a” using antibodies against transcription regulators (phosphorylated SMAD2/3 (pSMAD2/3) and c-Ski), methyl DNA-binding proteins (MECP2 and MBD2), or DNMTs. (E) Restoration of TTP expression in SK-Hep1 cells by double knock-down of DNMT1 and DNMT3A. Cells infected with lentiviruses encoding shRNA for DNMT1 and/or DNMT3A (MOI = 5, 72 hours) were treated with TGF-β1 (5 μg/L), and their TTP mRNA content was quantitatively determined by real-time RT-PCR. β-Actin was used as the normalization control. Error bars: mean ± SE. (F) Demethylation at the −500C by double knock-down of DNMT1 and DNMT3A. DNA methylation pattern of R1 region (Figure 2A) was determined by bisulfate sequencing of the genomic DNA from SK-Hep1 cells treated as in E.
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7. Figure 6
TTP-dependent cellular responses to the antiproliferative effect of TGF-β1. (A) DNA methylation-dependent dynamics of c-Myc mRNA level in response to TGF-β1. SK-Hep1 and PLC/PRF/5 cells were treated with TGF-β1 (5 μg/L) for the indicated time period with or without AzadC (2 μmol/L) pretreatment. Quantitative real-time RT-PCR results for c-Myc mRNA levels are presented as fold changes compared with those of untreated controls (right panel). (B) Differential responses of SK-Hep1 and PRC/PRF/5 cells to TGF-β1. Cells were treated with TGF-β1 for 3 hours (TTP and c-Myc) or 8 hours (p21Cip1) with/without AzadC pretreatment and analyzed for protein expression by Western blotting. Cell growth rates were measured by MTT assays at 96 hours after TGF-β1 treatment with/without AzadC. Images of cells stained with crystal violet are shown. (C) Effects of TTP overexpression on c-Myc and p21Cip1 protein expression and cell growth. TTP overexpression vector (pTTPOE) was transiently transfected into SK-Hep1 cells. Cell growth rates were measured by MTT assays at 96 hours after TGF-β1 treatment. (D) Effects of TTP knock-down on the dynamics of c-Myc mRNA level in response to TGF-β1. PLC/PRF/5 cells stably transfected with a lentiviral vector encoding short hairpin RNA for TTP were treated with TGF-β1 or actinomycin D (2 μg/L), a transcription inhibitor, for the indicated time and quantitatively analyzed for c-Myc mRNA content by real-time RT-PCR. (E) Effects of TTP knock-down on c-Myc and p21Cip1 protein expression and cell growth in response to TGF-β1. Growth rates of PLC/PRF/5 cells were measured by MTT assays at 96 hours after TGF-β1 treatment. (F) Relationship between the −500C methylation quantity and c-Myc mRNA level in HCC patient samples. c-Myc mRNA levels were quantitatively determined by real-time RT-PCR, and its relationship with the −500C methylation level was plotted against the −500C methylation data in Figure 3B. Correlation data between the levels of c-Myc mRNA and −500C methylation are shown for tumors exhibiting ≥30% of methylation rates at the −500C (right panel).
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8. Supplementary Figure 1
Representative bisulfite sequencing data are shown. After the bisulfite treatment, all cytosine residues were converted to thymine. The cytosine residue of the CpG site located at −500 bp from transcription start site (the CpG #2) is designated as “−500C.”
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9. Supplementary Figure 2
Representative pyrosequencing data for quantitation of methylated CpGs in the upstream TTP locus in HCC patient tissue samples. (A) Pyrosequencing of the −500C. The fifth “C” peak (arrowhead) represents the cytosine level detected at the −500C. The methylation percentage was calculated by comparing the magnitude of the “C” peak with that of the next “T” peak (gray box) subsequently read at the same nucleotide. (B and C) Pyrosequencing results for other CpG sites in the TTP promoter are presented for comparison. (B) Methylation percentage of the CpG sites at positions −113 through −88 bp from transcription start site (CpG sites #23–29). The percentage of the first “C” in each chart represents the experimental control for unconverted cytosines after bisulfite treatment. Each CpG site is denoted with a gray box. (C) Methylation percentage of the CpG sites at positions −75 through −45 bp from transcription start site (CpG sites #30–35). Sequences were read in the reverse order (ie, “CT” as “GA,” gray boxes). Accordingly, the percentage of the first “G” in each chart represents the experimental control.
