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Cell differentiation induces TIF1ß association with centromeric heterochromatin via an HP1 interaction

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP163, 67404 Illkirch-Cedex, France

View larger version (61K): [in a new window]   Fig. 1. F9 cell differentiation is accompanied by a specific change in the subnuclear distribution of TIF1ß. (A) Phase-contrast photomicrographs of F9 EC cultures showing undifferentiated F9 stem cells (control) and differentiated endoderm-like cells after 4 day exposure to 1 µM retinoic acid (RA). (B) Confocal images of single optical sections through the nucleus of vehicle-treated (control) and RA-treated F9 cells. The left panels show the Hoechst DNA staining, which highlights A/T-rich repeat sequences present in the centromeric heterochromatin (bright blue patches), and the right panels correspond to immunodetection with specific monoclonal antibodies (Abs), as indicated. Identical results were obtained using two different TIF1ß-specific Abs (PF64 and 1Tb3) raised against distinct epitopes. Bars, 5 µm. (C) TIF1ß colocalizes with all HP1-containing foci in the nuclei of RA-treated F9 cells. Cells were double-labeled with Abs against HP1 (green) and TIF1ß (red). (D) Time course of TIF1ß heterochromatin association following RA addition. F9 cells were plated at a density of 102 to 103 cells/cm2 in the presence of 1 µM RA and were harvested at the time points indicated. Each coverslip was stained for TIF1ß, and cells in 10 to 20 randomly selected confocal fields were scored for the presence or absence of TIF1ß nuclear foci. At least 1000 nuclei were scored per time point. Bars indicate the percentage of nuclei scoring positive for the focal TIF1ß staining pattern characteristic of heterochromatin association, plotted versus time after RA addition. The average results from four independent experiments are depicted.

View larger version (92K): [in a new window]   Fig. 2. TIF1ß is targeted to centromeric heterochromatin in ES-derived neurons. A and D show representative phase-contrast photomicrographs of undifferentiated ES cells growing on a layer of feeder cells in the presence of LIF (control) and ES-derived neurons taken 4 days after plating of the RA-treated embryoid bodies in defined medium (RA), respectively. Confocal images of single optical sections through the nucleus of undifferentiated and differentiated cells are shown. B and E show the Hoechst DNA staining, and C and F, the TIF1ß staining. Bars, 100 µm for (A) and (D) or 5 µm for others.

View larger version (42K): [in a new window]   Fig. 3. RA treatment triggers a subnuclear redistribution of TIF1ß in a subpopulation of F9 cells exhibiting a low proliferation rate and specific expression patterns for endodermal markers. (A-B) Bromodeoxyuridine preferentially labels the differentiated cells, exhibiting a diffuse nuclear staining of the TIF1ß protein. F9 cells treated with 1 µM RA for 6 days were pulse-labeled with BrdU, fixed and stained for TIF1ß (green) and BrdU (red) (see the Materials and Methods). The arrowhead points to a rare BrdU-positive cell with a TIF1ß nuclear dot pattern. (C-H) Endo A and laminin B1, but not fibronectin, are differentially expressed in the RA-treated F9 cells that exhibit a diffuse or a focal TIF1ß staining. F9 cells treated with RA for 6 days were processed for double-label immunofluorescence with antibodies against TIF1ß (C to H; green), fibronectin (D, red), Endo A (F, red) and laminin B1 (H, red). Bar, 20 µm.

View larger version (29K): [in a new window]   Fig. 4. Identification of two amino-acid residues in the HP1 box of TIF1ß, which are critical for the HP1-binding activity of TIF1ß. (A) A schematic representation of the conserved domains of TIF1ß. Numbers refer to amino-acid positions. An alignment of the HP1 interaction domains from TIF1ß, TIF1 and CAF-1 is shown. Invariant amino acids are shaded. Mutations introduced into the conserved hydrophobic residues of the TIF1ß HP1 box are indicated below the alignment. Database accession numbers: mouse TIF1ß (mTIF1ß, X99644); mouse TIF1 (mTIF1, S78219); human CAF-1 (hCAF-1, XP009408). (B) TIF1ßV488A/L490A has no HP1-binding activity in yeast. The indicated FLAG-epitope-tagged TIF1ß constructs, f:TIF1ß wildtype (f:TIF1ßWT) and f:TIF1ßV488A/L490A (f:TIF1ßVL/AA), were fused with the acidic activation domain (AAD) of VP16 and assayed for interaction with the `unfused' DNA-binding domain (DBD) of the oestrogen receptor ER or a DBD fusion containing HP1, HP1ß, HP1 or the KRAB transcriptional repression domain of KOX-1 in the yeast reporter strain PL3, which contains a URA3 reporter gene driven by three ER binding sites (Le Douarin et al., 1995b). Transformants were grown in liquid medium containing uracil. OMPdecase activities determined on each cell-free extracts are expressed in nmol substrate/min/mg protein. The values (±20%) are the average of at least three independent transformants. Note that expression of all fusion proteins was confirmed by western blotting. (C) TIF1ßV488A/L490A has no HP1 binding activity in mammalian cells. Whole cell extracts from COS-1 cells transfected with 5 µg of expression vector for unfused FLAG (control) or FLAG-TIF1ß were analyzed by western blotting either directly (input) or following immunoprecipitation with the M2 anti-FLAG antibody (FLAG IP). A western blot probed with a HP1 mAb is shown. Inputs correspond to 1/10 the amount of cell extract used for immunoprecipitation.

View larger version (87K): [in a new window]   Fig. 5. The double mutation V488A/L490A in the conserved HP1 box residues prevents the RA-induced accumulation of TIF1ß in heterochromatin. FLAG-tagged TIF1ß-expressing F9 EC-derived cell lines [F9(f:TIF1ßWT) and F9(f:TIF1ßVL/AA] untreated (control) or treated with 1 µM RA for 4 days (RA) were analyzed by confocal immunofluorescence microscopy. The left panels show the Hoechst DNA staining, and the right panels correspond to immunodetection with specific monoclonal antibodies (Abs), as indicated. Note that the mAb against TIF1ß also recognizes the FLAG-tagged proteins. Bars, 5 µm.