PMC

Chemical structure of mithramycin (MTR).

In vitro HAT assay to examine the effect of MTR on H4 acetylation at K5 and K8.

Time dependence and dose dependence Western blot analysis of MTR-treated HeLa cells. β-Actin has been used as loading control.

The percentage change in emission intensity at the emission maximum of MTR (540 nm) upon interaction with H2A, H2B, H3 and H4 tail peptides at 25 °C.

(A) Flow cytometry analysis of MTR-treated and untreated HeLa cells ([MTR] = 10 μM, treatment duration = 9 h). (B–D) MTT assay to assess HeLa cell viability upon 10 μM MTR treatment for 9, 12 and 15 h respectively.

CD spectra of 8 μM MTR in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl at 25 °C in absence (black) and in presence of 1 μM (red); 4 μM (green) histones H2A (A), H2B (B) and H4 (C) in the visible range.

ITC to examine MTR–histone interactions at high salt. Thermograms for the interaction of MTR with histones H2A (A), H2B (B), H3 (C) and H4 (D) at 25 °C in 10 mM Tris–HCl, pH 7.0 containing 1 M NaCl. The lower panels contain the background heat subtracted fitted isotherms.

ITC profiles for the interaction of MTR with histones H2A (A), H2B (B), H4 (C) and H1 (D) at 25 °C in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl. The lower panels contain the background heat subtracted fitted isotherms. H1 does not interact with MTR, so binding isotherm could not be obtained in (D).

Fluorimetric titration of MTR with histone dimer/tetramer. Emission spectra of 2 μM MTR in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl at 25 °C in presence of H2A-H2B dimer (A) and H3–H4 tetramer (B). λ_ex = 470 nm. Panels (C), (D) show the corresponding fitted isotherms obtained by non-linear curve fitting analysis.

Unsupervised hierarchical clustering of differentially expressed genes (A) using Pearson uncentered algorithm with average distance matrix shows distinct patterns of up and down regulated genes upon treatment in comparison to untreated samples. Venn diagram representation (B) of up and down regulated genes between two cell lines indicates less number of genes that are commonly regulated by MTR.

MTR-induced repression of H3K18 acetylation in HeLa cells as monitored by confocal microscopy. (A) Representative population of untreated and 10 μM MTR-treated HeLa cells for 9 h. The scale bar is 10 μM. (B) Quantification of the extent of repression of H3K18 acetylation upon increasing concentrations of MTR treatment (0.1–10 μM), using Image J software. Statistical analyses were performed over 300 cells in each case.

In vitro HAT assay to examine alteration of H3K18 acetylation (A), H3K9 acetylation (B) and H3K27 acetylation (C) by MTR in recombinant histone H3. (D) Modulation of H3K18 acetylation, ex vivo, probed by Western blot analysis of MTR-treated HeLa cells. Extent of MTR-induced repression of H3K18Ac has been quantified by Image J software. (E) Inhibition of H3K18 acetylation in HeLa cells by MTR monitored by confocal microscopy. The scale bar is 10 μm.

Binding isotherms obtained from fluorimetric titration of MTR with human recombinant histones H2A (A), H2B (B) H4 (C) at 25 °C. The insets show the emission spectra of 2 μM MTR in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl in presence of increasing concentration of the histones. λ_ex = 470 nm. (D) Emission spectra of 2 μM MTR in presence of increasing concentrations of linker histone H1. Inset shows the variation of fluorescence intensity of MTR at emission maximum with increasing concentration of H1.

BAN (Biological Analysis Network) of differentially expressed genes and biological processes. Network of differentially expressed genes and biological processes shows high degree of down regulation in (A) TC32 cell line treated with MTR, as compared to (B) TC71. Biological processes are colored in (violet blocks) and genes are colored (green for down regulated and orange for up regulated) by their fold change upon treatment compared with untreated cell line. Among them a good number of differentially expressed genes are associated with chromatin regulation, DNA binding, histone modification, cell cycle, development, diseases, miRNA, neuronal, signal transduction and transcription.

(A) Dynamic Light Scattering measurements to study the effect of MTR on the hydrodynamic diameter (Z_av) of chromatin. Intensity statistics of 10 measurements each are plotted for each sample. Error bars indicate standard deviations. Standard deviations of Z_av values obtained from three independent sets of experiment are within 5%. (B) Electron Microscopy images of untreated and MTR treated chromatin. The scale bar is 2000 nm. Chromatin was treated with MTR for 3 h at MTR/DNA base ratio of 0.2

(A) Interaction of MTR with core octamer in 10 mM Tris–HCl, pH 7.0 containing 2 M NaCl at 25 °C: Binding isotherm obtained using non-linear curve fitting analysis. Inset: Emission spectra of MTR (2 μM) in absence (black) and in presence (red) of 3.3 μM core octamer. λ_ex = 470 nm. (B) Near UV CD spectra of 3.3 μM histone octamer in 10 mM Tris–HCl, pH 7.0 containing 2 M NaCl at 25 °C in absence (black) and in presence of increasing MTR concentrations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Emission spectra of 20 μM MTR in 10 mM Tris–HCl, pH 7.0 containing 15 mM NaCl at 25 °C in presence of increasing concentrations of chromatin (A) and chromatosome (D). λ_ex = 470 nm. Panels (B) and (E) show corresponding binding isotherms for MTR–chromatin/chromatosome interactions. Circular dichroism spectra of 15 μM MTR monitored at 25 °C in absence (black) and presence of chromatin (C) and chromatosome (F). Red curves represent 45 μM and blue curves represent 120 μM of chromatin/chromatosome respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(A) Binding isotherm for the interaction of MTR with histone H3 in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl at 25 °C obtained from steady state fluorescence spectroscopy. Inset shows the emission spectra of 2 μM MTR in absence (black) and presence of increasing concentrations (0.5 μM, red; 1 μM, blue; 2 μM, green) of human recombinant histone H3. λ_ex = 470 nm. (B) Circular dichroism spectra of 8 μM MTR in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl at 25 °C in absence (black) and presence of 1 μM (red) and 4 μM (green) histone H3 in the visible range. (C) ITC profile for the association of MTR with histone H3 at 25 °C in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl. The lower panel contains the background heat subtracted fitted isotherm. Emission spectra of 2 μM MTR in 10 mM Tris–HCl, pH 7.0 containing 150 mM NaCl at 25 °C in absence (black) and in presence of (2 μM, red; 4 μM, green; 9 μM, blue) N-terminal tail peptide H3 (residues 1–21). λ_ex = 470 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)