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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 interacts with the XAF1 promoter.(a) ACHN cells were pre-treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with either TNF-α or IFN-α. Non-demethylated cells were stimulated with either TNF-α or IFN-α. mRNA expression of XAF1, CTCF and HPRT was analysed by qPCR. Results are presented in terms of fold change. The means from three independent experiments were plotted with ±SEM, *P < 0.05. (b) MCF-7 cells were either treated or not treated with demethylating agents, as shown in (a). Bisulphite sequencing was then performed. A schematic representation of the XAF1 promoter shows the locations of 11 CpG-dinucleotides sites from −22 to −500 bp relative to the TSS. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively (c) MCF-7 cells were treated as shown in (a). ChIP assays were performed using a specific antibody against CTCF protein. The CTCF-binding site in the XAF1 promoter was analysed by PCR in the DNA recovered after ChIP (Left panel). As positive and negative controls of CTCF-DNA interaction, three specific sets of primers were included. Two of them were directed to previously validated CTCF binding sites (c-Myc and IGF2) as positive controls, and one was a negative control. The input represents soluble chromatin that was reversed cross-linked and amplified by PCR (central panel). RT-PCR was performed from cells used for ChIP assays. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA).
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f2: CTCF interacts with the XAF1 promoter.(a) ACHN cells were pre-treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with either TNF-α or IFN-α. Non-demethylated cells were stimulated with either TNF-α or IFN-α. mRNA expression of XAF1, CTCF and HPRT was analysed by qPCR. Results are presented in terms of fold change. The means from three independent experiments were plotted with ±SEM, *P < 0.05. (b) MCF-7 cells were either treated or not treated with demethylating agents, as shown in (a). Bisulphite sequencing was then performed. A schematic representation of the XAF1 promoter shows the locations of 11 CpG-dinucleotides sites from −22 to −500 bp relative to the TSS. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively (c) MCF-7 cells were treated as shown in (a). ChIP assays were performed using a specific antibody against CTCF protein. The CTCF-binding site in the XAF1 promoter was analysed by PCR in the DNA recovered after ChIP (Left panel). As positive and negative controls of CTCF-DNA interaction, three specific sets of primers were included. Two of them were directed to previously validated CTCF binding sites (c-Myc and IGF2) as positive controls, and one was a negative control. The input represents soluble chromatin that was reversed cross-linked and amplified by PCR (central panel). RT-PCR was performed from cells used for ChIP assays. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA).

Mentions: As expected based on previous reports showing that XAF1 promoter is hypermethylated in cancer91217, here, pre-exposure to demethylating agents increased the transcriptional activation of XAF1 in basal conditions (Supplementary Fig.1a). To test XAF1 dynamic expression, we used two well-known XAF1 transcriptional activators, TNF-α and IFN-α293031 Demethylating conditions were required to display full transcriptional activation of XAF1 at both the mRNA and protein levels after TNF-α (Fig. 1a,b) or IFN-α (Fig. 1c,d) exposure. To extend these observations to another unrelated cancer cell line, we used ACHN cells, which have previously been shown to be responsive to IFN-α in demethylating conditions32. As observed with MCF-7 cells, we observed a dramatic increase in XAF1 responsiveness in demethylating conditions (Fig. 2a). As a positive control, we used the Colo205 cell line that presents an unmethylated XAF1 promoter9. Even without previous exposure to epigenetic modifiers, we observed a clear XAF1 transcriptional activation by TNF-α exposure (Supplementary Fig. 1b). We then reasoned that differential dinucleotide CpG methylation between control cells and cells treated with demethylating agents could help us to identify which DNA segments are important for the full responsiveness of XAF1. To this end, we performed bisulphite genomic sequencing using a specific set of primers to amplify the XAF1 promoter. Exposure to 5-aza-2′-deoxycytidine (5-A-DC) and trichostatin-A (TSA) induced consistent demethylation of three CpG dinucleotides in MCF-7 cells (Fig. 2b; A, B and C). These results indicate that full transcriptional activation of the XAF1 gene is associated with a specific CpG-dinucleotide methylation state of its promoter.


