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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: Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner.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

CTCF regulates transcriptional activation of the XAF1 gene.(a) ACHN cells were pre-treated with 5-aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days. After that, the cells were transiently transfected with CTCF siRNAs or control scramble siRNAs. qPCR analyses were performed to measure the expression of both XAF1 and CTCF mRNA. HPRT was used as loading control. The means from three independent experiments were plotted with +SEM, *P < 0.05. (b) MCF-7 cells were transitorily co-transfected with both Wild-type-XAF1-promoter-SEAP or Δ-CTCF-XAF1-promoter-SEAP constructs and pMetLuc, which was used for transfection normalization. Data are represented as the means + SEM from three independent experiments, *P < 0.05. (c) MCF-7 stable clones of CTCF/Tet-On were stimulated with tetracycline at the indicated concentrations. Using qPCR assays, XAF1 and CTCF mRNA expression was normalized to HPRT, used as a loading control. The mean and range were plotted from two independent stable cell lines. (d) MCF-7-CTCF/Tet-On and MCF-7 Empty/Tet-On cell lines were transitory co-transfected with Wild-type-XAF1-promoter-SEAP and pMetLuc. After 48 h, tetracycline was added for 24 h. Data are represented as the means + SEM from three independent experiments, *P < 0.05. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA); CTCF-binding site (CBS).
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f4: CTCF regulates transcriptional activation of the XAF1 gene.(a) ACHN cells were pre-treated with 5-aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days. After that, the cells were transiently transfected with CTCF siRNAs or control scramble siRNAs. qPCR analyses were performed to measure the expression of both XAF1 and CTCF mRNA. HPRT was used as loading control. The means from three independent experiments were plotted with +SEM, *P < 0.05. (b) MCF-7 cells were transitorily co-transfected with both Wild-type-XAF1-promoter-SEAP or Δ-CTCF-XAF1-promoter-SEAP constructs and pMetLuc, which was used for transfection normalization. Data are represented as the means + SEM from three independent experiments, *P < 0.05. (c) MCF-7 stable clones of CTCF/Tet-On were stimulated with tetracycline at the indicated concentrations. Using qPCR assays, XAF1 and CTCF mRNA expression was normalized to HPRT, used as a loading control. The mean and range were plotted from two independent stable cell lines. (d) MCF-7-CTCF/Tet-On and MCF-7 Empty/Tet-On cell lines were transitory co-transfected with Wild-type-XAF1-promoter-SEAP and pMetLuc. After 48 h, tetracycline was added for 24 h. Data are represented as the means + SEM from three independent experiments, *P < 0.05. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA); CTCF-binding site (CBS).

Mentions: To further define the role of CTCF on XAF1 mRNA expression, we used specific siRNAs to downregulate CTCF expression in a series of loss-of-function experiments. We verified the efficacy of these siRNAs at both mRNA and protein levels (Supplementary Fig. 1d). Because previous reports have shown that demethylating agents increase XAF1 induction by IFN in ACHN cells32, we used this cell line to analyse the effect of these siRNAs on XAF1 transcriptional responsiveness to TNF-α or IFN-α. As described above, demethylating conditions are necessary to uncover the CTCF-binding site (Fig. 2b,c). We clearly observed lower levels of XAF1 mRNA in cells transfected with the siRNAs against CTCF than those transfected with control siRNAs (Fig. 4a). Additionally, we confirmed the regulatory effect of CTCF on the XAF1 promoter using the secreted alkaline phosphatase (SEAP) reporter gene assays. In these assays, the enzymatic activity drove by the XAF1 promoter region comprising −3000 bp to +350 bp relative to the transcription start site (Wild-type-XAF1-promoter-SEAP) was compared with the same region with a deletion of the core CTCF binding site (Δ-CTCF-XAF1-promoter-SEAP). The absence of the CTCF binding site in the XAF1 promoter inhibits its basal transcriptional activation (Fig. 4b). To further support these results, we also conducted gain-of-function experiments by analysing the effects of CTCF overexpression on XAF1 mRNA expression. To achieve this, we engineered a Tet-on CTCF system in the MCF-7 cell line. In demethylating conditions, the overexpression of CTCF mediated by tetracycline addition induced transcriptional activation of XAF1 (Fig. 4c). Additionally, these cells were transfected with the Wild-type-XAF1-promoter-SEAP construct. After tetracycline addition, we observed a significant increase in the enzymatic activity of the reporter in cells overexpressing CTCF (Fig. 4d). On the other hand, we evaluated the role of CTCF over-expression in terms of transcriptional responsiveness of XAF1 in TNF-α- or IFN-α–treated cells. Although we detected an increase in XAF1 levels in cells over-expressing CTCF, the TNF-α or IFN-α-mediated transcriptional increase was not modified by CTCF overexpression (supplementary Fig. 2a). This points toward a shared signalling mechanism and supports the role of CTCF in the effects of these cytokines on XAF1 regulation, with additional factors needed for maximal responsiveness. Thus, both gain and loss of function approaches showed the participation of CTCF in XAF1 expression.