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10. Supplementary Figure 3
Representative quantitative RT-PCR (for TTP expression) and pyrosequencing (for quantitation of the −500C methylation in the TTP promoter) data from HCC patient tissue samples. The methylation percentage is indicated under the corresponding RT-PCR band.
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11. Supplementary Figure 4
Induction of TTP and TGF-β1 pathway marker gene expression in HCC cell lines whose TTP expression was epigenetically repressed. (A) TTP induction patterns in response to TGF-β1 with or without AzadC pretreatment. Cells were treated with TGF-β1 (5 μg/L) for 1 hour with or without AzadC pretreatment (2 μmol/L, 72 hours) and then analyzed for TTP mRNA expression levels by semiquantitative RT-PCR. Note that the basal-level TTP expression was restored by AzadC in all the HCC cell lines examined, but the TGF-β1-induced expression in the presence of AzadC was only observed in SK-Hep1, Hep3B, and SNU182 cells (“responder group”) but not in SNU475, SNU354, and SNU368 cells (“nonresponder group”). (B) TGF-β1-induced expression of TGF-β1 signaling pathway marker genes in HCC cell lines. The expression patterns of PAI-1 and SMAD7, 2 representative marker genes of TGF-β1 signaling pathway, were determined from the total RNA of the HCC cell lines by semiquantitative RT-PCR analyses. Note that both markers were clearly induced by TGF-β1 in the responder group (SK-Hep1, Hep3B, and SNU182 cells) even in the absence of AzadC pretreatment but only weakly in the nonresponder group (SNU475, SNU354, and SNU368 cells). These results suggest that the TGF-β1 signaling pathway is functionally intact in SK-Hep1, Hep3B, and SNU182 cells, although their TGF-β1-dependent TTP expression has been selectively repressed by DNA methylation.
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12. Supplementary Figure 5
Site-specific de novo DNA methylation at the −500C of exogenously provided TTP promoter in the nucleus of transfected cells. A plasmid containing the TTP promoter sequence used in the luciferase assay (Figure 4B) was transiently transfected into SK-Hep1 cells and examined for DNA methylation status by bisulfite sequencing. To discriminate the exogenously provided TTP promoter sequence from the endogenous copy, we used differential primer sequences for the PCR amplification of bisulfite-treated DNA, ie, one located in the pGL3 luciferase vector as the 5′-primer (5′-GTAAAATAGGTTGTTTTTAGTG-3′) and the other located in the TTP promoter as the 3′-primer (5′-AACAATCAAATCCATAATATAAC-3′). In line with the observations in HCC cell lines and tumor samples, de novo DNA methylation occurred exclusively at the −500C, whereas the other CpG sites of TTP promoter remained unmethylated. The −500C methylation rates were much higher in the DNAs extracted from the nucleus than from whole cells; the −500C methylation rates in the nuclear DNA from 3 independent experiments were 45.4%, 33.3%, and 41.6%, respectively.
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13. Supplementary Figure 6
Involvement of DNA methylation-dependent histone deacetylation in the epigenetic repression of TTP locus. Four portions of SK-Hep1 cells were cultured with or without AzadC (2 μmol/L) for 3 days, whereas 2 portions of them were additionally treated with trichostatin A (histone deacetylase inhibitor; 1 μmol/L) during the last 24 hours of culture. The cells were then treated with TGF-β1 (5 μg/L) for 1 hour, and their TTP mRNA content was analyzed by quantitative real-time RT-PCR (n = 5). Note that TSA treatment, by itself, did not increase the TTP mRNA level but augmented the AzadC-mediated TTP induction. The results suggest that the epigenetic inactivation of TTP involves histone deacetylation, which is dependent on the MECP2/HDAC complex recruited to the site by DNA methylation.
