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First published online October 27, 2005
doi: 10.1242/10.1242/jcs.02621

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Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases

Eva Bártová¹, Jií Pacherník², Andrea Harniarová¹, Ale Kovaík¹, Martina Kovaíková¹, Jirina Hofmanová¹, Magdalena Skalníková³, Michal Kozubek³ and Stanislav Kozubek^1,*

¹ Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65, Brno, Czech Republic
² Center for Cell Therapy and Tissue Repair, Charles University, Vúvalu 84, 150 06 Prague 5, Czech Republic, and Laboratory of Molecular Embryology, Mendel University Brno, Zemdlská 1, 613 00 Brno, Czech Republic
³ Faculty of Informatics, Masaryk University Brno, Botanická 68a, Czech Republic

View larger version (40K): [in a new window]   Fig. 1. (A) Changes in nuclear volume induced by HDACi (TSA and NaBt) determined in two human cell types (A549 and HT29). Average nuclear volume ± s.e.m. was calculated for each treatment and was related to the control values (100%). Asterisks indicate results significantly different from the control; P<0.05. (B) Chromatin decondensation of classical satellites of chromosome 9 in control and TSA-treated A549 cells. Bar, 2 µm. (C) The induction of apoptosis by HDACi was confirmed in A549 and HT29 cells by western blots that show the cleavage of PARP (120 kDa) into 80 kDa fragments and cleavage of lamin B (65 kDa) into 45 kDa fragments.

View larger version (31K): [in a new window]   Fig. 2. (A) Flow cytometric detection of the cell-cycle profiles in TSA- and NaBt-treated A549 and HT29 cells. Modfit software analysis shows G1 (red), S (hatched) and G2 (green) stages of the cell cycle. NaBt induced accumulation of cells in G1 while TSA increased the number of cells in G2. (B) Western blot detection of dimethylated H3(K9) (17 kDa), acetylated H3(K9) (17 kDa) and dimethylated H3(K4) (17 kDa) in A549 cells treated with TSA and NaBt. The western blot data were related to the selected band (arrow) of total nuclear protein levels. The figures show an illustration of representative blots.

View larger version (75K): [in a new window]   Fig. 3. Immunocytochemical analyses of (A) H3(K9) dimethylation, (B) H3(K9) acetylation and (C) H3(K4) dimethylation patterns in A549 cells treated with TSA or NaBt. (Top row) Overlays of TO-PRO-3 counterstained images (middle row) with immunocytochemical staining (bottom row). Nucleolar regions are indicated by yellow arrows, the nuclear periphery by white arrows, and interchromatin compartments by blue arrows. The red arrow in C indicates the non-uniform nature of the peripheral shell. (D) Quantitative analysis of the distribution of H3(K9) dimethylation, H3(K9) acetylation and H3(K4) dimethylation across interphase nuclei. The fluorescence intensity was evaluated across the region indicated by the rectangle in each panel using Andor iQ software. The images represent the histone modification patterns induced by HDACi in the majority of the cell population. Bars, 1.5 µm.

View larger version (62K): [in a new window]   Fig. 4. The levels of H3(K9) dimethylation (17 kDa), H3(K9) acetylation (17 kDa) and H3(K4) dimethylation (17 kDa) analysed by western blots in (A) the human HT29 colon cancer cell line, and (B) normal colon cells FHC. In order to compare the effects of HDACi on normal and tumour cells, the following treatments were tested: sodium butyrate (HT29/NaBt; FHC/NaBt), TNF (HT29/TNF; FHC/TNF) and a combination of NaBt with TNF (HT29/NaBt+TNF; FHC/NaBt+TNF). In both cell types, in comparison with the control, there was a substantial increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation after treatments involving NaBt. Apoptotic cleavage of lamin B (65 kDa) into a 45 kDa fragment was enhanced after the combined treatment with NaBt and TNF (see A and B). Protein levels for all treatments were normalized to the selected band (arrow) of total nuclear protein levels. (C) Comparison of the effects of TSA and NaBt on H3(K9) dimethylation (17 kDa), H3(K9) acetylation (17 kDa) and H3(K4) dimethylation (17 kDa) of HT29 cells. (D) Immunocytochemical analyses of H3(K9) dimethylation, H3(K9) acetylation and H3(K4) dimethylation patterns in HT29 cells treated with TSA and NaBt. TO-PRO-3 was used as a counterstain. The images represent the histone modification patterns induced by HDACi in the majority of the cell population. White and yellow arrows indicate the nuclear periphery and the nucleoli, respectively. Bars, 1 µm.

View larger version (53K): [in a new window]   Fig. 5. (A) Western blot analyses of HP1, ß and proteins in A549 and HT29 cells treated with HDAC inhibitors. Protein levels for all treatments were normalized to the selected band (arrow) of total nuclear protein levels. (B) Interphase patterns of HP1, ß (overlays) and (counterstain, immunostaining and overlay) studied by immunocytochemistry in A549 cells treated with TSA and NaBt. In order to demonstrate the location of HP1 in interchromatin compartments (white arrows) colour separations are also shown. Bars, 1 µm. (C) Nuclear radial distributions of HP1 foci (, ß, ) in control (black line), TSA-treated cells (red line) and NaBt-treated cells (green line), showing the repositioning of HP1 proteins towards the nuclear interior. Average fluorescence intensities across the nucleus were determined using Andor iQ software.

View larger version (63K): [in a new window]   Fig. 6. (A) Nuclear patterns of CENP-A in maximum image (Max. image) and in a central section (Mid. section) for control and TSA-treated A549 cells, showing the repositioning of centromeres towards the nuclear periphery after HDACi treatment. Note that the peripheral location of CENP-A foci can be seen in the central section, which cannot be easily distinguished in the maximum image. (B) Immunocytochemistry with anti-CENP-A and anti-HP1 in control and TSA-treated A549 cells. Colocalization of centromeres (green signals) with HP1 protein (red signals) was observed in the control cell population whereas TSA-induced dissociation of both regions, mostly at the nuclear periphery. DAPI was used as a counterstain; yellow arrows indicate the location of nucleoli. Bar, 1 µm.

View larger version (98K): [in a new window]   Fig. 7. Confocal sections of cell nuclei at different z-positions (S1, S2 and S3) and maximum images (bottom) are shown for nuclei of A549 cells. The H3(K4) dimethylation pattern (green signals) was determined by immunocytochemistry, and centromeric regions (classical satellites of chromosome 9; red signals) were stained using the FISH technique. In both control and TSA-treated cells the centromeres were located in regions lacking H3(K4) dimethylation. Bar, 1 µm.