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Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors.

Renaud S, Pugacheva EM, Delgado MD, Braunschweig R, Abdullaev Z, Loukinov D, Benhattar J, Lobanenkov V - Nucleic Acids Res. (2007)

Bottom Line: We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter.Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters.These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Pathology, Laboratory of Immunopathology, NIAID, NIH, Rockville, MD 20815, USA.

ABSTRACT
BORIS, like other members of the 'cancer/testis antigen' family, is normally expressed in testicular germ cells and repressed in somatic cells, but is aberrantly activated in cancers. To understand regulatory mechanisms governing human BORIS expression, we characterized its 5'-flanking region. Using 5' RACE, we identified three promoters, designated A, B and C, corresponding to transcription start sites at -1447, -899 and -658 bp upstream of the first ATG. Alternative promoter usage generated at least five alternatively spliced BORIS mRNAs with different half-lives determined by varying 5'-UTRs. In normal testis, BORIS is transcribed from all three promoters, but 84% of the 30 cancer cell lines tested used only promoter(s) A and/or C while the others utilized primarily promoters B and C. The differences in promoter usage between normal and cancer cells suggested that they were subject to differential regulation. We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter. Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters. These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

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Regulation of BORIS expression by CTCF. (A) Real-time PCR analyses of RNA extracted from NHDF (Normal Human Dermal Fibroblasts) treated with siRNA against CTCF show the decrease of CTCF expression and the increase of BORIS expression coming from the three promoters at different levels. Data were analyzed by comparative Ct method and normalized to the untreated control. Results are represented for each set of primers used in real-time PCR. (B) Schematic map of the overlapping fragments used as EMSA probes for in vitro detection of CTCF-binding sites and results of EMSA analyses showing binding of CTCF within promoters A, B and C in red (first panel). Results of EMSA analyses of SssI-methylated probes showing insensitivity of CTCF binding in promoter A and B, and methylation sensitive sites in promoter B and C within the CpG island (second panel). The third panel showed the methylation-sensitive enzymatic restriction of probes used in methylation-sensitive EMSA. (C) CTCF in vivo occupancy on BORIS promoters via ChIP in normal fibroblasts BJ, Ovcar-8 and Ovcar-3 cell lines. Real-time PCR analysis of the fold difference for the presence of DNA with the CTCF-binding site in the input chromatin versus in the CTCF chromatin fraction is obtained by ChIP. The fold difference between immunoprecipitated DNA with specific CTCF antibody or rabbit serum (used as negative control) is represented for each set of primers. hMYC-N primers are used as positive control for good enrichment after ChIP and c-MYC non-binding site G is used as control for the specificity of the enrichment. Controls are represented in the right graph for each cell lines.
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Figure 4: Regulation of BORIS expression by CTCF. (A) Real-time PCR analyses of RNA extracted from NHDF (Normal Human Dermal Fibroblasts) treated with siRNA against CTCF show the decrease of CTCF expression and the increase of BORIS expression coming from the three promoters at different levels. Data were analyzed by comparative Ct method and normalized to the untreated control. Results are represented for each set of primers used in real-time PCR. (B) Schematic map of the overlapping fragments used as EMSA probes for in vitro detection of CTCF-binding sites and results of EMSA analyses showing binding of CTCF within promoters A, B and C in red (first panel). Results of EMSA analyses of SssI-methylated probes showing insensitivity of CTCF binding in promoter A and B, and methylation sensitive sites in promoter B and C within the CpG island (second panel). The third panel showed the methylation-sensitive enzymatic restriction of probes used in methylation-sensitive EMSA. (C) CTCF in vivo occupancy on BORIS promoters via ChIP in normal fibroblasts BJ, Ovcar-8 and Ovcar-3 cell lines. Real-time PCR analysis of the fold difference for the presence of DNA with the CTCF-binding site in the input chromatin versus in the CTCF chromatin fraction is obtained by ChIP. The fold difference between immunoprecipitated DNA with specific CTCF antibody or rabbit serum (used as negative control) is represented for each set of primers. hMYC-N primers are used as positive control for good enrichment after ChIP and c-MYC non-binding site G is used as control for the specificity of the enrichment. Controls are represented in the right graph for each cell lines.

Mentions: The fact that expression of CTCF and BORIS in normal testis is almost mutually exclusive raised the possibility that CTCF may negatively regulate BORIS transcription. To examine this possibility, we used siRNA to suppress CTCF expression in NHDF and then tested the cells for expression of CTCF and BORIS by qPCR (Figure 4A). We observed that the level of CTCF transcript was reduced more than 4-fold in cells expressing the siRNA while the levels of BORIS transcripts in the same cells were increased at least 8-fold (Figure 4A). To evaluate the contribution of each promoter to expression of BORIS in the siRNA-treated cells, we performed qPCR with primers and probes specific for each of the spliced isoforms. The results showed that in cells expressing the CTCF-specific siRNA, expression of BORIS from all three promoters was enhanced, but to differing extents—expression from promoter A was increased 8–fold, expression from promoter B was increased 2.5-fold, and expression from promoter C was increased 4.5-fold (Figure 4A). These results indicated that under normal circumstances, CTCF contributes to suppression of BORIS expression by acting on all three promoters.Figure 4.


Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors.

