QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 May 2006
doi: 10.1242/jcs.02965

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Luciani, J. J. Articles by Mattei, M.-G. Search for Related Content PubMed PubMed Citation Articles by Luciani, J. J. Articles by Mattei, M.-G.

PML nuclear bodies are highly organised DNA-protein structures with a function in heterochromatin remodelling at the G2 phase

¹ Inserm, Université de la Méditerranée, UMR491, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille, France
² TIMC UMR5525 CNRS, Faculté de Médecine, 38706 La Tronche, France
³ Département de Génétique Médicale, Université Saint Joseph, 5076 Beirut, Lebanon
⁴ Service de Génétique Médicale, Hôpital Arnaud de Villeneuve, 34059 Montpellier, France
⁵ Heinrich Pette Institute for Experimental Virology and Immunology, 20206 Hamburg, Germany
⁶ Département de Génétique Médicale, Hôpital d'enfants de la Timone, 13385 Marseille, France
⁷ Laboratoire de Génétique Oncologique, Institut Gustave Roussy, 94805 Villejuif, France

View larger version (68K): [in a new window]   Fig. 1. Distribution of HP1 protein after immunostaining of ICF and control cells. (A) ICF nuclei and (B) control nuclei at G1 and G2 phases. At the G2 phase, there is an aberrant giant HP1 body in ICF nuclei (HP1 alpha, red; nuclei, blue with DAPI).

View larger version (75K): [in a new window]   Fig. 2. Protein distribution in ICF and control G2 nuclei. (A) ATRX and PML proteins colocalise in the giant HP1-PML body and normal PML-NBs. A weak diffuse ATRX signal is also detectable in the nucleoplasm of ICF and control nuclei (PML, green; ATRX, red; nuclei, blue with DAPI). (B) Other proteins colocalise within the giant body as shown on ICF nuclei immunostained with antibodies against PML and DAXX, PML and BLM, PML and TOPOIII, CBP and SUMO.

View larger version (37K): [in a new window]   Fig. 3. The 1qh satellite DNA is included in the giant HP1-PML body in ICF cells. (A) Two-dimensional fluorescent microscopy: a giant body, identified with anti-PML antibody (blue), colocalises with 1qh satellite DNA (1qh sat, green), adjacent to the centromeric DNA (cen 1, red); (B) schematic representation. (C) Three-dimensional reconstruction: transverse section of three giant bodies after immuno-FISH with anti-PML antibody (red) and 1qh satellite DNA probe (green).

View larger version (62K): [in a new window]   Fig. 4. In control G2 cells, the normal PML-NBs colocalise with juxtacentromeric satellite DNA, adjacent to the centromeric DNA. Immuno-FISH experiments demonstrate that PML protein signals (red) (A) are adjacent to the pan-centromeric DNA signals (cen, green) and (B) colocalise with the 1qh and 9qh satellite (1qh sat and 9qh sat) DNA signals (green). (C) Histogram showing the percentage of 1qh (M=24.64), 9qh (M=25.47), 16qh (M=6.5) and 15ph (M=4.44) satellite DNAs that colocalise with PML-NBs in three control subjects (C1, C2, C3) at G2 phase. The colocalisations have been scored from the analysis of 100 G2 cells and compared with those occurring between PML-NBs and chromosome 1 centromeric DNA, which is excluded from PML-NBs; P values were calculated by using Fisher's exact test, one sided (http://www.matforsk.no/ola/fisher.htm). M, mean.

View larger version (29K): [in a new window]   Fig. 5. In the giant body, protein signals are round shaped. Either they have a pale central core (PML, BLM, TOPOIII, SP100, DAXX, ATRX) or they are homogeneously stained (HP1ß). These different patterns can be observed by decomposition into colours and enlargement (x2.8) of the protein signals colocalising in the giant body. (A) Triple immunostaining of an ICF nucleus showing that the DAXX layer (red) is larger than the ATRX layer (blue), which in turn is larger than the HP1ß layer (green). (B-D) Triple immunostaining showing respectively that BLM, TOPOIII and SP100 layers (red) are located between PML (green) and DAXX (blue) layers.

View larger version (39K): [in a new window]   Fig. 6. Three-dimensional reconstruction of giant HP1-PML bodies. (A) Triple immunostaining showing PML (blue), HP1ß (green) and SP100 (red). In the middle row, the left reconstruction shows the body viewed with a slight pitch, the centre reconstruction shows the equatorial plane corresponding to the transverse sections shown in the other rows, and the right reconstruction shows the transverse section with simultaneous rendering of the three fluorescent signals. The top row shows the transverse section with the various combinations of pairs of signals. The bottom row shows the transverse section with the signals alone: left, PML sphere; centre, SP100 smaller sphere; right, HP1 small bowl. (B) Triple immunostaining showing DAXX (red), ATRX (blue) and HP1ß (green), in transverse section showing the various combinations of pairs of signals.

View larger version (43K): [in a new window]   Fig. 7. Model of DNA-protein organisation in the PML-NBs at the G2 phase. (A) Transverse section of a PML-NB. (B) Detail of the different protein layers. The numbers indicate the following references: 1 (Paull et al., 2001; Cable et al., 2003); 2 (Zhao et al., 2000); 3 (Le Douarin et al., 1996; McDowell et al., 1999); 4 (Xue et al., 2003; Tang et al., 2004); 5 (Jang et al., 2002); 6 (Sternsdorf et al., 1997b); 7 (Eladad et al., 2005); 8 (Müller et al., 1998). The arrows for the references correspond to protein-protein or protein-DNA interactions (straight bi-headed arrows) and to protein-sumoylation (curved arrows).

View larger version (70K): [in a new window]   Fig. 8. In ICF cells, the integrity of the giant PML body is not affected by DNAse treatment. (A) Two pale DAPI-stained digested nuclei immunostained with anti-PML (green) and then with anti-lamin B (red) Abs to be easily detected. (B) One pale digested nucleus co-immunostained with anti-PML (green) and anti-BRCA1 (red) Abs. (C) One digested nucleus co-immunostained with anti-PML (green) and anti-HP1 (red) Abs.

View larger version (75K): [in a new window]   Fig. 9. Distribution of the BRCA1 protein in the giant body and normal PML-NBs. (A) BRCA1 colocalises with all giant bodies, varying from a dot to a full bowl. (B) BRCA1 colocalises with a small number of normal PML-NBs (BRCA1, red; PML, green; nuclei, blue with DAPI).

View larger version (29K): [in a new window]   Fig. 10. Schematic representation of the chromatin-remodelling function exerted by PML-NBs on the juxtacentromeric satellite DNA. Chromatin-remodelling proteins and satellite DNA transiently colocalise within the PML-NBs during G2 phase. The mean percentages of colocalisation, obtained from the analysis of 100 cells of three control subjects (C1, C2, C3) at the S, G2 and M phases are respectively 8.5%, 24.6%, 6.1% for 1qh, and 8.3%, 25.4%, 5.8% for 9qh. These results support a chromatin-remodelling function of PML-NBs specific to the G2 phase (sat DNA, satellite DNA; cen, centromeric DNA).