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First published online 12 September 2007
doi: 10.1242/jcs.012914

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Plasticity of HP1 proteins in mammalian cells

¹ The Stem Cell and Chromatin Group, Laboratory of Biology, The University of Ioannina, School of Medicine and The Institute of Biomedical Research (FORTH/BRI), 45 110 Ioannina, Greece
² Advanced Light Microscopy Facility (ALMF), European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
³ Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609, USA
⁴ Tissue Fibrosis and Remodelling Group MRC/University of Edinburgh Centre for Inflammation Research, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK
⁵ The Division of Tumor Biology, Department of Immunology and Cell Biology, Forschungszentrum Borstel, D-23845 Borstel, Germany

View larger version (42K): [in this window] [in a new window]   Fig. 1. Distribution of endogenous HP1 proteins in human and mouse cell lines. (A) Staining of HeLa and C127 cells with antibodies (Ab) to HP1, HP1 and HP1. Propidium iodide (PI) staining of the cells is also shown. Arrows indicate major heterochromatic foci. (B) Double staining of the same cells with anti-HP1 (red) and anti-HP1 (green) antibodies. (C) Double immunofluorescence as in B using anti-HP1 (green) and anti-me3K9-H3 (red) antibodies. Arrows in the enlarged images indicate lack of colocalization. (D) Detail of HP1 and HP1 foci (green) counter-stained with PI (red) at high contrast and higher magnification. nu, nucleolus. (E) Relative fluorescence intensity per unit of nuclear surface (I/A; in arbitrary units) in antibody-stained cells. (F) Average number of HP1 and HP1 foci (3 µm or greater in diameter) in HeLa and C127 cells as detected after morphometric analysis. Successive optical sections and `projections' from 15 cells were analyzed in each case. Bars, 5 µm. Note: In this and all subsequent figures, merged images are displayed in color, whereas individual red and green profiles are reproduced in grayscale to minimize differences in the visual perception of the red and green color.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Differential stability of HP1 assemblies to detergent extraction and RNase digestion. (A) Partitioning of HP1 proteins after permeabilization with Triton X-100 and subsequent treatment with RNase A, as assessed by western blotting. Lanes: W, whole cell lysate; Pt, insoluble residue; S1, material released by Triton; S2, material released by the subsequent RNase digestion. (B) Distribution of the nucleolar protein B23 (control) after treatment with Triton alone or Triton followed by RNase. Note that the protein is not removed from the nucleoli by the detergent alone, but is fully removed after a combined Triton and RNAse treatment. Only merged images are shown (green: antibody staining; red: PI). (C-E) Distribution of HP1 proteins under conditions similar to those described in (B). A gallery of merged images in `projection' mode are shown on the left (green: antibody staining; red: PI), and selected and highly magnified sections are depicted on the right. Arrows indicate the cells from which these sections were taken and asterisks denote the position of nucleoli. (F) In situ binding of recombinant HP1 proteins (0.25 µg/ml) to Triton-ghosts and Triton/RNase A-ghosts. For each protein, antibody staining (anti-GST) is presented on the left, and the merged image after counter-staining with PI is depicted on the right. The profiles are of C127 cells. Analogous experiments with HeLa cells yield similar results. Bar, 5 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 3. Distribution of HP1, HP1, HP1 and me3K9-H3 in mouse ES cells. (A) The colored panels on the left show alkaline phosphatase staining, while the gallery on right depicts propidium iodide (PI) and antibody stained (Ab) E14 cells maintained in the presence (+) or absence (–) of LIF. Arrows indicate cells with a speckled pattern. (B) Some of the cells depicted in A magnified 3x. SP, speckled pattern; D, diffuse, `microgranular' pattern. (C) High contrast and high magnification images of HP1, HP1 and HP1 foci (green) counter-stained with PI (red). (D) Morphometric data depicting the proportion of cells exhibiting a speckled phenotype, the average number of foci per cell and the relative size of foci (ratio of particles measuring 2 µm to particles measuring 3-6 µm) in each