Renaud S, Pugacheva EM, Delgado MD, Braunschweig R, Abdullaev Z, Loukinov D, Benhattar J, Lobanenkov V - Nucleic Acids Res. (2007)

Regulation of BORIS expression by CTCF. (A) Real-time PCR analyses of RNA extracted from NHDF (Normal Human Dermal Fibroblasts) treated with siRNA against CTCF show the decrease of CTCF expression and the increase of BORIS expression coming from the three promoters at different levels. Data were analyzed by comparative Ct method and normalized to the untreated control. Results are represented for each set of primers used in real-time PCR. (B) Schematic map of the overlapping fragments used as EMSA probes for in vitro detection of CTCF-binding sites and results of EMSA analyses showing binding of CTCF within promoters A, B and C in red (first panel). Results of EMSA analyses of SssI-methylated probes showing insensitivity of CTCF binding in promoter A and B, and methylation sensitive sites in promoter B and C within the CpG island (second panel). The third panel showed the methylation-sensitive enzymatic restriction of probes used in methylation-sensitive EMSA. (C) CTCF in vivo occupancy on BORIS promoters via ChIP in normal fibroblasts BJ, Ovcar-8 and Ovcar-3 cell lines. Real-time PCR analysis of the fold difference for the presence of DNA with the CTCF-binding site in the input chromatin versus in the CTCF chromatin fraction is obtained by ChIP. The fold difference between immunoprecipitated DNA with specific CTCF antibody or rabbit serum (used as negative control) is represented for each set of primers. hMYC-N primers are used as positive control for good enrichment after ChIP and c-MYC non-binding site G is used as control for the specificity of the enrichment. Controls are represented in the right graph for each cell lines.
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Related In: Results  -  Collection

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Figure 4: Regulation of BORIS expression by CTCF. (A) Real-time PCR analyses of RNA extracted from NHDF (Normal Human Dermal Fibroblasts) treated with siRNA against CTCF show the decrease of CTCF expression and the increase of BORIS expression coming from the three promoters at different levels. Data were analyzed by comparative Ct method and normalized to the untreated control. Results are represented for each set of primers used in real-time PCR. (B) Schematic map of the overlapping fragments used as EMSA probes for in vitro detection of CTCF-binding sites and results of EMSA analyses showing binding of CTCF within promoters A, B and C in red (first panel). Results of EMSA analyses of SssI-methylated probes showing insensitivity of CTCF binding in promoter A and B, and methylation sensitive sites in promoter B and C within the CpG island (second panel). The third panel showed the methylation-sensitive enzymatic restriction of probes used in methylation-sensitive EMSA. (C) CTCF in vivo occupancy on BORIS promoters via ChIP in normal fibroblasts BJ, Ovcar-8 and Ovcar-3 cell lines. Real-time PCR analysis of the fold difference for the presence of DNA with the CTCF-binding site in the input chromatin versus in the CTCF chromatin fraction is obtained by ChIP. The fold difference between immunoprecipitated DNA with specific CTCF antibody or rabbit serum (used as negative control) is represented for each set of primers. hMYC-N primers are used as positive control for good enrichment after ChIP and c-MYC non-binding site G is used as control for the specificity of the enrichment. Controls are represented in the right graph for each cell lines.
Mentions: The fact that expression of CTCF and BORIS in normal testis is almost mutually exclusive raised the possibility that CTCF may negatively regulate BORIS transcription. To examine this possibility, we used siRNA to suppress CTCF expression in NHDF and then tested the cells for expression of CTCF and BORIS by qPCR (Figure 4A). We observed that the level of CTCF transcript was reduced more than 4-fold in cells expressing the siRNA while the levels of BORIS transcripts in the same cells were increased at least 8-fold (Figure 4A). To evaluate the contribution of each promoter to expression of BORIS in the siRNA-treated cells, we performed qPCR with primers and probes specific for each of the spliced isoforms. The results showed that in cells expressing the CTCF-specific siRNA, expression of BORIS from all three promoters was enhanced, but to differing extents—expression from promoter A was increased 8–fold, expression from promoter B was increased 2.5-fold, and expression from promoter C was increased 4.5-fold (Figure 4A). These results indicated that under normal circumstances, CTCF contributes to suppression of BORIS expression by acting on all three promoters.Figure 4.

Bottom Line: We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter.Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters.These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Pathology, Laboratory of Immunopathology, NIAID, NIH, Rockville, MD 20815, USA.

ABSTRACT
BORIS, like other members of the 'cancer/testis antigen' family, is normally expressed in testicular germ cells and repressed in somatic cells, but is aberrantly activated in cancers. To understand regulatory mechanisms governing human BORIS expression, we characterized its 5'-flanking region. Using 5' RACE, we identified three promoters, designated A, B and C, corresponding to transcription start sites at -1447, -899 and -658 bp upstream of the first ATG. Alternative promoter usage generated at least five alternatively spliced BORIS mRNAs with different half-lives determined by varying 5'-UTRs. In normal testis, BORIS is transcribed from all three promoters, but 84% of the 30 cancer cell lines tested used only promoter(s) A and/or C while the others utilized primarily promoters B and C. The differences in promoter usage between normal and cancer cells suggested that they were subject to differential regulation. We found that DNA methylation and functional p53 contributes to the negative regulation of each promoter. Moreover, reduction of CTCF in normally BORIS-negative human fibroblasts resulted in derepression of BORIS promoters. These results provide a mechanistic basis for understanding cancer-related associations between haploinsufficiency of CTCF and BORIS derepression, and between the lack of functional p53 and aberrant activation of BORIS.

Show MeSH
Related in: MedlinePlus