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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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Effect of demethylating agent on BORIS expression in cancer cell lines and normal fibroblasts. (A) BORIS expression and methylation patterns of BORIS promoter B in two BORIS-negative cell lines treated with 5-aza-dC. Lanes 1–2: MS-SSCA unmethylated and fully methylated controls respectively; lane 3: HeLa cells; lane 4: HeLa cells after 5-aza-dC treatment; lane 5: Co115 cells; lane 6: Co115 cells after 5-aza-dC treatment. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Real-time PCR on HeLa and Co115 before and after 5-aza-dC treatment. Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers. (C) and (D) Determination of BORIS promoters A, B and C activities after treatment with 5-aza-dC of NHDFs by RT-PCR (C) and real-time PCR (D). Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers.
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Figure 7: Effect of demethylating agent on BORIS expression in cancer cell lines and normal fibroblasts. (A) BORIS expression and methylation patterns of BORIS promoter B in two BORIS-negative cell lines treated with 5-aza-dC. Lanes 1–2: MS-SSCA unmethylated and fully methylated controls respectively; lane 3: HeLa cells; lane 4: HeLa cells after 5-aza-dC treatment; lane 5: Co115 cells; lane 6: Co115 cells after 5-aza-dC treatment. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Real-time PCR on HeLa and Co115 before and after 5-aza-dC treatment. Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers. (C) and (D) Determination of BORIS promoters A, B and C activities after treatment with 5-aza-dC of NHDFs by RT-PCR (C) and real-time PCR (D). Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers.

Mentions: To confirm the regulation of promoter B by DNA methylation, we treated two weakly BORIS-positive cancer cell lines (HeLa and Co115) with 5-aza-dC. Treatment resulted in demethylation of the CpG island and strong activation of BORIS expression in both lines (Figure 7A). Moreover, the real-time PCR analyses showed that BORIS expression detected before 5-aza-dC treatment was coming from promoters A and C, but after 5-aza-dC treatment the BORIS expression switched to promoter B in both cell lines (Figure 7B). The decrease of expression from promoters A and C after 5-aza-dC treatment might be explained by the effect of 5-aza-dC itself on other transcriptional factors that could also in their turn downregulate BORIS expression through promoters A and C. Expression of BORIS in normal cells was also induced after treatment with 5-aza-dC (Figure 7C and D) with BORIS transcripts originating from all three promoters. qPCR analyses showed that BORIS transcripts from promoter B were readily detected in cells treated for just 6 h and that transcripts from all three promoters could be detected in cells treated for 24 h with promoter B being the strongest (Figure 7D). Remarkably, we were not able to detect any demethylation of the CpG island at the 24 h time point, suggesting that a mechanism other than demethylation was responsible for the effects of 5-aza-dC on the BORIS promoters.Figure 7.


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)

Effect of demethylating agent on BORIS expression in cancer cell lines and normal fibroblasts. (A) BORIS expression and methylation patterns of BORIS promoter B in two BORIS-negative cell lines treated with 5-aza-dC. Lanes 1–2: MS-SSCA unmethylated and fully methylated controls respectively; lane 3: HeLa cells; lane 4: HeLa cells after 5-aza-dC treatment; lane 5: Co115 cells; lane 6: Co115 cells after 5-aza-dC treatment. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Real-time PCR on HeLa and Co115 before and after 5-aza-dC treatment. Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers. (C) and (D) Determination of BORIS promoters A, B and C activities after treatment with 5-aza-dC of NHDFs by RT-PCR (C) and real-time PCR (D). Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers.
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Figure 7: Effect of demethylating agent on BORIS expression in cancer cell lines and normal fibroblasts. (A) BORIS expression and methylation patterns of BORIS promoter B in two BORIS-negative cell lines treated with 5-aza-dC. Lanes 1–2: MS-SSCA unmethylated and fully methylated controls respectively; lane 3: HeLa cells; lane 4: HeLa cells after 5-aza-dC treatment; lane 5: Co115 cells; lane 6: Co115 cells after 5-aza-dC treatment. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Real-time PCR on HeLa and Co115 before and after 5-aza-dC treatment. Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers. (C) and (D) Determination of BORIS promoters A, B and C activities after treatment with 5-aza-dC of NHDFs by RT-PCR (C) and real-time PCR (D). Real-time PCR data were analyzed using the comparative Ct method between treated and untreated cells and for each set of primers.
Mentions: To confirm the regulation of promoter B by DNA methylation, we treated two weakly BORIS-positive cancer cell lines (HeLa and Co115) with 5-aza-dC. Treatment resulted in demethylation of the CpG island and strong activation of BORIS expression in both lines (Figure 7A). Moreover, the real-time PCR analyses showed that BORIS expression detected before 5-aza-dC treatment was coming from promoters A and C, but after 5-aza-dC treatment the BORIS expression switched to promoter B in both cell lines (Figure 7B). The decrease of expression from promoters A and C after 5-aza-dC treatment might be explained by the effect of 5-aza-dC itself on other transcriptional factors that could also in their turn downregulate BORIS expression through promoters A and C. Expression of BORIS in normal cells was also induced after treatment with 5-aza-dC (Figure 7C and D) with BORIS transcripts originating from all three promoters. qPCR analyses showed that BORIS transcripts from promoter B were readily detected in cells treated for just 6 h and that transcripts from all three promoters could be detected in cells treated for 24 h with promoter B being the strongest (Figure 7D). Remarkably, we were not able to detect any demethylation of the CpG island at the 24 h time point, suggesting that a mechanism other than demethylation was responsible for the effects of 5-aza-dC on the BORIS promoters.Figure 7.

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