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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 maintains an open-chromatin configuration in the XAF1 promoter in transgene assays.(a) The timeline for stable transgenic cell line generation using peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 plasmids. The detection of GFP expression for each cell line was performed using FACS. Single-cell clones were generated at day 60. (b) Right, bisulphite sequencing was performed from single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs. The exogenous XAF1 promoter was specifically amplified using a nested-PCR strategy in which the first amplification was performed using primers recognizing plasmid sequences. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively. Left, histograms from each single cell clones are showed (c) ChIP assays were performed from stable single-cell clones using specific antibodies against H3K4-2me or H3K9-3me. Data are represented as the means ± SEM from three single-cell clones. Red triangle symbols the deletion of the CTCF binding site.
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f5: CTCF maintains an open-chromatin configuration in the XAF1 promoter in transgene assays.(a) The timeline for stable transgenic cell line generation using peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 plasmids. The detection of GFP expression for each cell line was performed using FACS. Single-cell clones were generated at day 60. (b) Right, bisulphite sequencing was performed from single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs. The exogenous XAF1 promoter was specifically amplified using a nested-PCR strategy in which the first amplification was performed using primers recognizing plasmid sequences. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively. Left, histograms from each single cell clones are showed (c) ChIP assays were performed from stable single-cell clones using specific antibodies against H3K4-2me or H3K9-3me. Data are represented as the means ± SEM from three single-cell clones. Red triangle symbols the deletion of the CTCF binding site.

Mentions: The insulating action of CTCF protects several genes from epigenetic silencing2834. In particular, it has been described that the absence of CTCF in tumour suppressor gene promoters induces their epigenetic silencing, which supports the role of CTCF in cancer353637. To test the possible epigenetic-mediated regulation of CTCF on the XAF1 gene, we compared the XAF1 promoter activity in a genomic integrated context by measuring a GFP reporter gene. For this, we compared the wild type XAF1 promoter with the CTCF-deletion (Δ-CTCF-XAF1) construct. Supporting the insulating role of CTCF on the XAF1 gene, cells with the Δ-CTCF-XAF1 promoter showed lower GFP levels than those with the wild-type XAF1 promoter after 60 days of continuous culture (Fig. 5a). To further support this finding, single-cell clones for each transfection were isolated and propagated for an additional 35 days. As expected by the previous result, silencing of GFP expression levels was mainly observed in single-cell clones with the integrated Δ-CTCF-XAF1-promoter (Fig. 5a), pointing toward an epigenetic-protective effect of the CTCF binding site. A possible alternative explanation for the difference in GFP expression levels between transfections could be attributed to a distinct number of integration events. To exclude this possibility, the transgene copy number was measured by real-time PCR as previously reported38. The difference between GFP expression levels driven by the Δ-CTCF-XAF1-promoter and wild-type-XAF1-promoter was independent of the transgene copy number (Supplementary Fig. 2b). To gain insight into the epigenetic mechanism involved in GFP silencing of the Δ-CTCF-XAF1-promoter, we hypothesized that loss of the CTCF-binding site could promote 1) accelerated CpG methylation or 2) acquisition of a repressive chromatin configuration based on HPMs (or both). To test the first hypothesis, sequencing of the sodium bisulphite-modified genomic DNA from three single-cell clones for each transfection was performed. To discriminate endogenous XAF1 promoter amplification, a nested-PCR strategy was performed in which the first set of primers annealed to plasmid sequences surrounding the exogenous XAF1 promoter (Fig. 5b and Supplementary Fig. 2c). We observed that the Δ-CTCF-XAF1-promoter is more susceptible to dinucleotide-CpG methylation than the wild-type-XAF1-promoter in a genomic-integrated context (Fig. 5b). To test our second hypothesis, ChIP assays were performed using specific antibodies directed to H3K4-2me or H3K9-3me posttranslational modifications in single-cell clones from each stable transfection. To interrogate the relative enrichment of repressive or active HPMs in the transgene, we designed a pair of primers that anneal to the plasmid sequence immediately after the exogenous XAF1 promoter (Fig. 5c and Supplementary Fig. 2c). Lower levels of the H3K4-2me posttranslational modification, a marker for transcription activity, were observed in cell single clones from the Δ-CTCF XAF1-promoter (Fig. 5c). As expected, the Δ-CTCF XAF1 promoter was enriched with the repressive H3K9-3me modification (Fig. 5c). Overall, these findings support the notion that CTCF regulates DNA methylation in the XAF1 promoter; thus, loss of CTCF in its cognate-binding site induces DNA-methylation and polarization from active to repressive HPM, which in turn induces transcriptional repression.


