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First published online 3 August 2004
doi: 10.1242/jcs.01293

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Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin

¹ Kirchhoff-Institut für Physik, AG Molekulare Biophysik, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
² Molekulare Genetik, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
³ Biomedizinische Strukturanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
⁴ Cytometrie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
⁵ Intelligente Bioinformatiksysteme, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
⁶ Genregulation und DNA-Topologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

View larger version (30K): [in a new window]   Fig. 1. Effect of TSA on the cell cycle as analyzed by FACS. Black bars, control cells; dark gray bars, cells treated with 50 ng/ml TSA; light gray bars, cells treated with 100 ng/ml TSA. The overall changes in the distribution after (A) 12 hours and (C) 24 hours, and the change weighted with the fraction of cells in the given cell cycle stage after (B) 12 hours and (D) 24 hours are shown.

View larger version (17K): [in a new window]   Fig. 2. Concentration dependence of TSA induced apoptosis. TSA caused an increase in apoptosis in a concentration-dependent manner after incubation for 24 hours, as revealed by the FACS analysis. A fit of the data to a model in which apoptosis reflects the binding of TSA to a single class of independent binding sites (Eqn 2, =1) showed a systematic error (dashed line). By contrast, a good fit of the data was obtained with a cooperativity factor of =3.2 (solid line).

View larger version (27K): [in a new window]   Fig. 3. Effects of TSA on H2A-YFP expression. (A) H2A-YFP fluorescence intensity increased upon TSA treatment. Cells were incubated with the indicated TSA concentrations for 24 hours and H2A-YFP fluorescence was measured by FACS analysis. The fluorescence intensity of untreated cells was set to 1 and the relative fluorescence increase was plotted. (B) Western blot analysis of H2AYFP protein expression relative to that of lamin. The H2A-YFP concentration increased with longer incubation times (left) and higher TSA concentrations (right).

View larger version (53K): [in a new window]   Fig. 4. TSA-induced changes of interphase chromatin morphology. HeLa cells stably expressing H2A-YFP were treated with 100 ng/ml TSA for 24 hours. 28 µmx28 µm (400x400 pixel) images are shown. Image intensities were normalized to the same average value. (A) Nuclei of control cells imaged in vivo with H2A-YFP. Dense chromatin areas were seen as brighter spots around the nucleolus and close to the nuclear membrane. (B) Fixed and DAPI-stained images of the same nuclei as in A. (C) TSA-treated cells imaged in vivo with H2AYFP. The chromatin distribution was more homogeneous and most of the dense chromatin disappeared. (D) Fixed and DAPI-stained images of the same nuclei as in C.

View larger version (58K): [in a new window]   Fig. 5. Reversibility of TSA-induced chromatin decondensation. CLSM images of the same cell nuclei before treatment with TSA (A), after incubation with 100 ng/ml TSA for 4 hours (B), and after changing to a medium without TSA and incubation for another 4 hours (C). The effect of TSA is reversible since dense chromatin regions reappeared after washing out TSA.

View larger version (84K): [in a new window]   Fig. 6. Determination of the cell cycle stage of investigated cells. (A) In vivo CLSM images of HeLa cell nuclei expressing H2A-YFP. (B) Phase contrast image of fixed, DAPI-stained cells were taken with an inverse fluorescence microscope. The white circle shows the cell imaged in A. (C) DAPI image of the same area as in B. (D) Comparative histogram of cells analyzed by FACS (solid line) and on coverslips (dashed line). DAPI intensities of the imaged nuclei were plotted and the cell cycle stage of single cells was determined from their position in the histogram.

View larger version (20K): [in a new window]   Fig. 7. ICS analysis of TSA-induced decondensation. Both for and The spatial radial autocorrelation function was calculated and averaged over 60 control cells (dashed line) and 30 TSA-treated cells (solid line).

View larger version (19K): [in a new window]   Fig. 8. Spatially resolved scaling analysis (SRSA) of TSA-induced chromatin decondensation. The averaged distribution of the local fractal dimension Df was computed for both control and TSA-treated cells.

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