sample. The data represent averages from at least 50, optically sectioned cells. (E) High contrast and high magnification images of double staining of undifferentiated and differentiated E14 cells with antibodies to HP1 (green) and me3K9-H3 (red). Bars, 5 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Fate and dynamics of newly expressed HP1-GFP proteins in human and mouse cell lines. (A) Distribution of HP1-GFP proteins expressed in HeLa and C127 cells. Representative examples of the `diffuse' (D, white asterisks) and the `speckled' (SP, blue asterisks) phenotypes are shown. (B) Relative distribution of HP1-GFP and me3K9-histone H3 (as detected by a specific antibody) in the two cell types. Only merged images are shown (HP1-GFP: green, me3K9-H3: red). Arrows indicate sites where the two markers do not coincide. (C) Relative fluorescence intensity per unit of nuclear surface (I/A; in arbitrary units) in samples of transfected cells. Successive optical sections and `projections' from at least 15 cells were analyzed in each case. (D) Profiles of HeLa and C127 cells transfected with a chromodomain containing (CD-GFP) and a chromodomain-lacking (CD-GFP) mutant of HP1. Observe that CD-GFP assembles correctly in mouse cells, but remains diffuse in human cells. (E) Distribution of HP1-GFP proteins in the same cell lines used in (A) after S phase block with hydroxyurea. Note that HP1/HP1 are diffuse in human cells, but continue to form large foci in mouse C127 cells. Also notice that the targeting of HP1-GFP in the mouse system is not affected by S phase blocking. (F) FRAP data from transfected HeLa and C127 cells. Fluorescence recovery half-times (t1/2) are in seconds. rt1/2 is the ratio of the half-times in the two cell types; im. fr. is the immobile fraction. Bars, 5 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Effect of HAT and HDAC inhibitors on HP1 localization. (A) Distribution of HP1-GFP and HP1-GFP in C127 cells treated with buffer (NT), curcumin (Cu), Trichostatin A (TSA) and sodium butyrate (NaBu). Note that HDAC inhibitors cause randomization of HP1 proteins in a subset (30%) of cells. The same results are obtained with HeLa cells (not shown). (B) FRAP data from cells treated with NaBu and exhibiting the diffuse (D) phenotype. Fluorescence recovery half-times (t1/2) are in seconds; im. fr. is the immobile fraction. Note that the immobile fraction disappears and the fluorescence recovery half-times become shorter in comparison to those of non-treated cells (see Fig. 4F). Bar, 5 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 6. Distribution and dynamics of HP1 proteins in wild-type and mutant MEFs. (A) Localization of endogenous HP1, HP1, HP1 and me3K9-histone H3 in wild-type MEFs (WT) and HP1-HP1 double null mutants (–/–). Propidium iodide (PI) and antibody staining (Ab) are shown. (B) Western blot on WT and –/– cells with the three anti-HP1 antibodies. (C) Distribution of HP1-GFP protein variants in the two cell types used above. Propidium iodide (PI) and GFP fluorescence are shown. Notice the different distribution of HP1-GFP in WT and –/– cells. (D) The average number of HP1 foci (2 µm or greater in diameter) in transfected cells. (E) FRAP data from transfected WT and –/– cells. rt1/2 is the ratio of the half-times in the two cell types; im. fr. is the immobile fraction. Bar, 5 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Hypothetical model explaining HP1 plasticity. (A) Characteristic patterns of HP1 proteins in different cell types and developmental states that could be used as tools for epigenetic cell typing. Images collected in the course of this study have been artistically modified to highlight the variations in HP1 localization. (B) Potential microscopic states and equilibria involving HP1 proteins. The central concept in this model is that HP1 proteins are involved in multiple interactions, including self-association and binding to chaperones or assembly factors (gray circle S). A small fraction of HP1 (black circles with inner green circle) binds stably to chromatin, whereas a greater fraction (red, blue and green circles) associates weakly with chromatin territories through me3K9-H3 or pre-existing HP1. A sub-population of HP1 molecules `scan' chromatin in a perpetual fashion, whereas another proportion remain freely diffusible in the nucleoplasm. For more details see Discussion.

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