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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03297

Journal of Cell Science 119, 5098-5105 (2006)
Published by The Company of Biologists 2006
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Functional interaction between the ZO-1-interacting transcription factor ZONAB/DbpA and the RNA processing factor symplekin

Emma Kavanagh1,*, Michael Buchert2,*, Anna Tsapara1, Armelle Choquet2, Maria S. Balda1,{ddagger}, Frédéric Hollande2 and Karl Matter1,{ddagger}

1 Division of Cell Biology, Institute of Ophthalmology, University College London, London, UK
2 Institut de Génomique Fonctionnelle, Département d'Oncologie Cellulaire et Moléculaire, CNRS UMR5203/INSERM U661/Universités Montpellier I&II, Montpellier, France


Figure 1
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  		Fig. 1. Localisation of symplekin and ZONAB in epithelial cells. HT29-16E cells (A,B), wild-type MDCK cells (C,D), or MDCK cells stably transfected with a cDNA construct encoding flag-tagged full-length symplekin (E), were fixed and permeabilised and then stained with either anti-ZONAB and symplekin antibodies (A-D) or anti-flag antibodies (E). A, C, D and E are epifluorescence images and B shows images taken with a confocal microscope. Note, junctional staining for symplekin was often absent (C) and only observed occasionally (D) in MDCK cells.

 

Figure 2
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  		Fig. 2. Association of ZONAB with symplekin. (A) Detergent extracts of MDCK and Caco-2 cells were precipitated with anti-ZONAB immunobeads or control beads. After washing, the precipitates were analysed by immunoblotting with anti-ZONAB, anti-symplekin and anti-ZO-1 antibodies. (B) Detergent extracts of HT29-16E cells were precipitated using an anti-symplekin antibody, an anti-ZO-1 antibody, or pre-immune serum (control IP) and protein G agarose beads. After washing, the precipitates were analysed by immunoblotting with anti-symplekin and anti-ZO-1 antibodies. (C) A fraction enriched in nuclei was purified by density centrifugation, solubilised and the presence of ZONAB/symplekin complexes was assayed by co-immunoprecipitation as in A. (D) MDCK cell extracts were loaded on beads carrying either a GST-ZONAB fusion protein or GST alone. After washing, the presence of symplekin was analysed by immunoblotting. (E) Recombinant His6-tagged ZONAB was loaded onto beads with bound GST-symplekin or GST. After washing, pull down of recombinant ZONAB was tested by immunoblotting. All extract and input lanes represent 10% of total inputs.

 

Figure 3
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  		Fig. 3. Symplekin modulates transcriptional activity of ZONAB. MDCK cells at 90% density were transfected with the ZONAB-specific reporter constructs together with the indicated expression and RNAi plasmids. (A) Cells were transfected with increasing amounts of a symplekin expression vector. The total DNA concentration was kept constant by adding corresponding amounts of empty expression vector. Luciferases were assayed either 1 or 2 days after transfection. Because ZONAB functions as a repressor in this assay, reduced promoter activity represents increased ZONAB activity. (B,C) The luciferase constructs were transfected together with the indicated expression and RNAi plasmids and the luciferases were assayed 1 day after transfection. Analogous results were obtained when the transfections were left for 2 days. Note that normal ZONAB expression is required for symplekin to affect the promoter (C). Values are derived from representative experiments performed in triplicates. (*Values that are statistically significantly different from control transfections using a t-test.)

 

Figure 4
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  		Fig. 4. Downregulation of symplekin in HT29-16E cells. (A) Regulated depletion of symplekin in the intestinal adenocarcinoma cell line HT29-16E. Clones derived from transfections with either control plasmid or the symplekin RNAi plasmid were analysed after 4 days of culture in tetracycline. The cells were lysed and the expression of the indicated proteins was analysed by immunoblotting. Actin was blotted as a loading control. Note that partial depletion of symplekin led to reduced expression of ZONAB-B. (B) Expression of ZONAB mRNA in HT29-16E cells. Control and symplekin RNAi HT29-16E cells were grown for 4 days in the presence or absence of tetracycline. Total RNA was purified and equal amounts were used for the synthesis of the first strand cDNA according to the manufacturer's instructions (Invitrogen). The level of ZONAB-B mRNA was analysed using semi-quantitative RT-PCR.

 

Figure 5
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  		Fig. 5. Localisation of symplekin and ZONAB in HT29-16E cells. Control and symplekin-depleted HT29-16E cells were fixed and stained for symplekin and ZONAB. Note that in non-depleted cells, much of the stainings for both proteins was nuclear. In depleted cells, ZONAB was primarily in the cytosol and there was only a small amount of nuclear and junctional staining.

 

Figure 6
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  		Fig. 6. Symplekin depletion inhibits ZONAB function. (A) A control HT29-16E clone and two clones expressing symplekin shRNA were grown without (white bars) or with tetracycline (grey bars) and then transfected with the ZONAB-specific luciferase reporter plasmids. Note the stimulation of the promoter upon symplekin depletion, indicating inhibition of the transcriptional activity of ZONAB. Values represent the mean ± s.e.m. of triplicates from three experiments (*P<0.05, Student's t-test compared with untreated clones). (B) Symplekin RiT-A5 clone and a control clone were grown in the absence or presence of the indicated concentrations of tetracycline for the indicated duration. Cells were transfected with equal amounts of reporter plasmids. 24 hours later, cells were lysed and luciferases were assayed. Ratios of the two luciferase activities were then calculated and compared between the different samples. Values are the mean ± s.e.m. of a representative experiment performed in quadruplicate. The results obtained with the symplekin RiT-A5 clone are expressed relative to the activity in the respective (untreated or tetracycline-treated) control sample.

 

Figure 7
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  		Fig. 7. Symplekin depletion inhibits cyclin D1 expression and intestinal cell proliferation. Control and Symplekin RiT cells were grown for 4 days with (grey bars) or without (white bars) addition of tetracycline. (A) Cyclin D1 mRNA expression was quantified using qRT-PCR from total RNA, relative to the expression of GAPDH mRNA. Values are the average from three experiments performed on two symplekin-depleted clones. Since addition of tetracycline in control clones resulted in a slight increase of cyclin D1 mRNA expression, results are expressed relative to the expression in their respective (untreated or tetracycline-treated) control (*P<0.05 compared with untreated cells, Student's t-test). (B) Proliferation was determined by measuring absorbance at 595 nm after cells were stained with Crystal Violet and solubilised in 1% SDS. Shown is a representative experiment performed in quadruplicates (*P<0.05 compared with untreated clones, Student's t-test). (C) Apoptosis was measured using the TUNEL assay (Roche Diagnostics) in Sym RiT cells before (-TC) and after (+TC) treatment with tetracycline, as well as DNase-treated positive controls (pos). Bar, 20 µm.

 

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© The Company of Biologists Ltd 2006