1. Volume 46, Issue 5, Pages 636-649 (June 2012)
TGF-β-Dependent Active Demethylation and Expression of the p15ink4b Tumor Suppressor Are Impaired by the ZNF217/CoREST Complex 
Gobi Thillainadesan, Jennifer Mary Chitilian, Majdina Isovic, Jailal Nicholas George Ablack, Joe Stephen Mymryk, Marc Tini, Joseph Torchia 
Molecular Cell 
Volume 46, Issue 5, Pages (June 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
Transcriptional Repression of the p15ink4b Gene by the ZNF217/CoREST Complex
(A) ChIP-seq tracings for CtBP1 and ZNF217 at the ink 4 locus. The square indicates enrichment of ZNF217 and CtBP1 to the same region of the p15ink4b (CDKN2B) promoter. Genes have been correlated to fit the UCSC annotations.
(B) ZNF217 is required for targeting the CoREST complex. MCF7 cells were transfected with the indicated siRNA, and ChIP-qPCR analysis of the p15ink4b promoter was performed using the indicated antibodies. Error bars indicate standard error of the mean.
(C) Western blotting analysis of specific proteins (as indicated on the left) from MCF7 cells following transfection with siRNAs for ZNF217 or CtBP1.
(D) Knockdown of ZNF217 causes an increase in the percentage of cells found in G1. Cells were transfected with the indicated siRNAs and treated with nocodazole for 24 hr. The percentage of cells in G1 was then determined by flow cytometry. Shown is a representative experiment from three independent experiments as depicted in Figure S3. Error bars indicate standard deviation (SD).
(E) Knockdown of ZNF217 causes a decrease in phosphorylated Rb. MCF7 cells were transfected with the indicated siRNAs. Endogenous Rb status was analyzed by western blotting using the indicated antibodies as shown on the left. The amount of phosphorylated Rb (pRb) was quantified using densitometry and normalized to total Rb and tubulin.
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3. Figure 2
Knockdown of ZNF217 or DNMT3A Causes DNA Demethylation of the p15ink4b Promoter
(A) Knockdown of DNMT3A using siRNA. MCF7 cells were transfected with the indicated siRNA, and after 72 hr cell extracts were prepared and western blot analysis was performed with the antibodies as indicated on the left.
(B) DNA methylation analysis of the p15ink4b promoter. MCF7 cells were transfected with the indicated siRNAs, DNA was extracted, and the core CpG region of the p15ink4b promoter was analyzed by sodium bisulphite sequencing. The human p15ink4b promoter with the CpGs indicated in roman numerals is shown. White and black circles indicate unmethylated and methylated cytosines, respectively.
(C) Methylation-specific PCR of the p15ink4b promoter. Samples were treated as described in (A) and then analyzed by PCR using specific oligonucleotides recognizing the methylated sequence contained within the core CpG island. Error bars indicate standard error of the mean.
(D) ZNF217 and DNMT3A co-occupy the p15ink4b promoter in MCF7 cells. ChIP or sequential ChIP (ChIP-ReChIP) was performed with the indicated antibodies. The IgG control values for ChIP were subtracted from values obtained using specific antibodies. Error bars indicate standard error of the mean.
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4. Figure 3 TGF-β-Dependent Demethylation of the p15ink4b Promoter
(A) TGF-β stimulates DNA demethylation of the p15ink4b promoter. HaCAT cells were treated with TGF-β for the indicated time periods, and the p15ink4b promoter was analyzed by sodium bisulphite sequencing. White and black circles indicate unmethylated and methylated CpGs, respectively. The human p15ink4b promoter with the CpGs indicated in roman numerals is shown.
(B) PCR analysis of the p15ink4b promoter. Samples were treated as described in (A) and then analyzed by PCR using specific oligonucleotides recognizing the unmethylated sequence within the core CpG island as indicated by the arrows in (A).
(C) TGF-β stimulates a coregulator switch at the p15ink4b promoter. HaCAT cells were treated with 150 pM TGF-β for 20 or 90 min, and ChIP-qPCR was performed with the indicated antibodies. IgG control values for ChIP were subtracted from values obtained using specific antibodies. Error bars indicate standard error of the mean.
(D) TDG associates with both CBP and SMAD 2/3 in response to TGF-β. HaCAT cells were treated as in (C), and ChIP was performed using a single antibody or the combination of antibodies (ChiP-ReChIP) indicated on the left.
(E) Coimmunoprecipitation of endogenous SMAD 2/3 and TDG. HaCAT cells were treated with TGF-β for 90 min, and whole-cell extracts were prepared. SMAD 2/3 was then immunoprecipitated and western blotting performed using the TDG antibody.
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5. Figure 4 TDG Is Required for TGF-β-Dependent DNA Demethylation
(A) Knockdown of TDG using siRNA. HaCAT cells were transfected with the indicated siRNA and after 72 hr cell extracts were prepared and western blot analysis performed.
(B) TDG knockdown inhibits TGF-β-dependent DNA demethylation. HaCAT cells were transfected with the indicated siRNAs and then treated with TGF-β for 90 min. The p15ink4b promoter was then analyzed by sodium bisulphite sequencing. White and black circles indicate unmethylated and methylated cytosines, respectively.
(C) Methylation-specific PCR of the p15ink4b promoter. HaCAT cells were treated as described in (A) and analyzed by methylation-specific PCR. Error bars indicate standard error of the mean.
