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First published online 4 April 2006
doi: 10.1242/jcs.02890

Journal of Cell Science 119, 1746-1759 (2006)
Published by The Company of Biologists 2006
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Targeting of CTCF to the nucleolus inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism

Verónica Torrano1, Joaquín Navascués2,*, France Docquier3, Ru Zhang4, Les J. Burke4, Igor Chernukhin3, Dawn Farrar3, Javier León1, María T. Berciano2, Rainer Renkawitz4, Elena Klenova3, Miguel Lafarga2 and M. Dolores Delgado1,{ddagger}

1 Grupo de Biología Molecular del Cáncer, Departamento de Biologia Molecular, Universidad de Cantabria, 39011-Santander, Spain
2 Departamento de Anatomía y Biologia Celular, Unidad de Biomedicina-CSIC, Universidad de Cantabria, 39011-Santander, Spain
3 Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK
4 Institute for Genetics, Justus-Liebig-Universitaet Giessen, Heinrich-Buff-Ring 58-62, D-35392 Giessen, Germany


Figure 1
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  		Fig. 1. Nucleolar localization of endogenous CTCF in human myeloid cells induced to differentiate. (A) Indirect immunofluorescence showing CTCF nucleolar localization during induced differentiation of K562 cells. (a-d) Control undifferentiated K562 cells; (e-h) K562 cells treated with 1 µM 1-ß-D-arabinofuranosylcytosine (Ara-C) for 3 days to induce erythroid differentiation; (i-l) K562 cells treated with 100 nM staurosporine (STA) for 3 days to induce megakaryocytic differentiation. After induction of differentiation, cells were immunostained with the anti-CTCF monoclonal antibody. The images at low magnification show accumulation of CTCF in nucleoli after induction with Ara-C or STA (a,e,i). The detailed images show co-localization of CTCF (b,f,j; green channel) and B23 (c,g,k; red channel); d,h,l are the merged images of b and c, f and g and j and k, respectively. Bars, 40 µm in the low magnification images and 10 µm in the high magnification images. (B) Differential interference contrast (DIC) images showing the morphology of the nucleus (Nu) and isolated nucleoli (NO) from K562 cells (left panel). The purity of the nucleolar fraction was assessed by immunostaining with the anti-UBF antibody (right panel). (C) Western analysis of nucleolar fractions isolated from undifferentiated K562 cells and K562 cells induced into erythroid differentiation. Cell fractions were obtained, resolved on the SDS-PAGE, blotted and probed. Western analysis of nucleolar fractions isolated from undifferentiated K562 cells (Cont) and cells treated with 1 µM Ara-C for 5 days was performed with the anti-CTCF antibody (upper panel) and the anti-UBF antibody (lower panel). The developed films were scanned and quantified. In the NO fractions, the ratio of the intensity of the CTCF bands over the intensity of the corresponding UBF bands revealed a 2.4-fold increase of CTCF expression in the Ara-C fraction with respect to control. Cyto, cytoplasmic fraction; Nu, nuclear fraction; NO, nucleolar fraction; Total, whole cell extract.

 

Figure 2
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  		Fig. 2. Nucleolar localization of endogenous CTCF in human breast cancer cells treated with sodium butyrate. (A) Indirect immunofluorescent staining showing CTCF nucleolar localization following treatment of MCF7 cells with sodium butyrate (NaBu). After induction with 5 mM NaBu for 8 hours, MCF7 cells were immunostained with the anti-CTCF rabbit polyclonal antibody (a,d) or stained with DAPI (b,e); (c,f) merged images of CTCF and DAPI staining. (a-c) Untreated MCF7 cells (control); (d-f) MCF7 cells treated with NaBu. Bar, 10 µm. (B) Nucleolar fractionation and western analysis of MCF7 cells treated with NaBu. MCF7 cells were treated with 5 mM sodium butyrate for 8 hours, nucleolar fractions were isolated and western analysis with the rabbit polyclonal anti-CTCF antibody was performed. Total, whole cell extract; Np, nucleoplasmic fraction; NO, nucleolar fraction; Cyto, cytoplasmic fraction.

 

Figure 3
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  		Fig. 3. Nucleolar localization of endogenous CTCF in rat neurons in vivo. (A) Confocal laser microscopy following double immunofluorescent staining with anti-CTCF rabbit polyclonal antibody (left panel) and anti-B23 antibody (right panel) in differentiated neurons from rat trigeminal ganglia. Bar, 10 µm. (B) Immunoelectron localization of CTCF in the nucleolus of trigeminal ganglion neurons. Note the typical reticulated configuration of the nucleolus with its granular component (GC), dense fibrillar component (DFC) and fibrillar centres (FC). Gold particles indicating CTCF immunoreactivity preferentially decorated the dense fibrillar component of the nucleolus of rat neurons. Bar, 200 nm.

 

Figure 4
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  		Fig. 4. The zinc-finger region of CTCF targets it to the nucleolus. (A) The distribution of the various GFP-CTCF fusion proteins, as described in C, in K562 cells. K562 cells were transfected with the indicated GFP-CTCF truncated variants, cells were fixed 24 hours post-transfection and green fluorescence was analyzed by confocal microscopy. Bar, 5 µm. (B) The distribution of GFP-CTCF fusion proteins in UR61 cells. UR61 cells were transfected with the indicated GFP-CTCF truncated variants (as in C), cells were fixed 24 hours post-transfection and stained with propidium iodide. (a-d) green fluorescence from GFP-CTCF fusion proteins; (e-h) propidium iodide staining labelling nucleoli. Bar, 5 µm. (C) Schematic representation of the full-length and truncated versions of CTCF fused to the C terminus of GFP. Numbers denote amino acid positions. The summary of the nucleolar localization for the different GFP-CTCF fusion proteins in K562, UR61 and HeLa cells is shown. Fusion proteins were present (+) or not (-) in the nucleolus as indicated; (++), strong nucleolar accumulation; (±), weak signal; ND, not determined.

 

Figure 5
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  		Fig. 5. Nucleolar localization of CTCF is dependent on the transcription from rDNA. UR61 cells were transfected with the full-length CTCF fused with the GFP (GFP-CTCF) and either left untreated (A) or treated 24 hours post-transfection with 0.05 µg/ml actinomycin D (ActD) for 1 hour to specifically inhibit RNA pol I (B). Cells were fixed, immunostained for UBF, fibrillarin or B23 as indicated. Nucleic acids were visualized with propidium iodide (PI). The localization of the GFP-CTCF fusion protein (a,d,g,j; green channel), the markers for the different nucleolar compartments (b,e,h,k; red channel) and the merged images (c,f,i,l) are shown. Confocal images show that GFP-CTCF is released from the nucleolus upon inhibition of transcription by RNA pol I. Bar, 20 µm in a; 10 µm in b-l.

 

Figure 6
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  		Fig. 6. Nucleolar localization of the zinc-finger region of CTCF is independent of the transcription from rDNA. UR61 cells were transfected with the construct harbouring the central zinc-finger domain of CTCF fused with the GFP (GFP-ZF) and either left untreated (A) or treated 24 hours post-transfection with 0.05 µg/ml actinomycin D (ActD) (B) for 1 hour to specifically inhibit RNA pol I. Cells were fixed and immunostained for UBF, fibrillarin or B23 as indicated. Nucleic acids were visualized with propidium iodide (PI). The localization of the GFP-ZF fusion protein (a,d,g,j; green channel), the markers for the different nucleolar compartments (b,e,h,k; red channel) and the merged images (c,f,i,l) are shown. Confocal images show that GFP-ZF remains in the nucleolus following the inhibition of RNA pol I transcription. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Full-length CTCF, but not CTCF-ZF localization in the nucleolus requires protein synthesis. UR61 cells were transfected with the GFP-CTCF full length (GFP-CTCF; a,b,e,f) or with the GFP-CTCF-zinc-finger (GFP-ZF; c,d,g,h). 24 hours post-transfection cells were treated with 20 µg/ml cycloheximide (Chx) for 3 hours to inhibit protein synthesis. Untreated cells served as control. Cells were then fixed and analyzed by confocal microscopy for green fluorescence (a-d). Nucleoli were stained with propidium iodide (e-h). GFP-CTCF fusion protein, but not GFP-ZF, is delocalized from the nucleolus upon protein synthesis inhibition with cycloheximide. Bar, 5 µm.

 

Figure 8
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  		Fig. 8. Full-length CTCF, but not CTCF-ZF, inhibits nucleolar transcription. UR61 cells were transfected with the GFP-CTCF full length (GFP-CTCF; a-c,d-f), the GFP-CTCF-zinc-finger (GFP-ZF; g-i) or the pEGFP vector (GFP; j-l). 24 hours post-transfection cells were pulse-labelled with 5'-fluorouridine (FU) for 5 minutes (b) or 10 minutes (e,h,k). Cells were then fixed and the sites of 5'-FU incorporation revealed with an anti-BrdU antibody and a Texas Red-conjugated secondary antibody. The localization of the GFP-fusion proteins (a,d,g,j; green channel), the 5'-FU incorporation (b,e,h,k; red channel) and the merged images (c,f,i,l) are shown. Confocal images show that GFP-CTCF, but not GFP-ZF or GFP inhibits 5'-FU incorporation into nascent RNA. Bar, 10 µm.

 

Figure 9
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  		Fig. 9. Quantification of nucleolar localization of the GFP fusion proteins and FU incorporation in nucleoli, in untreated cells and cells treated with ABA. UR61 cells, untreated (Control, upper panel) and treated with 3-aminobenzamide for 8 hours (ABA, lower panel) were transfected with GFP-CTCF, GFP-ZF and GFP. 24 hours post-transfection, cells were pulse-labelled with 5'-FU for 10 minutes and cells showing nucleolar localization of the GFP fusion proteins (bars on the left) and cells showing 5'-FU incorporation in nucleoli (bars on the right) were scored. Data show the percentage of cells (mean ± s.e.m.) from three experiments.

 

Figure 10
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  		Fig. 10. Inhibition of PARPs impairs nucleolar localization of CTCF-full length, but not CTCF-ZF. UR61 cells were treated for 8 hours with 3-aminobenzamide (ABA) and then transfected with the GFP-CTCF full length (GFP-CTCF; a-c,d-f,g-i), or with the GFP-CTCF-zinc-finger domain (GFP-ZF; j-l). 24 hours post-transfection cells were analyzed by confocal microscopy for green fluorescence (a,d,g,j). Nucleoli were stained with propidium iodide (PI; b), or immunostained for UBF (e). Cells were also pulsed with 5'-FU for 10 minutes (h,k); merged images are also shown (c,f,i,l).

 

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© The Company of Biologists Ltd 2006