Nucleosome destabilization by polyamines

Abstract

The roles and molecular interactions of polyamines (PAs) in the nucleus are not fully understood. Here their effect on nucleosome stability, a key regulatory factor in eukaryotic gene control, is reported, as measured in agarose embedded nuclei of H2B-GFP expressor HeLa cells. Nucleosome stability was assessed by quantitative microscopy [1,2] in situ, in close to native state of chromatin, preserving the nucleosome constrained topology of the genomic DNA. A robust destabilizing effect was observed in the millimolar concentration range in the case of spermine, spermidine as well as putrescine, which was strongly pH and salt concentration-dependent, and remained significant also at neutral pH. The integrity of genomic DNA was not affected by PA treatment, excluding DNA break-elicited topological relaxation as a factor in destabilization. The binding of PAs to DNA was demonstrated by the displacement of ethidium bromide, both from deproteinized nuclear halos and from plasmid DNA. The possibility that DNA methylation patterns may be influenced by PA levels is contemplated in the context of gene expression and DNA methylation correlations identified in the NCI-60 panel-based CellMiner database: methylated loci in subsets of high-ODC1 cell lines and the dependence of PER3 DNA methylation on PA metabolism.

Keywords: Chromatin; Histone; NCI-60; Nucleosome; Polyamine; Stability.

Fig. 1.. The addition of PAs to agarose-embedded nuclei sensitizes nucleosomes to salt.

The stability of nucleosomes was analyzed in agarose-embedded nuclei of H2B-GFP expressor HeLa cells in the absence and presence of different PAs applied at a concentration of 10 mM. Spermine, spermidine and putrecsin are represented by green, blue and red lines, respectively; data obtained with PA-untreated, control samples are shown by the black line. The data points denote the means of green fluorescence intensities of ~1000 nuclei recorded by LSC. The G1 phase cells were gated for analyses according to the DNA distributions obtained by PI staining. The error bars are SEM values for 200–1000 G1 nuclei in one representative experiment.

Fig. 2.. Spermidine augments the eviction of histones induced by NaCl alone at the pH set by the PA.

Nuclei of H2B-GFP expressor HeLa cells suspended in PBS/EDTA were treated with a concentration series of NaCl alone with the pH of the buffer set to the value of the corresponding solution containing also spermidine (solid lines), or in combination with spermidine used at three different concentrations (dashed lines). The pH values of each solution are shown in Supplementary Fig. 1. The NaCl/PBS/EDTA solutions containing no spermidine were set to the pH of the corresponding 2.5, 5 and 10 mM spermidine containing solutions and are represented by blue, green and red lines, respectively. The samples containing also 2.5, 5 or 10 mM spermidine are represented by the same colours but with dashed lines. The PBS/EDTA + NaCl solutions were alkalized to the pH of the corresponding spermidine-containing solutions with NaOH. Means of the green fluorescence intensities of ~1000 nuclei recorded by LSC are plotted. The bars show SEM values.

Fig. 3.. Spermidine-induced histone eviction is pH-dependent.

Nuclei of H2B-GFP expressor HeLa cells were treated with 5 or 10 mM spermidine alone, in PBS/EDTA, without the addition of extra salt (filled blue and red symbols connected with solid lines, respectively) or with spermidine in combination with 550 mM (at 5 mM spermidine) or 350 mM (at 10 mM spermidine) NaCl (dashed lines, empty blue and red symbols, respectively). The black line and filled circles denote data obtained by treatment of nuclei with PBS-EDTA supplemented with 400 mM NaCl. The pH of the samples was adjusted to the values indicated on the X-axis. Means of the green fluorescence intensities of ~1500 nuclei were recorded by LSC and plotted as a function of the pH. The bars show SEM values.