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14. Supplementary Figure 7
Direct binding of TTP to the 3′-UTR of c-Myc mRNA. (A) Genomic structure of the c-Myc locus and the nucleotide sequence of 3′-UTR. The sequences corresponding to the 32P-labeled RNA probe used in RNA-protein ultraviolet (UV) cross-linking assay are highlighted as bold characters, and AU-rich elements (AREs), the putative TTP binding sites, are underlined. #1, #2, and #3 Denote the PCR-amplified regions in ribonucleoprotein (RNP) immunoprecipitation (RIP) assay. Empty boxes correspond to exons. (B) Analysis of TTP binding to c-Myc mRNA by RIP assay. HEK293T cells were transfected with a Myc-tagged TTP overexpression vector, and, 48 hours after the transfection, cytoplasmic mRNA-protein complexes were immunoprecipitated with an antibody against Myc epitope. The presence of c-Myc transcript in the immunoprecipitated mRNAs was determined by a quantitative real-time RT-PCR using the primers for the regions #1 (+79 to +284 bp from transcription start site), #2 (+927 to bp), and #3 (+1814 to bp). Cytoplasmic extracts (1/200) were used as the input control and immunoglobulin G (IgG) as the negative control. (C) Analysis of TTP binding to c-Myc mRNA by RNA-protein ultraviolet (UV) cross-linking assays. The cytoplasmic extracts of HEK293T cells described in B were incubated with 32P-labeled RNA probe containing the 3′-UTR of c-Myc mRNA, and the reaction mixtures were UV-irradiated to induce RNA-protein cross-linking. After digestion of the mixtures with RNase T1, the RNase-resistant RNA-protein complexes were analyzed by SDS-PAGE (8%) followed by autoradiography. Multiple TTP-RNA probe complexes with unidentified proteins, whose radioactive signals were specifically competed away by the cold self RNA probe, were detected in TTP overexpressed HEK293T cell extracts (left panel). As a positive experimental control, we performed similar RNA-protein UV cross-linking assays using the 3′-UTR of TNF-α mRNA (right panel). N.S., Cold non-self RNA probe derived from the ORF region of c-Myc or TNF-α; Self, Cold self RNA probe derived from the 3′-UTR of c-Myc or TNF-α; ×20 and ×100, 20-, and 100-fold quantity in excess of the 32P-labeled probe.
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15. Supplementary Figure 8
Transcription initiation pattern of c-Myc in response to TGF-β1. PLC/PRF/5 cells stably transfected with a lentiviral shTTP vector were treated with TGF-β1 (5 μg/L) for indicated times. The transcription initiation rates at the c-Myc locus were measured by chromatin immunoprecipitation (ChIP) assays using antibodies against phospho-S5 RNA polymerase II (S5; phosphorylated at serine 5 of carboxy terminal domain). Phospho-S5 RNA polymerase II binding to the c-Myc promoter was gradually decreased after the TGF-β1 treatment, but its gross pattern was relatively similar in TTP knock-down (shTTP) and control (shGFP) cells. The PCR region used in the ChIP analysis of c-Myc transcription initiation was the regions #1 (+79 to +284 bp from transcription start site) as shown Supplementary Figure 7A.
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16. Supplementary Figure 9
Fluorescent-activated cell sorter (FACS)-based cell cycle pattern analysis of PLC/PRF/5 cells expressing shRNA for TTP or green fluorescent protein (GFP) control. Stably transfected cell lines were treated with TGF-β1 (5 μg/L) for 72 hours and then subjected to FACS analysis. Subdivided areas representing the size of the cell population in different phases of cell cycle were quantified and graphically depicted on the right.
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