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Epigenetic silencing of the XAF1 gene is mediated by the loss of CTCF binding.

Victoria-Acosta G, Vazquez-Santillan K, Jimenez-Hernandez L, Muñoz-Galindo L, Maldonado V, Martinez-Ruiz GU, Melendez-Zajgla J - Sci Rep (2015)

Bottom Line: By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications.In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators.We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

View Article: PubMed Central - PubMed

Affiliation: Functional Cancer Genomics Laboratory, National Institute of Genomic Medicine, Mexico D.F., 14610, Mexico.

ABSTRACT
XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

No MeSH data available.


Related in: MedlinePlus

The CTCF binding site in the XAF1 promoter mediates XAF1 responsiveness to activators in apoptotic conditions.(a) MCF-7 cells were treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with TNF-α in the presence of cycloheximide (TNF-α + CHX) (left panel). qPCR analyses of XAF1 and CTCF mRNA expression were performed. HPRT mRNA was used as loading control. Results are presented as fold change. Data are represented as the means ± SEM from three independent experiments, *P < 0.05. (b) MCF-7 cells were pre-treated as shown in (A) before the addition of IFN-α in the presence of TRAIL (IFN-α + TRAIL) (right panel). The expression of XAF1 and CTCF and HPRT was determined by qPCR. HPRT was used as loading control. (c) Stable single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs were stimulated with either TNF-α + CHX or IFN-α + TRAIL. After, GFP protein levels were measured using FACS. Data are represented as the mean SD of four single-cell clones from each transfection, *P < 0.05.
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f6: The CTCF binding site in the XAF1 promoter mediates XAF1 responsiveness to activators in apoptotic conditions.(a) MCF-7 cells were treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with TNF-α in the presence of cycloheximide (TNF-α + CHX) (left panel). qPCR analyses of XAF1 and CTCF mRNA expression were performed. HPRT mRNA was used as loading control. Results are presented as fold change. Data are represented as the means ± SEM from three independent experiments, *P < 0.05. (b) MCF-7 cells were pre-treated as shown in (A) before the addition of IFN-α in the presence of TRAIL (IFN-α + TRAIL) (right panel). The expression of XAF1 and CTCF and HPRT was determined by qPCR. HPRT was used as loading control. (c) Stable single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs were stimulated with either TNF-α + CHX or IFN-α + TRAIL. After, GFP protein levels were measured using FACS. Data are represented as the mean SD of four single-cell clones from each transfection, *P < 0.05.

Mentions: It has been well described that XAF1 expression reactivation has a crucial role in apoptosis induced by TNF-α/cycloheximide (CHX) or IFN-α/TNF-related apoptosis-inducing ligand (TRAIL)622. To test if CTCF could regulate XAF1 expression in apoptotic conditions, MCF-7 cells were exposed to either TNF-α/CHX or IFN-α/TRAIL. Cytotoxicity induced by the co-treatment of either TNF-α/CHX or IFN-α/TRAIL was analysed by cell viability assays (Supplementary Fig. 2d). As expected, we observed the transcriptional activation of XAF1 after exposure to both regimens (Fig. 6a,b). To assess the biological relevance of CTCF-mediated XAF1 transcription, single-cell clones with the wild type- or Δ-CTCF-XAF1 promoter were exposed to inducers of apoptosis. After that, FACS was used to measure GFP-reporter gene activity. Whereas the wild-type promoter activity correlated with the XAF1 transcriptional activation, the Δ-CTCF-XAF1 promoter did not present any transcriptional activity (Fig. 6c). Several reports have shown that XAF1 is an IFN-stimulated gene in cancer cells303940. Because its promoter is commonly hypermethylated in these cells, transcriptional activation of the XAF1 gene could be dependent on IFN-α-mediated demethylation and could thus rely on CTCF20. Supporting this hypothesis, we found that single-cell clones with the Δ-CTCF-XAF1 promoter were unable to respond to IFN-α, indicating that CTCF could be relevant in the IFN-α-mediated induction of XAF1 (Fig. 6c).


