Flow Cytometric and Laser Scanning Microscopic Approaches in Epigenetics Research

Székvölgyi, Lóránt; Imre, László; Minh, Doan Xuan Quang; Hegedüs, Éva; Bacsó, Zsolt; Szabó, Gábor

doi:10.1007/978-1-60327-414-2_7

Cited by 3 publications

(3 citation statements)

References 27 publications

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“…Similar, TSS-proximal accumulation of nicks was observed using wild-type cells at different metabolic conditions and alpha factor synchronized (G1) bar1Δ cells ( Supplementary Figure S8D and data not shown). The results of the microarray analyses were confirmed in an independent ‘chip-on-beads’ experiment according to ( 72 , 73 ): We demonstrated enrichment of Ser5-phosphorylated (i.e. initiating and elongating; see ( 74 )) RNAP II in the <500 bp vicinity of nicks, whereas no such an accumulation of the Ser2-phosphorylated, elongating and terminating species was observed ( Supplementary Figure S9 ).…”

Section: Resultssupporting

confidence: 58%

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

Hegedüs

Kókai

Nánási

et al. 2018

Nucleic Acids Research

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Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3′OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3′OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

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Section: Resultssupporting

confidence: 58%

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

Hegedüs

Kókai

Nánási

et al. 2018

Nucleic Acids Research

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“…Quantitative microscopy has proved useful in the epigenetics field as a tool of global analysis 85 , 86 ; the method described here greatly extends its utility yielding data of direct functional relevance. Further examples of possible applications are described in the Supplementary Discussion.…”

Section: Discussionmentioning

confidence: 99%

Nucleosome stability measured in situ by automated quantitative imaging

Imre

Simándi

Horváth

et al. 2017

Sci Rep

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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.

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“…Появляются новые технологии, способные анализировать эпигенетические изменения (уровень метилирования ДНК, экспрессию микроРНК, посттрансляционные модификации гистонов и др. ), такие как иммунопреципитация хроматина (CHIP), проточная цитометрия и лазерное сканирование, это дает основания полагать, что в ближайшее время будут выявлены биомаркеры для изучения мультифакториальных заболеваний, редких (орфанных болезней) и злокачественных новообразований и внедрены в качестве методов лабораторной диагностики [8].…”

Section: Toksikologicheskiy Vestnik (Toxicological Review) Volume 29 • Issue 4 • 2021unclassified

On the issue of nongenotoxic cancerogenes

Ревазова

Ilyushina

2021

Toksikol. vestn.

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Introduction. Chemical, physical or biological factors that can cause the formation and expansion of cancer cells are diverse in terms of both activity and mechanisms of action, which leads to the complexity of assessing the risk of developing malignant neoplasms. The aim. Discussion of the classification of carcinogens based on their ability to interact with DNA cell and possible mechanisms of genetic control of carcinogenesis processes induced by non-genotoxic carcinogens. Core content. The article draws attention to some controversial points related to the attribution of factors acting on the body to genotoxic or non-genotoxic carcinogens. The terminology used in the literature to describe genotoxic (mutagenic) and carcinogenic factors is presented. The mechanisms of action of non-genotoxic carcinogens are discussed. The important role of experts determining the danger to public health of factors with potential genotoxicity and carcinogenicity is noted. Conclusion. Non-genotoxic carcinogens are capable of inducing malignant growth through mechanisms not associated with direct damage to genetic structures in the cell. However, the realization of carcinogenic effects caused by such factors is determined by various mechanisms of genetic control.

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Flow Cytometric and Laser Scanning Microscopic Approaches in Epigenetics Research

Cited by 3 publications

References 27 publications

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae

Nucleosome stability measured in situ by automated quantitative imaging

On the issue of nongenotoxic cancerogenes

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