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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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Detection of BORIS expression from different alternative promoters and stability of BORIS alternative transcripts. (A) Unique BORIS cDNA sequences attached to promoters A, B and C. To detect BORIS expression from different promoters, forward primer was designed on basis of unique cDNA sequence for every promoter. Reverse primer was designed on the basis of BORIS coding exon 2 sequence. Expected RT-PCR fragment was 720 bp for BORIS promoter A expression, 760 bp for BORIS promoter B expression and 810 bp for BORIS promoter C expression. All RT-PCR fragments contain splice site allowing distinguishing them from genomic DNA. (B) K562 and Ovcar-8 cells were treated with Actinomycin D for various durations to block transcription. Total RNA was extracted at the indicated time points following the addition of actinomycin D. Quantitative real-time PCR amplification were performed as described in Materials and Methods section to determine copy number of each BORIS alternative transcript. Each data point represents the average of three amplification reactions. RNA degradation curves were obtained by setting at 100% the maximum of mRNA expression at Time 0 before Act D treatment.
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Figure 2: Detection of BORIS expression from different alternative promoters and stability of BORIS alternative transcripts. (A) Unique BORIS cDNA sequences attached to promoters A, B and C. To detect BORIS expression from different promoters, forward primer was designed on basis of unique cDNA sequence for every promoter. Reverse primer was designed on the basis of BORIS coding exon 2 sequence. Expected RT-PCR fragment was 720 bp for BORIS promoter A expression, 760 bp for BORIS promoter B expression and 810 bp for BORIS promoter C expression. All RT-PCR fragments contain splice site allowing distinguishing them from genomic DNA. (B) K562 and Ovcar-8 cells were treated with Actinomycin D for various durations to block transcription. Total RNA was extracted at the indicated time points following the addition of actinomycin D. Quantitative real-time PCR amplification were performed as described in Materials and Methods section to determine copy number of each BORIS alternative transcript. Each data point represents the average of three amplification reactions. RNA degradation curves were obtained by setting at 100% the maximum of mRNA expression at Time 0 before Act D treatment.

Mentions: The fact that BORIS transcription initiated from three different promoters suggested that there would likely be a series of 5′ splice variants. Indeed, we identified five major transcripts that differed in their 5′ noncoding regions but encoded the same protein sequence (Figure 2). The BORIS A1 and BORIS A3 isoforms are expressed from promoter A and have first 180 and 548 bp noncoding exons Ea and Ea+Ea1+Eb, respectively that splice directly to the first 530 bp coding exon, E1. The BORIS A2 splice variant also originates from promoter A, but has two more noncoding exons, Ea1 and Eb, than the BORIS A1 isoform. The BORIS variant that comes from promoter B contains a first 80 bp noncoding exon, Eb, which splices to first 530 bp coding exon, E1. The BORIS C1 isoform is expressed from promoter C and is transcribed as an unspliced first 1190 bp coding exon (Figure 2A). To investigate whether the alternative 5′ UTRs affect mRNA stability, we blocked RNA synthesis by treating cells with ActD. Two cell lines were used in those experiments: K562 cells that express all five alternative 5′ UTRs and Ovcar-8 cells that express only two of five. As shown in Figure 2 (panel B), the half-lives of BORIS, BORIS A1, BORIS A2, BORIS A3 and BORIS C1 transcripts in K562 cells were 4.7, 7.4, 6.9, 7.9 and 6.4 h, respectively, indicating that BORIS mRNAs in cultured cells are relatively stable and long-lived. The alternative transcript expressed from promoter B, BORIS, was 1.5-fold less stable than BORIS transcripts expressed from promoters A and C. The relative turnover of the three alternative 5′ UTRs expressed from promoter A—BORIS A1, BORIS A2 and BORIS A3—was comparable and the rates of degradation were approximately 1.2-fold slower than for the BORIS C1 transcript expressed from promoter C. Similar half-lives of BORIS (4.7 h) and BORIS C1 (6.6 h) were obtained in Ovcar-8 cell line (Figure 2B). In both cell lines, GAPDH mRNA degradation was similar to published values for that gene (34).Figure 2.