(D) Knockdown of TDG inhibits p15ink4b expression. HaCAT cells were transfected with the indicated siRNA and then stimulated with TGF-β for 6 hr. RNA was then extracted and analyzed by qPCR.
(E) Dot blot analysis of genomic 5mC. HaCAT cells were treated with TGF-β for 90 min. Bulk genomic DNA was isolated, sonicated, crosslinked to nitrocellulose membrane, and probed with a monoclonal antibody specific for 5mC. Recognition of DNA by the anti-5mC antibody is shown in the top panel, and loading control is shown by the methylene blue stain in the bottom panel. The blot was quantitated by densitometry, and a representative experiment is shown from two independent experiments.
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6. Figure 5
TGF-β-Dependent Demethylation of the p15ink4b Promoter Involves Conversion of 5mC to 5hmC
(A) Schematic diagram of the p15 promoter with the CpG islands indicated as a closed ball and stick.
(B) DNA immunoprecipitation analysis of the p15ink4b promoter. HaCAT cells were transfected with the indicated siRNAs and then treated with TGF-β or vehicle for 90 min. DNA was isolated and DNA immunoprecipitation assays were performed using 5mC- or 5hmC-specific antibodies. Aliquots from the same DNA samples were used to perform MeDIP and hMeDIP. The data are expressed as the ratio of the percentage of input DNA from the TGF-β-treated and vehicle-treated cells from three independent experiments.
(C) AID is recruited to the p15ink4b promoter. HaCAT cells were treated with 150 pM TGF-β for 20 or 90 min, and ChIP-ReChIP assays followed by qPCR were performed with the indicated antibodies. Error bars indicate standard error of the mean.
(D) Components of the BER machinery are recruited to the p15ink4b promoter. Experiments were performed as described in (B), and ChIP assays were performed with the indicated antibodies. Shown are the ChIP assays from samples treated with TGF-β for 90 min. No significant change in recruitment was observed 20 min following TGF-β treatment (data not shown). Error bars indicate standard error of the mean.
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7. Figure 6 MBD4 Is Required for TGF-β-Dependent DNA Demethylation
(A) Knockdown of MBD4 using siRNA. HaCAT cells were transfected with the indicated siRNA, and after 72 hr cell extracts were prepared and western blot analysis performed.
(B) Knockdown of MBD4 inhibits TGF-β-dependent DNA demethylation. HaCAT cells were transfected with the indicated siRNAs and then stimulated with TGF-β for 90 min. The p15ink4b promoter was analyzed by sodium bisulphite sequencing. White and black circles indicate unmethylated and methylated cytosines, respectively.
(C) Methylation-specific PCR of the p15ink4b promoter. HaCAT cells were treated as described in (A) and analyzed by methylation-specific PCR. Error bars indicate standard error of the mean.
(D) MBD4 and TDG do not co-occupy the p15ink4b promoter. HaCAT cells were treated with 150 pM TGF-β for 90 min, and then ChIP or ChIP-ReChIP assays were performed with the indicated antibodies. Error bars indicate standard error of the mean.
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8. Figure 7
Overexpression of the ZNF217 Oncogene Inhibits TGF-β-Dependent DNA Demethylation of the p15ink4b Promoter
(A) Overexpression of HA-tagged ZNF217 in HaCAT cells. HaCAT cells were infected with the indicated adenoviruses (GFP or ZNF217). After 24 hr, extracts were prepared, and western blotting was performed with the indicated antibodies.
(B) Overexpression of ZNF217 inhibits p15ink4b protein expression. (Left) HaCAT cells were infected with the indicated adenoviruses, and after 24 hr cells were stimulated with TGF-β and western blotting performed using the indicated antibodies shown on the left. (Right) The amount of p15ink4b protein expression was quantified using densitometry and normalized to tubulin.
(C) Overexpression of ZNF217 blocks TGF-β-dependent demethylation. Cells were infected with the GFP or ZNF217 adenoviruses, then stimulated with TGF-β as described for 90 min, and the p15ink4b promoter was analyzed by methylation-specific PCR. Error bars indicate standard error of the mean.
(D) Overexpression of ZNF217 inhibits recruitment of SMAD2/3 and TDG to the the p15ink4b promoter. HaCAT cells were infected with the GFP or ZNF217 adenoviruses and then stimulated with TGF-β for 90 min. ChIP-qPCR was performed using antibodies recognizing SMAD 2/3 or TDG. Error bars indicate standard error of the mean.
(E) Model depicting the mechanism of TGF-β-dependent demethylation of the p15ink4b promoter. In normal proliferating epithelial cells, the ZNF217/CoREST/DNMT3A complex is bound to the p15ink4b promoter along with c-myc, and the promoter is hypermethylated (filled circles). Stimulation with TGF-β causes release of the ZNF217/CoREST/DNMT3A complex and c-myc and a concomitant binding of activating transcription factors in association with CBP/p300, TDG, and AID. 5mC is then oxidized to 5hmC and may undergo deamination by AID/APOBEC proteins to generate 5 hydroxyuracil (5hU), which is then processed by TDG and repaired by the BER enzymes (APE1, DNA polymerase β, and DNA ligase), which reintroduce an unmethylated cytosine (open circles). Alternatively, TGF-β may stimulate recruitment of MBD4, which triggers active demethylation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions