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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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Related in: MedlinePlus

Transfection of BORIS confirms enrichment in the nucleolus.Live cell imaging of HEK293T cells transiently transfected with GFP-tagged BORIS (green), GFP-tagged CTCF (green) or empty vector (green), counterstained with Hoechst 333412 (blue). Images are shown at 40x magnification.
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pone-0022399-g006: Transfection of BORIS confirms enrichment in the nucleolus.Live cell imaging of HEK293T cells transiently transfected with GFP-tagged BORIS (green), GFP-tagged CTCF (green) or empty vector (green), counterstained with Hoechst 333412 (blue). Images are shown at 40x magnification.

Mentions: To confirm these findings, HEK293T cells were transfected with GFP-tagged BORIS or GFP-tagged CTCF constructs. Using live-cell imaging, GFP was found to accumulate progressively in nucleoli of cells transfected with GFP-BORIS. 71.3% +/− 1.7 of GFP-BORIS transfected cells showed clear nucleolar localization (873 cells analysed in total from two independent experiments). In contrast, cells transfected with GFP-CTCF showed an even distribution of GFP throughout the nucleus, and those cells transfected with an empty GFP vector showed mainly GFP in the cytoplasm (Fig. 6). Furthermore, immunostaining of cells 72 hours after transfection of HEK293T cells with BORIS specific shRNA plasmids, but not empty vector, significantly (p<0.05) reduced the nucleolar immunostaining of BORIS (Fig. S6A, B). This was accompanied by a corresponding reduction in BORIS transcript levels (Fig S7).


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)

Transfection of BORIS confirms enrichment in the nucleolus.Live cell imaging of HEK293T cells transiently transfected with GFP-tagged BORIS (green), GFP-tagged CTCF (green) or empty vector (green), counterstained with Hoechst 333412 (blue). Images are shown at 40x magnification.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022399-g006: Transfection of BORIS confirms enrichment in the nucleolus.Live cell imaging of HEK293T cells transiently transfected with GFP-tagged BORIS (green), GFP-tagged CTCF (green) or empty vector (green), counterstained with Hoechst 333412 (blue). Images are shown at 40x magnification.
Mentions: To confirm these findings, HEK293T cells were transfected with GFP-tagged BORIS or GFP-tagged CTCF constructs. Using live-cell imaging, GFP was found to accumulate progressively in nucleoli of cells transfected with GFP-BORIS. 71.3% +/− 1.7 of GFP-BORIS transfected cells showed clear nucleolar localization (873 cells analysed in total from two independent experiments). In contrast, cells transfected with GFP-CTCF showed an even distribution of GFP throughout the nucleus, and those cells transfected with an empty GFP vector showed mainly GFP in the cytoplasm (Fig. 6). Furthermore, immunostaining of cells 72 hours after transfection of HEK293T cells with BORIS specific shRNA plasmids, but not empty vector, significantly (p<0.05) reduced the nucleolar immunostaining of BORIS (Fig. S6A, B). This was accompanied by a corresponding reduction in BORIS transcript levels (Fig S7).

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