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First published online 30 January 2007
doi: 10.1242/jcs.03357

Journal of Cell Science 120, 638-647 (2007)
Published by The Company of Biologists 2007
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hCAF1, a new regulator of PRMT1-dependent arginine methylation

Yannis Robin-Lespinasse, Stéphanie Sentis, Chloé Kolytcheff, Marie-Claude Rostan, Laura Corbo* and Muriel Le Romancer

Inserm Unit U590, Centre Léon Bérard, 28 Rue Laënnec, 69373 Lyon Cedex 08, France and Université Claude Bernard Lyon 1, Lyon, France


Figure 1
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  		Fig. 1. Distribution of PRMT1 and hCAF1 in MCF-7 cells. Confocal fluorescence microscopy experiments showing the subcellular distribution of endogenous PRMT1 and hCAF1. (A) PRMT1 was visualized using a monoclonal anti-PRMT1 antibody in MCF-7 cells (a) control or transfected with (b) scramble siRNA duplexes or (c) specific siRNA PRMT1 duplexes. The corresponding phase-contrast micrographs are shown in panels a'-c'. (B) Suppression of endogenous PRMT1 was checked by western blotting with polyclonal anti-PRMT1. A loading control was performed with an anti-GAPDH antibody. (C) Immunofluorescence analysis using a rabbit polyclonal anti-hCAF1 antibody in MCF-7 cells (a) control or transfected with (b) scramble siRNA duplexes or (c) specific siRNA hCAF1 duplexes. The corresponding phase-contrast micrographs are shown in panels a'-c'. (D) Suppression of endogenous hCAF1 was checked by western blotting. A loading control was performed with an anti-GAPDH antibody.

 

Figure 2
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  		Fig. 2. PRMT1 colocalizes and interacts with hCAF1. (A) Laser-scanning confocal microscopy was used to compare the distributions of (a) hCAF1 and (b) PRMT1 in MCF-7 cells using the polyclonal anti-hCAF1 and the monoclonal anti-PRMT1 antibodies. The corresponding overlay is shown in panel c and the corresponding phase-contrast micrograph is shown in panel d. (B) MCF-7 cell extract was immunoprecipitated using anti-hCAF1 polyclonal antibody or non-immune serum (NIS). Total cell lysate (input) as well as immunoprecipitants (IP) were analyzed by western blotting using the polyclonal anti-PRMT1 or anti-hCAF1 antibodies. Input represents 4% of the total cell extract used for immunoprecipitation experiments. (C) Direct interaction between hCAF1 and PRMT1 was analyzed by GST-pull-down experiments. 35S-labeled in vitro translated hCAF1, BTG1 and luciferase (left) or deletion mutants (right) were incubated with GST-PRMT1–glutathione-Sepharose beads. The eluted proteins and 1/50 of input radiolabeled proteins were analyzed by SDS-PAGE and visualized by autoradiography.

 

Figure 3
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  		Fig. 3. Colocalization of hCAF1 and PRMT1 with nuclear speckle marker proteins is sensitive to AMD treatment. Laser-scanning confocal microscopy of double-labeling experiments before and after treatment with AMD (5 µg/ml). Distribution in MCF-7 cells of endogenous hCAF1 (a) and PRMT1 (g) along with specific compartment proteins such as SC35 (b) and Sf3b (h) was analyzed by double-labeling experiments using laser-scanning confocal microscopy. Panels (c) and (i) show overlays of the two signals. Distribution of hCAF1 (d) and PRMT1 (j) along with SC35 (e) and Sf3b (k) on MCF-7 cells treated with AMD (5 µg/ml). The corresponding overlays are shown in panels (f) and (l).

 

Figure 4
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  		Fig. 4. Effect of hCAF1 and BTG1 on PRMT1 enzymatic activity in vitro. GST-PRMT1 (1 µg) was incubated with 1 µg of (A) GST-Sam68P3, (B) GST-hnRNPA1 or (C) recombinant histone H4 in the presence of [3H]AdoMet without or with increasing amounts of GST-hCAF1, GST-BTG1 or GST for 90 minutes at 37°C. The reaction mixtures were resolved on SDS-PAGE and visualized by autoradiography. (D) GST-PRMT1 (1 µg) was incubated with 1 µg of recombinant histone H4 in the presence of cold AdoMet without or with increasing amounts of GST-hCAF1, GST-BTG1 or GST for 90 minutes at 37°C. The reaction mixtures were resolved on SDS-PAGE and revealed by western blot using the anti-dimethyl-Histone H4 (Arg3) antibody. Results are representative of several individual experiments. (E) GST-PRMT1 (1 µg) was incubated with [3H]AdoMet, 20 µg of hypomethylated extracts prepared from MCF-7 cells without or with increasing amounts of GST-hCAF1 or GST as described above. The reaction mixtures were resolved on SDS-PAGE and visualized by autoradiography. A longer exposition of the gel is shown in the right-hand panel.

 

Figure 5
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  		Fig. 5. hCAF1 regulates asymmetric dimethylation of proteins in MCF-7 cells. (A) Lysates of MCF-7 cells transfected either with control (scramble) or specific hCAF1 or PRMT1 siRNA duplexes were analyzed by western blotting using the pan antibody ASYM24. Results are representative of several individual experiments. (B) Quantification of arginine dimethylated bands revealed with ASYM24 antibody. Data represent the changes in arginine dimethylated bands after hCAF1 and PRMT1 knockdown, expressed as the ratio of the quantity present in knockdown cells to that found in cells treated with scrambled control siRNA. Data representing the mean of three independent experiments are shown with error bars. (C) Western blot analysis showing the expression of the indicated proteins in MCF-7 cell extracts after transfection with control (scramble) or hCAF1 or PRMT1 siRNA duplexes. GAPDH served as a loading control. (D) Confocal fluorescence microscopy experiments showing the subcellular distribution of arginine dimethylated proteins in MCF-7 cells transfected either with control (scramble) (a) or specific hCAF1 (b) or PRMT1 (c) siRNA duplexes revealed with ASYM24 antibody.

 

Figure 6
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  		Fig. 6. hCAF1 regulates Sam68 and histone H4 methylation in MCF-7 cells. (A) Extracts from MCF-7 cells transfected with scramble or specific hCAF1 siRNA duplexes or treated with Adox were immunoprecipitated using anti-Sam68 antibody, then analyzed by western blotting with ASYM24 after stripping with anti-Sam68 antibody. (B) Acid-extracted histones from MCF-7 cells transfected either with scramble or specific hCAF1 or PRMT1 siRNA duplexes were resolved on SDS-PAGE and revealed by western blotting using the anti-dimethyl-Histone H4 (Arg3) antibody.

 




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