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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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Analyses of DNA methylation of BORIS promoter B. (A) BORIS expression and methylation patterns of BORIS promoter B in human tissues and cell lines. Lanes 1–2: MS-SSCA and MS-DBA unmethylated and fully methylated controls respectively, obtained from plasmids containing BORIS promoter B sequences; lanes 3–4: NCCIT and OVCAR-3 cell lines; lanes 5–8: normal tissues, respectively colon, skin, and two testis; lanes 9–13: tumor tissues, respectively bladder, testis, ovary, breast, colon. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Genomic bisulfite sequencing of 16/32 CpGs within the CpG island covering promoters B in normal fibroblast NHDF, NCCIT (group A), Ovcar-3 (group A), Ovcar-8 (group B) and K562 (group B) cell lines.
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Figure 6: Analyses of DNA methylation of BORIS promoter B. (A) BORIS expression and methylation patterns of BORIS promoter B in human tissues and cell lines. Lanes 1–2: MS-SSCA and MS-DBA unmethylated and fully methylated controls respectively, obtained from plasmids containing BORIS promoter B sequences; lanes 3–4: NCCIT and OVCAR-3 cell lines; lanes 5–8: normal tissues, respectively colon, skin, and two testis; lanes 9–13: tumor tissues, respectively bladder, testis, ovary, breast, colon. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Genomic bisulfite sequencing of 16/32 CpGs within the CpG island covering promoters B in normal fibroblast NHDF, NCCIT (group A), Ovcar-3 (group A), Ovcar-8 (group B) and K562 (group B) cell lines.

Mentions: To further analyze the molecular mechanisms involved in regulating the activity of the BORIS promoters, we examined the effect of CpG methylation. The fact that promoters B and C co-localize within a CpG island raised the possibility that their activities might be affected by methylation (Figure 1A). DNA samples from 2 cell lines, 24 human normal tissue samples and 26 tumor tissue samples were extracted and analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA) and methylation-sensitive dot blot assay (MS-DBA) after sodium bisulfite modification (31,32). The CpG island was found to be completely methylated in samples of all normal tissues with the exception of testis, the tissue in which BORIS is normally expressed. Studies of the CpG island in tumors revealed demethylation in testicular tumors (6/6), ovarian tumors (1/3), breast cancers (1/6) and endometrial tumors (1/3) (Table 4). Representative examples of results from studies of the cell lines and tissues are shown in Figure 6A. The partial methylation observed in normal testis tissue is likely to be due to the presence of both germ cells and non-germ cells in this tissue. As might be expected, BORIS was expressed in almost all the tumors and cell lines that contained a hypomethylated CpG island. Moreover, it has been shown that demethylation of BORIS promoter B in tumor tissues, versus methylated BORIS promoter B in normal tissues, is correlated with the expression of BORIS in tumors (Table 4). However, among the 26 cell lines which belong to group A/C, only 2 showed partial demethylation of promoters B and C. Full methylation of these promoters in all the other lines was correlated with inhibition of BORIS transcription from promoter B (Table 1). In the case of cell lines from group B, promoters B and C were found to be unmethylated or partially methylated in the case of COLO-205. Nine out of 32 CpGs within promoters B and C were analyzed by genomic bisulfite sequencing in NHDF, 2 cell lines from group A/C (NCCIT and Ovcar-3) and 2 cell lines from group B (Ovcar-8 and K562) (Figure 6B). In normal fibroblasts, 98% of the CpGs analyzed were found to be methylated and BORIS expression could not be detected. In NCCIT and Ovcar-3, 98% and 96% of CpGs were methylated (Figure 6B). In these cells, BORIS was expressed primarily from promoter A, in association with minimal expression from promoter C and no expression from promoter B (Table 1). In Ovcar-8 and K562, sequencing showed that 0.9% and 0.45% of CpGs were methylated (Figure 6B). In these two lines, expression of BORIS comes almost exclusively from promoter B, 96% in Ovcar-8 and 80% in K562 (Table 1).Figure 6.


