The DNA intercalators ethidium bromide and propidium iodide also bind to core histones

Abstract

Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.

Keywords: Chromatin compaction; DLS, dynamic light scattering; DNA release; EB, ethidium bromide; HAT, histone acetyl transferase; HMG, high mobility group; Histone acetylation; Histone and DNA binding; ITC, isothermal titration calorimetry; MTT, (3-(4-5 dimethylthiozol-2-yl) 2-5diphenyl-tetrazolium bromide); Oct, octamer; PI, propidium iodide.

Graphical abstract

Fig. 1

Association of propidium iodide and ethidium bromide with hierarchical levels of chromatin and core histones monitored by steady state fluorescence spectroscopy and isothermal titration calorimetry: (a) Chemical structure of propidium iodide (PI). (b) Emission spectra of PI in absence (black) and presence of increasing concentrations (9.5, 19, 33, 47, 82, and 143 μM) of long chromatin. λ_ex = 536 nm. (c) Emission spectra of core histone octamer in absence (black) and presence of increasing concentrations (1.5, 4.2, 8.4, 17.2, and 40 μM) of PI. λ_ex = 278 nm. (d) ITC profile for the association of PI with long chromatin. (e) ITC profile for the association of PI with core histones. (f) Chemical structure of ethidium bromide (EB). (g) Emission spectra of EB in absence (black) and presence of increasing concentrations (9.5, 19, 33, 47, 82, and 143 μM) of long chromatin. λ_ex = 520 nm. (h) Emission spectra of core histone octamer in absence (black) and presence of increasing concentrations (1.5, 4.2, 8.4, 17.2, and 40 μM) of EB. λ_ex = 278 nm. (i) ITC profile for the association of EB with long chromatin. (j) ITC profile for the association of EB with core histones. (k) Binding isotherms for the interaction of PI and EB with hierarchical levels of chromatin obtained from steady state fluorescence spectroscopy. Data points for long chromatin, chromatosome and chromosomal DNA are denoted by circles, triangles and squares respectively. The experimental set for PI shows black fills and the set with no fill shapes correspond to EB. The concentration of PI/EB was taken 3 μM in these fluorimetric titrations. (l) Binding isotherms for the interaction of histone octamer [1.5 μM] with PI (circles) and EB (squares) as obtained from steady state fluorescence spectroscopy. (m) Binding energetics of the interaction of PI with chromosomal DNA (left) and core histones (right). (n) Binding energetics of the interaction of EB with chromosomal DNA (left) and core histones (right). The enthalpy change for the ligand-macromolecule association is denoted by ΔH, the entropy factor is denoted by TΔS and the free energy change for the interaction is denoted by ΔG in each case in (m) and (n). The upper panels in the ITC profiles in (d, e, i and j) show the heat of association for ligand-macromolecule interactions. The lower panels show the heat exchanged per mole of injectant as a function of molar ratio of ligand to macromolecule. The solid lines represent the fitted isotherms obtained using “one set of sites” binding model. The thermograms have been fitted after subtracting appropriate controls. The heat changes for the controls are shown in the supplementary section. All the experiments in Fig. 1 were performed at 25 °C.

Fig. 2

Effect of the ligands on the structure of chromatin and its components monitored by dynamic light scattering (DLS) and agarose gel electrophoresis: Panels (a–f) depict the results obtained from DLS employed to study the influence of PI and EB on the hydrodynamic size of chromatin and histone octamer at 25 °C: Intensity statistics of 10 measurements are plotted for chromatin (400 μM DNA base) in presence of increasing concentrations of PI (a) and EB (b). The corresponding polydispersity index is plotted against input ratio of PI (c) and EB (d). The number statistics of 10 measurements each are plotted for 5 μM histone octamer (Oct) in presence of increasing concentrations of PI (e) and EB (f). Error bars indicate standard deviations. Panels (g) and (h) show the results of chromatosome stability assay performed to study the effect of PI and EB on chromatosome: Chromatosome samples (400 μM DNA base) were incubated with PI (g) and EB (h) at 37 °C for 30 min at ligand/DNA base ratio indicated, post-stained with Sybr green and analyzed on 1.5% agarose gel. Fresh chromatosome sample (lane 1) and chromatosome incubated with buffer at 37 °C for 30 min (lane 2) serve as negative controls in each case. The bands around 500 bp correspond to intact chromatosome and the bands around 200 bp correspond to the DNA released from the chromatosome as a result of incubation with the ligands. The 100 bp DNA ladder has been used as the marker.

Fig. 3

Modulation of acetylation levels of histones H3 and H4 by the ligands (PI and EB) monitored by western blot analyses: (a) Effect of PI/EB on histone H3 acetylation at lysine 9 and 14 in HeLa cells. β-actin and histone H3 have been used as loading controls. (b) Ligand induced alteration of histone H4 acetylation at lysine 5 and 8 in HeLa cells. β-actin and histone H4 have been used as loading controls. (c) Histone acetyltransferase (HAT) assay performed in absence and presence of the ligands using purified HeLa core histones (2 μg) and processed for immunoblot analysis. No enzyme (lane 1 and 6) and no ligand (lanes 2 and 7) controls are shown. Alteration of histone H3K9Ac status in presence of increasing concentrations (2, 4, and 8 μM) of PI and EB are shown in lanes 3–5 and lanes 8–10, respectively. Loading and transfer of equal amounts of core histones were confirmed by immunodetection of histone H3. (d) Quantification of the extent of ligand induced hypoacetylations. The extent of repression of acetylation marks (H3K9Ac, H4K5Ac and H4K8Ac) in presence of the ligands has been quantified using Image J software and represented in the 3D plot against different concentrations of the ligands. The autoradiograms were quantified using Image J software after normalizing with respective histone H3 and H4 loading controls. Then the fold change was calculated in Origin. The error bars were estimated from three independent sets of experiments.