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 maintains an open-chromatin configuration in the XAF1 promoter in transgene assays.(a) The timeline for stable transgenic cell line generation using peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 plasmids. The detection of GFP expression for each cell line was performed using FACS. Single-cell clones were generated at day 60. (b) Right, bisulphite sequencing was performed from single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs. The exogenous XAF1 promoter was specifically amplified using a nested-PCR strategy in which the first amplification was performed using primers recognizing plasmid sequences. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively. Left, histograms from each single cell clones are showed (c) ChIP assays were performed from stable single-cell clones using specific antibodies against H3K4-2me or H3K9-3me. Data are represented as the means ± SEM from three single-cell clones. Red triangle symbols the deletion of the CTCF binding site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: CTCF maintains an open-chromatin configuration in the XAF1 promoter in transgene assays.(a) The timeline for stable transgenic cell line generation using peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 plasmids. The detection of GFP expression for each cell line was performed using FACS. Single-cell clones were generated at day 60. (b) Right, bisulphite sequencing was performed from single-cell clones containing either peGFPN1-XAF1 or peGFPN1-Δ-CTCF-XAF1 constructs. The exogenous XAF1 promoter was specifically amplified using a nested-PCR strategy in which the first amplification was performed using primers recognizing plasmid sequences. Methylated and unmethylated CpGs are depicted as filled and open circles, respectively. Left, histograms from each single cell clones are showed (c) ChIP assays were performed from stable single-cell clones using specific antibodies against H3K4-2me or H3K9-3me. Data are represented as the means ± SEM from three single-cell clones. Red triangle symbols the deletion of the CTCF binding site.
Mentions: The insulating action of CTCF protects several genes from epigenetic silencing2834. In particular, it has been described that the absence of CTCF in tumour suppressor gene promoters induces their epigenetic silencing, which supports the role of CTCF in cancer353637. To test the possible epigenetic-mediated regulation of CTCF on the XAF1 gene, we compared the XAF1 promoter activity in a genomic integrated context by measuring a GFP reporter gene. For this, we compared the wild type XAF1 promoter with the CTCF-deletion (Δ-CTCF-XAF1) construct. Supporting the insulating role of CTCF on the XAF1 gene, cells with the Δ-CTCF-XAF1 promoter showed lower GFP levels than those with the wild-type XAF1 promoter after 60 days of continuous culture (Fig. 5a). To further support this finding, single-cell clones for each transfection were isolated and propagated for an additional 35 days. As expected by the previous result, silencing of GFP expression levels was mainly observed in single-cell clones with the integrated Δ-CTCF-XAF1-promoter (Fig. 5a), pointing toward an epigenetic-protective effect of the CTCF binding site. A possible alternative explanation for the difference in GFP expression levels between transfections could be attributed to a distinct number of integration events. To exclude this possibility, the transgene copy number was measured by real-time PCR as previously reported38. The difference between GFP expression levels driven by the Δ-CTCF-XAF1-promoter and wild-type-XAF1-promoter was independent of the transgene copy number (Supplementary Fig. 2b). To gain insight into the epigenetic mechanism involved in GFP silencing of the Δ-CTCF-XAF1-promoter, we hypothesized that loss of the CTCF-binding site could promote 1) accelerated CpG methylation or 2) acquisition of a repressive chromatin configuration based on HPMs (or both). To test the first hypothesis, sequencing of the sodium bisulphite-modified genomic DNA from three single-cell clones for each transfection was performed. To discriminate endogenous XAF1 promoter amplification, a nested-PCR strategy was performed in which the first set of primers annealed to plasmid sequences surrounding the exogenous XAF1 promoter (Fig. 5b and Supplementary Fig. 2c). We observed that the Δ-CTCF-XAF1-promoter is more susceptible to dinucleotide-CpG methylation than the wild-type-XAF1-promoter in a genomic-integrated context (Fig. 5b). To test our second hypothesis, ChIP assays were performed using specific antibodies directed to H3K4-2me or H3K9-3me posttranslational modifications in single-cell clones from each stable transfection. To interrogate the relative enrichment of repressive or active HPMs in the transgene, we designed a pair of primers that anneal to the plasmid sequence immediately after the exogenous XAF1 promoter (Fig. 5c and Supplementary Fig. 2c). Lower levels of the H3K4-2me posttranslational modification, a marker for transcription activity, were observed in cell single clones from the Δ-CTCF XAF1-promoter (Fig. 5c). As expected, the Δ-CTCF XAF1 promoter was enriched with the repressive H3K9-3me modification (Fig. 5c). Overall, these findings support the notion that CTCF regulates DNA methylation in the XAF1 promoter; thus, loss of CTCF in its cognate-binding site induces DNA-methylation and polarization from active to repressive HPM, which in turn induces transcriptional repression.

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