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Loss of Heterochromatin Protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death

Ilaria Filesi1, Alessio Cardinale1, Sjaak van der Sar2, Ian G. Cowell2, Prim B. Singh2,* and Silvia Biocca*,1

1 Department of Neuroscience, University of Roma, Tor Vergata, Via Montpellier 1, 00133 Roma, Italy
2 Nuclear Reprogramming Laboratory, Division of Gene Expression and Development, Roslin Institute (Edinburgh), Midlothian, Scotland, EH25 9PS, UK



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  		Fig. 6. Anti-CD scFvs induce cell death in mammalian cells. (A) The number of Annexin-V- and Hoechst-33342-positive cells were counted in non-transfected cell (nt) population and cells transfected with different scFv fragments (as indicated). Each histogram shows the results obtained by transfection of different cell lines as indicated. The figure shows the average of three different experiments for each cell line used. At least 60 positively transfected cells for each plasmid were counted. (B) The left hand column shows a field of cells transfected with the anti-ßgal scFv and then stained with Annexin V and Hoechst 33342. Arrow points to a single Annexin-V/Hoechst-33342-positive cell that is not transfected. The right hand column shows a field of cells transfected with the 373cyt scFv. Many more cells were positive for Annexin V and Hoechst 33342. Scale bars, 20 µm.

 


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  		Fig. 7. Anti-CD scFvs induce p53-independent cell death, which is partly prevented by Z-VAD fmk treatment. (A) Non-transfected cells (nt) and cells transfected with anti-ßgal, 372nuc and 373nuc scFv fragments (as indicated) were counted for their positive staining with Annexin V and Hoechst 33342. The figure shows the average of three different experiments. At least 60 positively transfected cells for each plasmid were counted and checked. (B) Hoechst-33342-positive cells were counted from non-transfected cells incubated with H2O2 and cells transfected with different scFvs (as indicated) in the presence or in the absence of the caspase inhibitor Z-VAD fmk.

 


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  		Fig. 2. Expression of the 372 and 373 scFvs in E. coli. (A) Western blot analysis of the purified His6-tagged scFv 372 (lane 1) and 373 (lane 2) obtained by affinity chromatography. The arrow points to the scFv protein. The migration of molecular weight markers (in kDa) is shown on the left. (B) An ELISA to measure and compare the binding activity of purified scFv fragments and the parental Ig. A solution of 10 µg/ml of HP1ß-GST protein (dark columns) and 3 mg/ml of lysozyme (light columns) was used for coating. As a negative control, a purified anti-lysozyme D1.3 scFv fragment was used. Values obtained by coating the wells with GST alone have been subtracted. (C) An ELISA to determine the fine specificity of the scFv fragments against recombinant chromodomain-containing proteins HP1ß, HP1{gamma}, HP1{alpha}, mPc1 and the chromodomain (CD) peptide, all fused to GST. Values obtained by coating the wells with GST alone have been subtracted. (D) An ELISA to test competition of anti-chromodomain scFv fragments against the same epitope. Miniwells were coated with soluble purified 372 (upper panel) or the 373 scFv fragment (lower panel). HP1ß-GST protein, which had been previously incubated for 30 minutes at room temperature with or without the purified anti-CD scFv fragments or the non relevant anti-lysozyme D1.3 scFv at a 1:10 molar ratio, was added to the coated wells. The HP1ß-GST bound protein was determined by further incubation with anti-GST antibodies and anti-goat-peroxidase.

 


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  		Fig. 1. The amino-acid sequences of 372 and 373 scFv fragments. The deduced amino-acid sequences of the VK and VH variable regions of 372 and 373 antibodies assembled in a VK-VH single-chain format are shown. Complementarity determining regions (CDRs) and the linker peptides are boxed.

 


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  		Fig. 3. Expression of anti-CD scFvs in mammalian cells. COS cells were transiently transfected with the 372 scFv fragment targeted to the nucleus (372nuc) (A), 372 targeted to the cytoplasm (372cyt) (C) and with the 373nuc (E) and 373cyt (G). Their nuclear localization was determined using anti-myc antibodies. In the corresponding right panel (B,D,F,H) are shown the same cells counterstained with the dye Hoechst 33258, which highlights the A/T-rich repeat sequences present in the prominent heterochromatic regions. Scale bars, 5 µm.

 


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  		Fig. 4. In vivo interaction of anti-CD scFvs with endogenous HP1ß protein. (A) COS cells were transiently transfected with DNA encoding scFv fragments and analysed after 48 hours. A western blot analysis was performed with anti-HP1ß MAC 353 of the insoluble pool from COS fibroblasts transfected with anti-ßgal scFv (lane 1). Western blot of soluble proteins from COS cells transfected with anti-ßgal (lane 2), 372nuc (lane 3) and 373nuc (lane 4) immunopurified using anti-myc 9E10 and then probed with anti-HP1ß monoclonal antibody MAC 353 is shown. The arrow points to the HP1ß protein. (B) Western blot analysis of anti-ßgal (lane 1), 372nuc (lane 2) and 373nuc (lane 3) scFv fragments immunopurified from transfected COS cells, viewed with anti-myc IgG. The migration of molecular weight markers (in kDa) is shown on the left and the heavy ({gamma}) and light (k) chains are indicted on the right. The arrow points to the anti-ßgal scFv fragment (lane 1) and the arrowhead points to the anti-CD scFv fragments (lanes 2 and 3).

 


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  		Fig. 5. Colocalization analysis of anti-CD scFvs with endogenous HP1ß, HP1{alpha}, HP1{gamma} and mPc1 (M33) proteins. Panels (A,E,I,M,Q,U) depict nuclei stained with Hoechst 33258, which preferentially stains the heterochromatin blocks present in mouse nuclei. Panels (B,F,J,N,R,V) are the same nuclei and depict the steady-state distributions of HP1 isotypes and mPc1 (M33) proteins. Panels (B) and (F) represent the distribution of HP1ß; panels (J) and (N) represent the distribution on HP1{alpha}; panel (R) represents the distribution of HP1{gamma} and (V) the distribution of mPc1 (M33). Panels (C,G,K,O,S,W) are the same nuclei and depict the distribution of the 372nuc scFv. Panels (D,H,L,P,T,X) represent the merged images. Scale bars, 5 µm.

 

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