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)

Analyses of DNA methylation of BORIS promoter B. (A) BORIS expression and methylation patterns of BORIS promoter B in human tissues and cell lines. Lanes 1–2: MS-SSCA and MS-DBA unmethylated and fully methylated controls respectively, obtained from plasmids containing BORIS promoter B sequences; lanes 3–4: NCCIT and OVCAR-3 cell lines; lanes 5–8: normal tissues, respectively colon, skin, and two testis; lanes 9–13: tumor tissues, respectively bladder, testis, ovary, breast, colon. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Genomic bisulfite sequencing of 16/32 CpGs within the CpG island covering promoters B in normal fibroblast NHDF, NCCIT (group A), Ovcar-3 (group A), Ovcar-8 (group B) and K562 (group B) cell lines.
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Figure 6: Analyses of DNA methylation of BORIS promoter B. (A) BORIS expression and methylation patterns of BORIS promoter B in human tissues and cell lines. Lanes 1–2: MS-SSCA and MS-DBA unmethylated and fully methylated controls respectively, obtained from plasmids containing BORIS promoter B sequences; lanes 3–4: NCCIT and OVCAR-3 cell lines; lanes 5–8: normal tissues, respectively colon, skin, and two testis; lanes 9–13: tumor tissues, respectively bladder, testis, ovary, breast, colon. BORIS mRNA was detected by RT-PCR, with β-actine as internal control. (B) Genomic bisulfite sequencing of 16/32 CpGs within the CpG island covering promoters B in normal fibroblast NHDF, NCCIT (group A), Ovcar-3 (group A), Ovcar-8 (group B) and K562 (group B) cell lines.
Mentions: To further analyze the molecular mechanisms involved in regulating the activity of the BORIS promoters, we examined the effect of CpG methylation. The fact that promoters B and C co-localize within a CpG island raised the possibility that their activities might be affected by methylation (Figure 1A). DNA samples from 2 cell lines, 24 human normal tissue samples and 26 tumor tissue samples were extracted and analyzed by methylation-sensitive single-strand conformation analysis (MS-SSCA) and methylation-sensitive dot blot assay (MS-DBA) after sodium bisulfite modification (31,32). The CpG island was found to be completely methylated in samples of all normal tissues with the exception of testis, the tissue in which BORIS is normally expressed. Studies of the CpG island in tumors revealed demethylation in testicular tumors (6/6), ovarian tumors (1/3), breast cancers (1/6) and endometrial tumors (1/3) (Table 4). Representative examples of results from studies of the cell lines and tissues are shown in Figure 6A. The partial methylation observed in normal testis tissue is likely to be due to the presence of both germ cells and non-germ cells in this tissue. As might be expected, BORIS was expressed in almost all the tumors and cell lines that contained a hypomethylated CpG island. Moreover, it has been shown that demethylation of BORIS promoter B in tumor tissues, versus methylated BORIS promoter B in normal tissues, is correlated with the expression of BORIS in tumors (Table 4). However, among the 26 cell lines which belong to group A/C, only 2 showed partial demethylation of promoters B and C. Full methylation of these promoters in all the other lines was correlated with inhibition of BORIS transcription from promoter B (Table 1). In the case of cell lines from group B, promoters B and C were found to be unmethylated or partially methylated in the case of COLO-205. Nine out of 32 CpGs within promoters B and C were analyzed by genomic bisulfite sequencing in NHDF, 2 cell lines from group A/C (NCCIT and Ovcar-3) and 2 cell lines from group B (Ovcar-8 and K562) (Figure 6B). In normal fibroblasts, 98% of the CpGs analyzed were found to be methylated and BORIS expression could not be detected. In NCCIT and Ovcar-3, 98% and 96% of CpGs were methylated (Figure 6B). In these cells, BORIS was expressed primarily from promoter A, in association with minimal expression from promoter C and no expression from promoter B (Table 1). In Ovcar-8 and K562, sequencing showed that 0.9% and 0.45% of CpGs were methylated (Figure 6B). In these two lines, expression of BORIS comes almost exclusively from promoter B, 96% in Ovcar-8 and 80% in K562 (Table 1).Figure 6.

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