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 interacts with the XAF1 promoter.(a) ACHN cells were pre-treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with either TNF-α or IFN-α. Non-demethylated cells were stimulated with either TNF-α or IFN-α. mRNA expression of XAF1, CTCF and HPRT was analysed by qPCR. Results are presented in terms of fold change. The means from three independent experiments were plotted with ±SEM, *P < 0.05. (b) MCF-7 cells were either treated or not treated with demethylating agents, as shown in (a). Bisulphite sequencing was then performed. A schematic representation of the XAF1 promoter shows the locations of 11 CpG-dinucleotides sites from −22 to −500 bp relative to the TSS. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively (c) MCF-7 cells were treated as shown in (a). ChIP assays were performed using a specific antibody against CTCF protein. The CTCF-binding site in the XAF1 promoter was analysed by PCR in the DNA recovered after ChIP (Left panel). As positive and negative controls of CTCF-DNA interaction, three specific sets of primers were included. Two of them were directed to previously validated CTCF binding sites (c-Myc and IGF2) as positive controls, and one was a negative control. The input represents soluble chromatin that was reversed cross-linked and amplified by PCR (central panel). RT-PCR was performed from cells used for ChIP assays. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: CTCF interacts with the XAF1 promoter.(a) ACHN cells were pre-treated with 5-Aza-2′-deoxycytidine (5 μM) and Trichostatin-A (0.2 μM) for 3 days before stimulation with either TNF-α or IFN-α. Non-demethylated cells were stimulated with either TNF-α or IFN-α. mRNA expression of XAF1, CTCF and HPRT was analysed by qPCR. Results are presented in terms of fold change. The means from three independent experiments were plotted with ±SEM, *P < 0.05. (b) MCF-7 cells were either treated or not treated with demethylating agents, as shown in (a). Bisulphite sequencing was then performed. A schematic representation of the XAF1 promoter shows the locations of 11 CpG-dinucleotides sites from −22 to −500 bp relative to the TSS. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively (c) MCF-7 cells were treated as shown in (a). ChIP assays were performed using a specific antibody against CTCF protein. The CTCF-binding site in the XAF1 promoter was analysed by PCR in the DNA recovered after ChIP (Left panel). As positive and negative controls of CTCF-DNA interaction, three specific sets of primers were included. Two of them were directed to previously validated CTCF binding sites (c-Myc and IGF2) as positive controls, and one was a negative control. The input represents soluble chromatin that was reversed cross-linked and amplified by PCR (central panel). RT-PCR was performed from cells used for ChIP assays. 5-Aza-2′-deoxycytidine (5-A-DC); Trichostatin-A (TSA).
Mentions: As expected based on previous reports showing that XAF1 promoter is hypermethylated in cancer91217, here, pre-exposure to demethylating agents increased the transcriptional activation of XAF1 in basal conditions (Supplementary Fig.1a). To test XAF1 dynamic expression, we used two well-known XAF1 transcriptional activators, TNF-α and IFN-α293031 Demethylating conditions were required to display full transcriptional activation of XAF1 at both the mRNA and protein levels after TNF-α (Fig. 1a,b) or IFN-α (Fig. 1c,d) exposure. To extend these observations to another unrelated cancer cell line, we used ACHN cells, which have previously been shown to be responsive to IFN-α in demethylating conditions32. As observed with MCF-7 cells, we observed a dramatic increase in XAF1 responsiveness in demethylating conditions (Fig. 2a). As a positive control, we used the Colo205 cell line that presents an unmethylated XAF1 promoter9. Even without previous exposure to epigenetic modifiers, we observed a clear XAF1 transcriptional activation by TNF-α exposure (Supplementary Fig. 1b). We then reasoned that differential dinucleotide CpG methylation between control cells and cells treated with demethylating agents could help us to identify which DNA segments are important for the full responsiveness of XAF1. To this end, we performed bisulphite genomic sequencing using a specific set of primers to amplify the XAF1 promoter. Exposure to 5-aza-2′-deoxycytidine (5-A-DC) and trichostatin-A (TSA) induced consistent demethylation of three CpG dinucleotides in MCF-7 cells (Fig. 2b; A, B and C). These results indicate that full transcriptional activation of the XAF1 gene is associated with a specific CpG-dinucleotide methylation state of its promoter.

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