Epigenetic silencing of the XAF1 gene is mediated by the loss of CTCF binding.

Victoria-Acosta G, Vazquez-Santillan K, Jimenez-Hernandez L, Muñoz-Galindo L, Maldonado V, Martinez-Ruiz GU, Melendez-Zajgla J - Sci Rep (2015)

The CTCF binding site in the XAF1 promoter mediates XAF1 responsiveness to activators in apoptotic conditions.(a) MCF-7 cells were treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with TNF-α in the presence of cycloheximide (TNF-α + CHX) (left panel). qPCR analyses of XAF1 and CTCF mRNA expression were performed. HPRT mRNA was used as loading control. Results are presented as fold change. Data are represented as the means ± SEM from three independent experiments, *P < 0.05. (b) MCF-7 cells were pre-treated as shown in (A) before the addition of IFN-α in the presence of TRAIL (IFN-α + TRAIL) (right panel). The expression of XAF1 and CTCF and HPRT was determined by qPCR. HPRT was used as loading control. (c) Stable single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs were stimulated with either TNF-α + CHX or IFN-α + TRAIL. After, GFP protein levels were measured using FACS. Data are represented as the mean SD of four single-cell clones from each transfection, *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595840&req=5

f6: The CTCF binding site in the XAF1 promoter mediates XAF1 responsiveness to activators in apoptotic conditions.(a) MCF-7 cells were treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with TNF-α in the presence of cycloheximide (TNF-α + CHX) (left panel). qPCR analyses of XAF1 and CTCF mRNA expression were performed. HPRT mRNA was used as loading control. Results are presented as fold change. Data are represented as the means ± SEM from three independent experiments, *P < 0.05. (b) MCF-7 cells were pre-treated as shown in (A) before the addition of IFN-α in the presence of TRAIL (IFN-α + TRAIL) (right panel). The expression of XAF1 and CTCF and HPRT was determined by qPCR. HPRT was used as loading control. (c) Stable single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs were stimulated with either TNF-α + CHX or IFN-α + TRAIL. After, GFP protein levels were measured using FACS. Data are represented as the mean SD of four single-cell clones from each transfection, *P < 0.05.
Mentions: It has been well described that XAF1 expression reactivation has a crucial role in apoptosis induced by TNF-α/cycloheximide (CHX) or IFN-α/TNF-related apoptosis-inducing ligand (TRAIL)622. To test if CTCF could regulate XAF1 expression in apoptotic conditions, MCF-7 cells were exposed to either TNF-α/CHX or IFN-α/TRAIL. Cytotoxicity induced by the co-treatment of either TNF-α/CHX or IFN-α/TRAIL was analysed by cell viability assays (Supplementary Fig. 2d). As expected, we observed the transcriptional activation of XAF1 after exposure to both regimens (Fig. 6a,b). To assess the biological relevance of CTCF-mediated XAF1 transcription, single-cell clones with the wild type- or Δ-CTCF-XAF1 promoter were exposed to inducers of apoptosis. After that, FACS was used to measure GFP-reporter gene activity. Whereas the wild-type promoter activity correlated with the XAF1 transcriptional activation, the Δ-CTCF-XAF1 promoter did not present any transcriptional activity (Fig. 6c). Several reports have shown that XAF1 is an IFN-stimulated gene in cancer cells303940. Because its promoter is commonly hypermethylated in these cells, transcriptional activation of the XAF1 gene could be dependent on IFN-α-mediated demethylation and could thus rely on CTCF20. Supporting this hypothesis, we found that single-cell clones with the Δ-CTCF-XAF1 promoter were unable to respond to IFN-α, indicating that CTCF could be relevant in the IFN-α-mediated induction of XAF1 (Fig. 6c).

Bottom Line: By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications.In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators.We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

View Article: PubMed Central - PubMed

Affiliation: Functional Cancer Genomics Laboratory, National Institute of Genomic Medicine, Mexico D.F., 14610, Mexico.

ABSTRACT
XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.

No MeSH data available.


Related in: MedlinePlus