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)

Detection of BORIS expression from different alternative promoters and stability of BORIS alternative transcripts. (A) Unique BORIS cDNA sequences attached to promoters A, B and C. To detect BORIS expression from different promoters, forward primer was designed on basis of unique cDNA sequence for every promoter. Reverse primer was designed on the basis of BORIS coding exon 2 sequence. Expected RT-PCR fragment was 720 bp for BORIS promoter A expression, 760 bp for BORIS promoter B expression and 810 bp for BORIS promoter C expression. All RT-PCR fragments contain splice site allowing distinguishing them from genomic DNA. (B) K562 and Ovcar-8 cells were treated with Actinomycin D for various durations to block transcription. Total RNA was extracted at the indicated time points following the addition of actinomycin D. Quantitative real-time PCR amplification were performed as described in Materials and Methods section to determine copy number of each BORIS alternative transcript. Each data point represents the average of three amplification reactions. RNA degradation curves were obtained by setting at 100% the maximum of mRNA expression at Time 0 before Act D treatment.
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Figure 2: Detection of BORIS expression from different alternative promoters and stability of BORIS alternative transcripts. (A) Unique BORIS cDNA sequences attached to promoters A, B and C. To detect BORIS expression from different promoters, forward primer was designed on basis of unique cDNA sequence for every promoter. Reverse primer was designed on the basis of BORIS coding exon 2 sequence. Expected RT-PCR fragment was 720 bp for BORIS promoter A expression, 760 bp for BORIS promoter B expression and 810 bp for BORIS promoter C expression. All RT-PCR fragments contain splice site allowing distinguishing them from genomic DNA. (B) K562 and Ovcar-8 cells were treated with Actinomycin D for various durations to block transcription. Total RNA was extracted at the indicated time points following the addition of actinomycin D. Quantitative real-time PCR amplification were performed as described in Materials and Methods section to determine copy number of each BORIS alternative transcript. Each data point represents the average of three amplification reactions. RNA degradation curves were obtained by setting at 100% the maximum of mRNA expression at Time 0 before Act D treatment.
Mentions: The fact that BORIS transcription initiated from three different promoters suggested that there would likely be a series of 5′ splice variants. Indeed, we identified five major transcripts that differed in their 5′ noncoding regions but encoded the same protein sequence (Figure 2). The BORIS A1 and BORIS A3 isoforms are expressed from promoter A and have first 180 and 548 bp noncoding exons Ea and Ea+Ea1+Eb, respectively that splice directly to the first 530 bp coding exon, E1. The BORIS A2 splice variant also originates from promoter A, but has two more noncoding exons, Ea1 and Eb, than the BORIS A1 isoform. The BORIS variant that comes from promoter B contains a first 80 bp noncoding exon, Eb, which splices to first 530 bp coding exon, E1. The BORIS C1 isoform is expressed from promoter C and is transcribed as an unspliced first 1190 bp coding exon (Figure 2A). To investigate whether the alternative 5′ UTRs affect mRNA stability, we blocked RNA synthesis by treating cells with ActD. Two cell lines were used in those experiments: K562 cells that express all five alternative 5′ UTRs and Ovcar-8 cells that express only two of five. As shown in Figure 2 (panel B), the half-lives of BORIS, BORIS A1, BORIS A2, BORIS A3 and BORIS C1 transcripts in K562 cells were 4.7, 7.4, 6.9, 7.9 and 6.4 h, respectively, indicating that BORIS mRNAs in cultured cells are relatively stable and long-lived. The alternative transcript expressed from promoter B, BORIS, was 1.5-fold less stable than BORIS transcripts expressed from promoters A and C. The relative turnover of the three alternative 5′ UTRs expressed from promoter A—BORIS A1, BORIS A2 and BORIS A3—was comparable and the rates of degradation were approximately 1.2-fold slower than for the BORIS C1 transcript expressed from promoter C. Similar half-lives of BORIS (4.7 h) and BORIS C1 (6.6 h) were obtained in Ovcar-8 cell line (Figure 2B). In both cell lines, GAPDH mRNA degradation was similar to published values for that gene (34).Figure 2.

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