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)

CTCF regulates transcriptional activation of the XAF1 gene.(a) ACHN cells were pre-treated with 5-aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days. After that, the cells were transiently transfected with CTCF siRNAs or control scramble siRNAs. qPCR analyses were performed to measure the expression of both XAF1 and CTCF mRNA. HPRT was used as loading control. The means from three independent experiments were plotted with +SEM, *P < 0.05. (b) MCF-7 cells were transitorily co-transfected with both Wild-type-XAF1-promoter-SEAP or Δ-CTCF-XAF1-promoter-SEAP constructs and pMetLuc, which was used for transfection normalization. Data are represented as the means + SEM from three independent experiments, *P < 0.05. (c) MCF-7 stable clones of CTCF/Tet-On were stimulated with tetracycline at the indicated concentrations. Using qPCR assays, XAF1 and CTCF mRNA expression was normalized to HPRT, used as a loading control. The mean and range were plotted from two independent stable cell lines. (d) MCF-7-CTCF/Tet-On and MCF-7 Empty/Tet-On cell lines were transitory co-transfected with Wild-type-XAF1-promoter-SEAP and pMetLuc. After 48 h, tetracycline was added for 24 h. Data are represented as the means + SEM from three independent experiments, *P < 0.05. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA); CTCF-binding site (CBS).
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f4: CTCF regulates transcriptional activation of the XAF1 gene.(a) ACHN cells were pre-treated with 5-aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days. After that, the cells were transiently transfected with CTCF siRNAs or control scramble siRNAs. qPCR analyses were performed to measure the expression of both XAF1 and CTCF mRNA. HPRT was used as loading control. The means from three independent experiments were plotted with +SEM, *P < 0.05. (b) MCF-7 cells were transitorily co-transfected with both Wild-type-XAF1-promoter-SEAP or Δ-CTCF-XAF1-promoter-SEAP constructs and pMetLuc, which was used for transfection normalization. Data are represented as the means + SEM from three independent experiments, *P < 0.05. (c) MCF-7 stable clones of CTCF/Tet-On were stimulated with tetracycline at the indicated concentrations. Using qPCR assays, XAF1 and CTCF mRNA expression was normalized to HPRT, used as a loading control. The mean and range were plotted from two independent stable cell lines. (d) MCF-7-CTCF/Tet-On and MCF-7 Empty/Tet-On cell lines were transitory co-transfected with Wild-type-XAF1-promoter-SEAP and pMetLuc. After 48 h, tetracycline was added for 24 h. Data are represented as the means + SEM from three independent experiments, *P < 0.05. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA); CTCF-binding site (CBS).
Mentions: To further define the role of CTCF on XAF1 mRNA expression, we used specific siRNAs to downregulate CTCF expression in a series of loss-of-function experiments. We verified the efficacy of these siRNAs at both mRNA and protein levels (Supplementary Fig. 1d). Because previous reports have shown that demethylating agents increase XAF1 induction by IFN in ACHN cells32, we used this cell line to analyse the effect of these siRNAs on XAF1 transcriptional responsiveness to TNF-α or IFN-α. As described above, demethylating conditions are necessary to uncover the CTCF-binding site (Fig. 2b,c). We clearly observed lower levels of XAF1 mRNA in cells transfected with the siRNAs against CTCF than those transfected with control siRNAs (Fig. 4a). Additionally, we confirmed the regulatory effect of CTCF on the XAF1 promoter using the secreted alkaline phosphatase (SEAP) reporter gene assays. In these assays, the enzymatic activity drove by the XAF1 promoter region comprising −3000 bp to +350 bp relative to the transcription start site (Wild-type-XAF1-promoter-SEAP) was compared with the same region with a deletion of the core CTCF binding site (Δ-CTCF-XAF1-promoter-SEAP). The absence of the CTCF binding site in the XAF1 promoter inhibits its basal transcriptional activation (Fig. 4b). To further support these results, we also conducted gain-of-function experiments by analysing the effects of CTCF overexpression on XAF1 mRNA expression. To achieve this, we engineered a Tet-on CTCF system in the MCF-7 cell line. In demethylating conditions, the overexpression of CTCF mediated by tetracycline addition induced transcriptional activation of XAF1 (Fig. 4c). Additionally, these cells were transfected with the Wild-type-XAF1-promoter-SEAP construct. After tetracycline addition, we observed a significant increase in the enzymatic activity of the reporter in cells overexpressing CTCF (Fig. 4d). On the other hand, we evaluated the role of CTCF over-expression in terms of transcriptional responsiveness of XAF1 in TNF-α- or IFN-α–treated cells. Although we detected an increase in XAF1 levels in cells over-expressing CTCF, the TNF-α or IFN-α-mediated transcriptional increase was not modified by CTCF overexpression (supplementary Fig. 2a). This points toward a shared signalling mechanism and supports the role of CTCF in the effects of these cytokines on XAF1 regulation, with additional factors needed for maximal responsiveness. Thus, both gain and loss of function approaches showed the participation of CTCF in XAF1 expression.

Bottom Line: Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner.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