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Widespread expression of BORIS/CTCFL in normal and cancer cells.

Jones TA, Ogunkolade BW, Szary J, Aarum J, Mumin MA, Patel S, Pieri CA, Sheer D - PLoS ONE (2011)

Bottom Line: Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS.While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable.These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

View Article: PubMed Central - PubMed

Affiliation: Queen Mary University of London, Centre for Neuroscience and Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry, London, United Kingdom.

ABSTRACT
BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a "cancer-testis" antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

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BORIS and CTCF expression in mouse tissues.A, BORIS and CTCF mRNA levels in selected normal mouse tissues. B, Western blotting for BORIS showing major bands (*) at 60–70 KDa and 45 KDa in mouse tissues. C, Western blotting for CTCF showing bands (*) between 70–100 KDa in mouse tissues. D, GAPDH was used as a control for loading differences. GE Healthcare Full Range Rainbow Marker, RPN800E, was used for determination of band size. Error bars in (A) represent the standard deviation
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pone-0022399-g003: BORIS and CTCF expression in mouse tissues.A, BORIS and CTCF mRNA levels in selected normal mouse tissues. B, Western blotting for BORIS showing major bands (*) at 60–70 KDa and 45 KDa in mouse tissues. C, Western blotting for CTCF showing bands (*) between 70–100 KDa in mouse tissues. D, GAPDH was used as a control for loading differences. GE Healthcare Full Range Rainbow Marker, RPN800E, was used for determination of band size. Error bars in (A) represent the standard deviation

Mentions: We then used real-time RT–PCR to determine the levels of BORIS expression in normal mouse tissues including cerebellum, gut, kidney, liver, ovary, spleen and testis. As in human tissues, we found a far lower level of BORIS compared to CTCF in mouse tissues with the exception of the testis (Fig. 3A). Western blotting on a selection of mouse tissues revealed several bands, the most abundant of which were at 60–70 kDa (Fig. 3B). We also detected a band of approximately 48 kDa in the kidney, hippocampus, gut and the cortex, whilst other less intense bands above 200 kDa were present in the testis, spleen, lungs and the liver (Fig. 3B). These higher molecular weight bands may correspond to homo/heterodimer of BORIS, or poly(ADP)-ribosylation as has been described for CTCF [20]. Again, no bands were detected when membranes were probed with BORIS antibodies pre-incubated with free BORIS peptides (Fig. S3), neither did we detect the same bands when membranes were probed with CTCF antibodies (Fig. 3C). Although BORIS protein is clearly detectable, we only detect low levels of BORIS transcripts, both in cells and tissues (Fig 1, Fig 2A and Fig 3A). However, discordance between mRNA level and protein abundance has been reported to be poor for certain other genes [21], [22], [23].


Widespread expression of BORIS/CTCFL in normal and cancer cells.

Jones TA, Ogunkolade BW, Szary J, Aarum J, Mumin MA, Patel S, Pieri CA, Sheer D - PLoS ONE (2011)

BORIS and CTCF expression in mouse tissues.A, BORIS and CTCF mRNA levels in selected normal mouse tissues. B, Western blotting for BORIS showing major bands (*) at 60–70 KDa and 45 KDa in mouse tissues. C, Western blotting for CTCF showing bands (*) between 70–100 KDa in mouse tissues. D, GAPDH was used as a control for loading differences. GE Healthcare Full Range Rainbow Marker, RPN800E, was used for determination of band size. Error bars in (A) represent the standard deviation
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139640&req=5

pone-0022399-g003: BORIS and CTCF expression in mouse tissues.A, BORIS and CTCF mRNA levels in selected normal mouse tissues. B, Western blotting for BORIS showing major bands (*) at 60–70 KDa and 45 KDa in mouse tissues. C, Western blotting for CTCF showing bands (*) between 70–100 KDa in mouse tissues. D, GAPDH was used as a control for loading differences. GE Healthcare Full Range Rainbow Marker, RPN800E, was used for determination of band size. Error bars in (A) represent the standard deviation
Mentions: We then used real-time RT–PCR to determine the levels of BORIS expression in normal mouse tissues including cerebellum, gut, kidney, liver, ovary, spleen and testis. As in human tissues, we found a far lower level of BORIS compared to CTCF in mouse tissues with the exception of the testis (Fig. 3A). Western blotting on a selection of mouse tissues revealed several bands, the most abundant of which were at 60–70 kDa (Fig. 3B). We also detected a band of approximately 48 kDa in the kidney, hippocampus, gut and the cortex, whilst other less intense bands above 200 kDa were present in the testis, spleen, lungs and the liver (Fig. 3B). These higher molecular weight bands may correspond to homo/heterodimer of BORIS, or poly(ADP)-ribosylation as has been described for CTCF [20]. Again, no bands were detected when membranes were probed with BORIS antibodies pre-incubated with free BORIS peptides (Fig. S3), neither did we detect the same bands when membranes were probed with CTCF antibodies (Fig. 3C). Although BORIS protein is clearly detectable, we only detect low levels of BORIS transcripts, both in cells and tissues (Fig 1, Fig 2A and Fig 3A). However, discordance between mRNA level and protein abundance has been reported to be poor for certain other genes [21], [22], [23].

Bottom Line: Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS.While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable.These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

View Article: PubMed Central - PubMed

Affiliation: Queen Mary University of London, Centre for Neuroscience and Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry, London, United Kingdom.

ABSTRACT
BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a "cancer-testis" antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

Show MeSH
Related in: MedlinePlus