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First published online 29 November 2005
doi: 10.1242/jcs.02689

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Histone acetylation increases chromatin accessibility

Sabine M. Görisch^1,*, Malte Wachsmuth^2,, Katalin Fejes Tóth², Peter Lichter¹ and Karsten Rippe^2,

¹ Division of Molecular Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
² Molecular Biophysics Group, Kirchhoff-Institut für Physik, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany

View larger version (77K): [in a new window]   Fig. 1. TSA-induced increase in chromatin accessibility. Examples of confocal sections of HeLa interphase cell nuclei (A-E) microinjected with 42, 77, 282, 464 and 2500 kDa and of mitotic cells microinjected with 464 kDa FITC-dextrans (F) are presented. The dextran signal is in green and the DNA counterstained with DAPI is in red. The left side shows control cells and the right side cells after 17 hours of TSA treatment. The dashed lines indicate the direction of the corresponding linescans that are shown next to the images. Scale bars, 10 µm.

View larger version (48K): [in a new window]   Fig. 2. Reversibility of chromatin accessibility changes due to hyperacetylation. (A) Time series of HeLa cell nuclei with microinjected 464 kDa dextrans (green) and Hoechst DNA stain (red) before TSA treatment, after 3 or 17 hours TSA incubation and 3 or 4 hours after removal of TSA. The dashed lines indicate the direction of the corresponding linescans, which are shown adjacent to the images. Scale bars, 10 µm. (B) Kinetics of chromatin decondensation due to TSA-induced histone acetylation. The increase of the chromatin correlation length represents the induced decondensation. The fit curve is a single-exponential reaction kinetics with a half-time of 3.5 hours.

View larger version (34K): [in a new window]   Fig. 3. Image cross-correlation spectroscopy analysis of dextran and chromatin distribution. (A) Example of the image correlation spectroscopy analysis of cells, into which 464 kDa FITC-dextrans were injected. The autocorrelation function of the DAPI-stained chromatin distribution, G1(r), and of the distribution of the 464 kDa FITC-dextrans, G2(r), as well as the cross-correlation function of the two distributions, Gx(r), were computed as described in the text (Eqn 3). The dashed lines are Gaussian fits to the shortest decay of each correlation function, from which the autocorrelation amplitudes G1(0) and G2(0) are obtained as indicated. These values are then used to calculate the normalized cross-correlation function ratioG(r) with its value at r=0, ratioG0, according to Eqn 4. This normalizes for intensity differences between images. The autocorrelation length lc that describes the chromatin condensation state is the length, after which the Gaussian fit to the DAPI-stained chromatin correlation function has decreased to half of its maximum value. Due to the exclusion of the dextrans from condensed chromatin areas, the cross-correlation curve and its Gaussian fit have a negative amplitude Gx(0). From the decay of the Gx(r) curve, the cross-correlation length lx for the exclusion is determined in analogy to the autocorrelation length. (B-D) Normalized cross-correlation curves for dextrans in control (broken line) and TSA-treated cells (solid line). (B) 77 kDa dextran during interphase. (C) 464 kDa during interphase. (D) 464 kDa dextrans during metaphase. (E) Measured dextran sizes given by the radius of gyration RG. As expected for a random coil, RG is proportional to the square root of the dextran mass m and a very good fit (solid line) to the expression RG=1.104 nm kDa–1/2 m1/2 was obtained. Error bars for the low molecular mass dextrans are smaller than the size of the data points. (F) Nuclear distribution of dextrans with respect to chromatin in dependence of dextran size for control (broken line) and TSA-treated cells (solid line) during interphase. The co-localization/exclusion of dextrans from chromatin is expressed as the normalized cross-correlation signal ratioG0 according to Eqn 3. Eqn 4 was fit to the data for the control cells (broken line) with values of r1=8.9 nm and r2=22.9 nm. For the TSA-treated cells a constant line (solid line) with the average value of ratioG0 is depicted.

View larger version (45K): [in a new window]   Fig. 4. Illustration of chromatin accessibility. The chromatin is shown in gray and macromolecules are represented by three differently sized spheres in green corresponding to the distribution of 42, 77 and 464 kDa dextrans. The left side illustrates an untreated cell, where small molecules can diffuse in all parts of the chromatin with a subfraction of heterochromatin being accessible only to the smallest particles. The large complexes are excluded from heterochromatin and restricted to more open chromatin regions in the nuclear center. The right side shows schematically the macromolecule distribution in a TSA-treated cell. The dense chromatin regions are disintegrated so that also the large complexes can distribute throughout the complete nucleus.