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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01606

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PARP-1 and PARP-2 interact with nucleophosmin/B23 and accumulate in transcriptionally active nucleoli

¹ UPR 9003 du Centre National de la Recherche Scientifique. Laboratoire conventionné avec le Commissariat à l'Energie Atomique, Université Louis Pasteur, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, BP10413, 67412 Illkirch, France
² Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch, France

View larger version (90K): [in a new window]   Fig. 1. Subnuclear distribution of endogenous PARP-2 in human and mouse cells. (A) Confocal sections showing the simultaneous immunodetection of PARP-2 and PARP-1 in HeLa cells fixed with 2% formaldehyde (FA, top), or HeLa cells (middle) and MEFs (bottom) fixed with 1% formaldehyde/0.1% Triton X-100. The morphology of the nucleus is illustrated by differential interference contrast (DIC) images. Merged images of stained PARP-2 and either stained PARP-1 or the DIC image are shown. Inset shows a 2x magnification of the area indicated by the square. To improve the resolution of final images, only the nuclei are displayed as no specific signal above background was detected in the cytoplasm. (B) Cellular fractionation of HeLa cells. 15 µg of each cellular fraction were analyzed by western blotting with antibodies against B23 (bottom), PARP-2 (middle) and PARP-1 (top). WCE, whole cell extract; Cyto, cytoplasmic fraction; Nu, nuclear fraction; Np, nucleoplasmic fraction; No, nucleolar fraction. (C) Confocal sections showing the simultaneous immunodetection of PARP-2 and B23 in HeLa cells (top) or MEFs (bottom) fixed with 1% formaldehyde/0.1% Triton X-100. Merged images of stained PARP-2 and either stained B23 or the DIC image are shown. Bar, 10 µm.

View larger version (50K): [in a new window]   Fig. 4. Identification of the NoLS of mouse PARP-2. (Top) Schematic representation of the recombinant proteins, as described in Fig. 3. The substitution of 4-RRQR-7 to four alanines is indicated in red. Residues 15-50 and 33-46, involved in nuclear targeting, are underlined or boxed in green, respectively. (Bottom) Localization in living PARP-2–/– MEFs of the GFP-fused recombinant proteins (left panels) each labelled as in the scheme above. The corresponding DIC images are presented on the right. Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 2. PARP-1 and PARP-2 accumulate in the nucleolus independently of each other. Wide-field views of the simultaneous immunodetection of PARP-2 and PARP-1 in wild type (top), PARP-1–/– (middle) and PARP-2–/– (bottom) MEFs fixed with 1% formaldehyde/0.1% Triton X-100. DNA is stained with DAPI. Co-immunodetection of PARP-2 and B23 was performed in parallel under the same conditions, only the B23 staining is shown in the right column. Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 3. Localization in living PARP-2–/– MEFs of full-length and truncated versions of mouse PARP-2 fused to the C-terminus of GFP. (Top) Schematic representation of mPARP-2 or truncated versions of mPARP-2 fused to GFP. When indicated, the NLS of SV40-T was inserted in frame between the coding sequence of GFP and the sequence from mPARP-2. Fusion proteins were present (+) or not (–) in the nucleus or the nucleolus. (Bottom) Localization in living PARP-2–/– MEFs of the GFP-fused recombinant proteins (left panels) labelled as in the scheme above. The corresponding DIC images are shown on the right. Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 5. B23 interacts constitutively with PARP-1 and PARP-2 through their DNA binding domains and PARP-1 BRCT domain. (A) Schematic representation of GST-hPARP-1 and GST-hPARP-2. DBD, DNA binding domain. GST (lane 7), GST-hPARP-1 (lanes 2-6) or GST-hPARP-2 (lanes 8-12) were overexpressed in HeLa cells either untreated (lanes 2, 5, 8 and 11) or treated with 1 mM H2O2 for 10 minutes (lanes 3 and 9), 4 mM 3-AB for 3 hours (lanes 4 and 10) or 0.2 µg/ml actinomycin D for 3 hours (ActD, lanes 6 and 12). 10 µg/ml ethidium bromide was added to the lysate (EtBr, lanes 5 and 11). Proteins were analyzed by GST pull down and western blot using anti-B23 and anti-GST antibodies as indicated in the lower panel. Lane 1: input corresponding to 1/30th of the lysate. (B) GST (lanes 2 and 9), GST mPARP-2 (lane 10) or GST-tagged deletion mutants of hPARP-1 (lanes 3-7) or of mPARP-2 (lanes 11-13) were expressed in HeLa cells. Interacting proteins were analyzed by GST pull down and western blotting with anti-B23 and anti-GST antibodies. Input (lanes 1 and 8): 1/30th of the GST-expressing cell lysate. (C) Immunoprecipitation with anti-GFP antibodies of GFP (lane 2), mPARP-21-69-GFP (lane 3) and mPARP-21-69A4-7GFP (lane 4) overexpressed in HeLa cells. Western blots were subsequently probed with a monoclonal anti-B23 antibody (top) and a polyclonal anti GFP antibody. Lane 1: crude extract from 105 HeLa cells.

View larger version (104K): [in a new window]   Fig. 6. PARP-1 and PARP-2 are delocalized from the nucleolus upon RNA pol I transcription inhibition. Wide field views of the simultaneous immunodetection of PARP-2 and PARP-1 in wild-type MEFs treated or not (NT, not treated) with 0.05 µg/ml actinomycin D for 2 hours, 50 µg/ml -amanitin for 2 hours, 100 µM DRB for 1 hour, and in one experiment, followed by two washes with PBS and a 1 hour incubation in fresh medium or 10µM camptothecin (CPT) for 30 minutes. Cells were fixed with 1% formaldehyde/0.1% Triton X-100. DNA is stained with DAPI. Co-immunodetection of PARP-2 and B23 was performed in parallel under the same conditions; only the B23 staining is shown in the right panels. Bar, 10 µm.

View larger version (64K): [in a new window]   Fig. 8. The absence or inhibition of PARP-1 and PARP-2 has no repercussion on nucleolar transcription. Northern blot analysis of total RNAs obtained from the wild type (WT), PARP-1–/– (P1–/–), PARP-2–/– (P2–/–) or wild-type MEFs cultivated for 2 hours in the presence of 5 mM 3-AB (WT+3AB). Cells were either treated or not with the indicated agents. A typical experiment is illustrated. The upper panel shows the autoradiogram after hybridization of a 47S pre-rRNA specific probe; lower panel, staining of the membrane with methylene blue, showing the 28S and 18S rRNA as a loading control.

View larger version (62K): [in a new window]   Fig. 7. PARP-1 and PARP-2 are only moderately delocalized from the nucleolus upon alkylated or oxidative DNA damage. (A) Wide field views of the immunodetection of PARP-2 and PARP-1 in wild-type MEFs treated or not (NT, not treated) with H2O2 (0.2 or 2 mM for 2 hours), MMS (0.2 or 2 mM for 2 hours) or 1 µg/ml 4NQO for 1 hour. Cells were fixed with 1% formaldehyde/0.1% Triton X-100. DNA is stained with DAPI. Co-immunodetection of PARP-2 and B23 in MEF cells treated in the same conditions. Only the B23 staining is shown in the panels on the right. (B) Western blot analyses with anti-PARP-1 or anti-PARP-2 antibodies performed on crude extracts of 2.5x105 MEFs submitted to the indicated treatments or not treated (–). Bar, 10 µm.

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