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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.

SUBMITTER: Imre L

PROVIDER: S-EPMC5630628 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Nucleosome stability measured in situ by automated quantitative imaging.

Imre László L Simándi Zoltán Z Horváth Attila A Fenyőfalvi György G Nánási Péter P Niaki Erfaneh Firouzi EF Hegedüs Éva É Bacsó Zsolt Z Weyemi Urbain U Mauser Rebekka R Ausio Juan J Jeltsch Albert A Bonner William W Nagy László L Kimura Hiroshi H Szabó Gábor G

Scientific reports 20171006 1

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 ...[more]

PMID: 28986581

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Project description:BackgroundWe have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features.ResultsHere we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue.ConclusionsIn summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.

Project description:Background Automated analysis of cardiovascular magnetic resonance images provides the potential to assess aortic distensibility in large populations. The aim of this study was to compare the prediction of cardiovascular events by automated cardiovascular magnetic resonance with those of other simple measures of aortic stiffness suitable for population screening. Methods and Results Aortic distensibility was measured from automated segmentation of aortic cine cardiovascular magnetic resonance using artificial intelligence in 8435 participants. The associations of distensibility, brachial pulse pressure, and stiffness index (obtained by finger photoplethysmography) with conventional risk factors was examined by multivariable regression and incident cardiovascular events by Cox proportional-hazards regression. Mean (±SD) distensibility values for men and women were 1.77±1.15 and 2.10±1.45 (P<0.0001) 10-3 mm Hg-1, respectively. There was a good correlation between automatically and manually obtained systolic and diastolic aortic areas (r=0.980 and r=0.985, respectively). In regression analysis, distensibility associated with age, mean arterial pressure, heart rate, weight, and plasma glucose but not male sex, cholesterol or current smoking. During an average follow-up of 2.8±1.3 years, 86 participants experienced cardiovascular events 6 of whom died. Higher distensibility was associated with reduced risk of cardiovascular events (adjusted hazard ratio [HR], 0.61 per log unit of distensibility; P=0.016). There was no evidence of an association between pulse pressure (adjusted HR 1.00; P=0.715) or stiffness index (adjusted HR, 1.02; P=0.535) and risk of cardiovascular events. Conclusions Automated cardiovascular magnetic resonance-derived aortic distensibility may be incorporated into routine clinical imaging. It shows a similar association to cardiovascular risk factors as other measures of arterial stiffness and predicts new-onset cardiovascular events, making it a useful tool for the measurement of vascular aging and associated cardiovascular risk.

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Nucleosome stability measured in situ by automated quantitative imaging.

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Nucleosome stability measured in situ by automated quantitative imaging.

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