Nucleosome stability measured in situ by automated quantitative imaging

Abstract

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells

“…dependent manner (see [13,15,20,23]). Through the spectacles of confocal microscopy the majority of H2A molecules detected by immunofluorescence becomes topologically separated from the DNA-containing regions upon treatment with >9 μM Dox and gets trapped inside the nuclei as measured in our LSC-based assay (Fig 1, S1 Fig).…”

“…The two members of the dimer can be released from the tetrasomes independently from each other (see S13 Fig of ref. [15]), so they could also be independently affected…”

“…Embedding was carried out according to Imre et al [15]. Briefly, the wells of 8-well chambers (Ibidi, Martinsried, Germany) were coated with 1% (m/v) low melting point (LMP) Agarose.…”

“…The DNA and/or chromatin-related effects may be explained by multitudes of molecular interactions: Anthracyclines intercalate between the neighboring base-pairs of the doublehelix [9], bind free histones [10], can form anthracycline-DNA covalent adducts [11] and are able to destabilize G-quadruplex structures [12]. Intercalation is accompanied by the release of histones and eventually with eviction of the complete nucleosome [13][14][15]. All this is not surprising considering that it relaxes the natural twist of the DNA double helix by −27˚/ intercalating molecule [16].…”

The cell nucleus. A study in Burgundy

“…Gabor Szabo (University of Debrecen, Hungary) reported a cytometric-based pipeline using GFPlabelled histones that enables the quantitative analysis of nucleosome stability in situ, and he quantified the effects of histone variants, cell cycle and posttranslational modifications on nucleosome stability using this novel method [12]. Alexey Onufriev (Virginia Commonwealth Technical University, USA) reported mathematical modeling of charge distributions within the DNA-histone interaction surface in the context of a nucleosome [13].…”