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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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p53 effect on BORIS promoters A, B and C activities. (A) Luciferase assays were performed by transient transfection of reporter constructs and p53 wild-type (WT) or mutant (mut) expression vectors, in the p53−/− K562 cell line. (B) Northern blot showing the expression of BORIS, CTCF and p53 in the K562 p53 thermo-inducible cell line. (C) Real-time PCR on RNA extracted from p53−/− H1299 parental cells or H1299 cells expressing p53 wild type. Real-time PCR data were analyzed using the comparative Ct method and normalized to the non-induced cells.
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Figure 5: p53 effect on BORIS promoters A, B and C activities. (A) Luciferase assays were performed by transient transfection of reporter constructs and p53 wild-type (WT) or mutant (mut) expression vectors, in the p53−/− K562 cell line. (B) Northern blot showing the expression of BORIS, CTCF and p53 in the K562 p53 thermo-inducible cell line. (C) Real-time PCR on RNA extracted from p53−/− H1299 parental cells or H1299 cells expressing p53 wild type. Real-time PCR data were analyzed using the comparative Ct method and normalized to the non-induced cells.

Mentions: In our analyses of BORIS expression in the 31 cancer cell lines, we noted that the lines expressing BORIS at the highest levels were known to have deletions or mutations of TP53, the gene encoding the p53 tumor suppressor (data not shown). This prompted us to ask if p53 might be involved in regulating BORIS expression. To examine this possibility, we transfected K562 cells that are p53 negative with luciferase reporters for each of the three BORIS promoters together with expression plasmids for wild-type or mutant p53 (Figure 5A). The results of these studies showed that the luciferase activity of all three BORIS promoters was significantly reduced in cells transfected with wild-type p53 versus mutant p53. With wild-type p53, the activity of promoters A and B was downregulated about 3.3- and 4.6-fold, respectively, and the activity of promoter C was repressed about 1.5-fold. We then performed northern blot analysis of K562 cells transfected with a vector encoding temperature-sensitive alleles of wild-type and mutant p53. The results showed that over-expression of the mutant p53 had no effect on the levels of either BORIS or CTCF transcripts. In contrast, activation of wild-type p53 was associated with a great reduction in expression of BORIS while expression of CTCF was unaffected (Figure 5B). To further examine the impact of p53 on BORIS expression in vivo, we compared p53-negative H1299 parental cells with H1299 cells stably infected with a virus expressing wild-type p53 (28). The results (Figure 5C) showed that the level of p53 expression was 12-fold higher in the virus-infected H1299 cells than in the parental cells. Analyses of CTCF transcripts showed that they were unchanged (data not shown). In contrast, p53 had a profound effect on each of the three BORIS promoters. Expression from promoters A and B was reduced 4.6- and 4.9-fold, respectively, while that from promoter C was down 17-fold.Figure 5.


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)

p53 effect on BORIS promoters A, B and C activities. (A) Luciferase assays were performed by transient transfection of reporter constructs and p53 wild-type (WT) or mutant (mut) expression vectors, in the p53−/− K562 cell line. (B) Northern blot showing the expression of BORIS, CTCF and p53 in the K562 p53 thermo-inducible cell line. (C) Real-time PCR on RNA extracted from p53−/− H1299 parental cells or H1299 cells expressing p53 wild type. Real-time PCR data were analyzed using the comparative Ct method and normalized to the non-induced cells.
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Figure 5: p53 effect on BORIS promoters A, B and C activities. (A) Luciferase assays were performed by transient transfection of reporter constructs and p53 wild-type (WT) or mutant (mut) expression vectors, in the p53−/− K562 cell line. (B) Northern blot showing the expression of BORIS, CTCF and p53 in the K562 p53 thermo-inducible cell line. (C) Real-time PCR on RNA extracted from p53−/− H1299 parental cells or H1299 cells expressing p53 wild type. Real-time PCR data were analyzed using the comparative Ct method and normalized to the non-induced cells.
Mentions: In our analyses of BORIS expression in the 31 cancer cell lines, we noted that the lines expressing BORIS at the highest levels were known to have deletions or mutations of TP53, the gene encoding the p53 tumor suppressor (data not shown). This prompted us to ask if p53 might be involved in regulating BORIS expression. To examine this possibility, we transfected K562 cells that are p53 negative with luciferase reporters for each of the three BORIS promoters together with expression plasmids for wild-type or mutant p53 (Figure 5A). The results of these studies showed that the luciferase activity of all three BORIS promoters was significantly reduced in cells transfected with wild-type p53 versus mutant p53. With wild-type p53, the activity of promoters A and B was downregulated about 3.3- and 4.6-fold, respectively, and the activity of promoter C was repressed about 1.5-fold. We then performed northern blot analysis of K562 cells transfected with a vector encoding temperature-sensitive alleles of wild-type and mutant p53. The results showed that over-expression of the mutant p53 had no effect on the levels of either BORIS or CTCF transcripts. In contrast, activation of wild-type p53 was associated with a great reduction in expression of BORIS while expression of CTCF was unaffected (Figure 5B). To further examine the impact of p53 on BORIS expression in vivo, we compared p53-negative H1299 parental cells with H1299 cells stably infected with a virus expressing wild-type p53 (28). The results (Figure 5C) showed that the level of p53 expression was 12-fold higher in the virus-infected H1299 cells than in the parental cells. Analyses of CTCF transcripts showed that they were unchanged (data not shown). In contrast, p53 had a profound effect on each of the three BORIS promoters. Expression from promoters A and B was reduced 4.6- and 4.9-fold, respectively, while that from promoter C was down 17-fold.Figure 5.

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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Related in: MedlinePlus