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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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Cellular distribution of BORIS in human cell lines and tissues.A, B Immunofluorescence imaging of BORIS or CTCF staining in green (Alexa 488) together with fibrillarin staining in red (Alexa 594) in MRC5 and colorectal cell line HCT116 showing enrichment of BORIS, but not CTCF, within the nucleolus. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. BORIS and fibrillarin are close neighbours with each other and the dense fibrillar component (DFC) of the nucleolus. Images are shown at 100x magnification. C Double immunostaining showing BORIS staining in green (Alexa 488) within the Nucleolin region (red, Alexa 594) in human neural stem cells and glioblastoma cell line CRL2365. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. Images are shown at 100x magnification.
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pone-0022399-g004: Cellular distribution of BORIS in human cell lines and tissues.A, B Immunofluorescence imaging of BORIS or CTCF staining in green (Alexa 488) together with fibrillarin staining in red (Alexa 594) in MRC5 and colorectal cell line HCT116 showing enrichment of BORIS, but not CTCF, within the nucleolus. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. BORIS and fibrillarin are close neighbours with each other and the dense fibrillar component (DFC) of the nucleolus. Images are shown at 100x magnification. C Double immunostaining showing BORIS staining in green (Alexa 488) within the Nucleolin region (red, Alexa 594) in human neural stem cells and glioblastoma cell line CRL2365. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. Images are shown at 100x magnification.

Mentions: Immunofluorescence staining showed that BORIS is located within the cytoplasm and the nucleus of several human cell types, including MRC5 fibroblast, HEK293T embyonic kidney cells, neural stem cells, colorectal, neuroblastoma, melanoma, prostate and glioblastoma cell lines. In all cell types examined, BORIS immunoreactivity was enriched in the nucleolus (Fig. 4 and Fig. S4). Overnight incubation of BORIS antibody with the peptide completely blocked the immunofluorescent signal further confirming the specificity of the BORIS antibodies (Fig. S5). No immunoreactivity was detected when cells were stained with non-specific IgG antibodies. Co-staining of cells for BORIS or CTCF and fibrillarin, a highly conserved protein that is associated with small nucleolar RNAs [24], confirmed enrichment of BORIS but not CTCF in the nucleolus (Fig. 4A,B). Further co-staining showed BORIS within the nucleolar space occupied by nucleolin, an abundant non-ribosomal nucleolar protein [25] (Fig. 4C). Confocal microscopy and 3D reconstruction confirmed that BORIS was within the nucleolus, adjacent to fibrillarin in both normal and cancer cells (Fig. 5A, B and Movies S1, S2, S3 and S4).


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)

Cellular distribution of BORIS in human cell lines and tissues.A, B Immunofluorescence imaging of BORIS or CTCF staining in green (Alexa 488) together with fibrillarin staining in red (Alexa 594) in MRC5 and colorectal cell line HCT116 showing enrichment of BORIS, but not CTCF, within the nucleolus. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. BORIS and fibrillarin are close neighbours with each other and the dense fibrillar component (DFC) of the nucleolus. Images are shown at 100x magnification. C Double immunostaining showing BORIS staining in green (Alexa 488) within the Nucleolin region (red, Alexa 594) in human neural stem cells and glioblastoma cell line CRL2365. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. Images are shown at 100x magnification.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022399-g004: Cellular distribution of BORIS in human cell lines and tissues.A, B Immunofluorescence imaging of BORIS or CTCF staining in green (Alexa 488) together with fibrillarin staining in red (Alexa 594) in MRC5 and colorectal cell line HCT116 showing enrichment of BORIS, but not CTCF, within the nucleolus. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. BORIS and fibrillarin are close neighbours with each other and the dense fibrillar component (DFC) of the nucleolus. Images are shown at 100x magnification. C Double immunostaining showing BORIS staining in green (Alexa 488) within the Nucleolin region (red, Alexa 594) in human neural stem cells and glioblastoma cell line CRL2365. Cells were counterstained with 4′, 6-Diamidino-2-phenylindole (DAPI) shown in blue. Images are shown at 100x magnification.
Mentions: Immunofluorescence staining showed that BORIS is located within the cytoplasm and the nucleus of several human cell types, including MRC5 fibroblast, HEK293T embyonic kidney cells, neural stem cells, colorectal, neuroblastoma, melanoma, prostate and glioblastoma cell lines. In all cell types examined, BORIS immunoreactivity was enriched in the nucleolus (Fig. 4 and Fig. S4). Overnight incubation of BORIS antibody with the peptide completely blocked the immunofluorescent signal further confirming the specificity of the BORIS antibodies (Fig. S5). No immunoreactivity was detected when cells were stained with non-specific IgG antibodies. Co-staining of cells for BORIS or CTCF and fibrillarin, a highly conserved protein that is associated with small nucleolar RNAs [24], confirmed enrichment of BORIS but not CTCF in the nucleolus (Fig. 4A,B). Further co-staining showed BORIS within the nucleolar space occupied by nucleolin, an abundant non-ribosomal nucleolar protein [25] (Fig. 4C). Confocal microscopy and 3D reconstruction confirmed that BORIS was within the nucleolus, adjacent to fibrillarin in both normal and cancer cells (Fig. 5A, B and Movies S1, S2, S3 and S4).

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