Nucleosome stability measured in situ by automated quantitative imaging

László Imre; Zoltán Simándi; Attila Horváth; György Fenyofalvi; Péter Nánási; Erfaneh Firouzi Niaki; Éva Hegedüs; Zsolt Bacsó; Urbain Weyemi; Rebekka Mauser; Juan Ausio; Albert Jeltsch; William Bonner; László Nagy; Hiroshi Kimura; Gábor Szabó

doi:10.1038/s41598-017-12608-9

Nucleosome stability measured in situ by automated quantitative imaging

László Imre, Zoltán Simándi, Attila Horváth, György Fenyofalvi, Péter Nánási, Erfaneh Firouzi Niaki, Éva Hegedüs, Zsolt Bacsó, Urbain Weyemi, Rebekka Mauser, Juan Ausio, Albert Jeltsch, William Bonner, László Nagy, Hiroshi Kimura, Gábor Szabó

School of Medicine

Research output: Contribution to journal › Article › peer-review

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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.

Original language	English (US)
Article number	12734
Journal	Scientific reports
Volume	7
Issue number	1
DOIs	https://doi.org/10.1038/s41598-017-12608-9
State	Published - Dec 1 2017

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@article{7631f8c7b4084c18972c9d68f8b26be3,

title = "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.",

author = "L{\'a}szl{\'o} Imre and Zolt{\'a}n Sim{\'a}ndi and Attila Horv{\'a}th and Gy{\"o}rgy Fenyofalvi and P{\'e}ter N{\'a}n{\'a}si and Niaki, {Erfaneh Firouzi} and {\'E}va Heged{\"u}s and Zsolt Bacs{\'o} and Urbain Weyemi and Rebekka Mauser and Juan Ausio and Albert Jeltsch and William Bonner and L{\'a}szl{\'o} Nagy and Hiroshi Kimura and G{\'a}bor Szab{\'o}",

note = "Funding Information: The authors thank Adel Nagy Vezendine for conscientious technical assistance and Katrina Good for a careful proofreading of the manuscript. We thank Tom Misteli, NIH, for providing the LacO array system. This work was supported by Hungarian National Science and Research Foundation (OTKA) grants [K72762]; [NK101337]; by T{\'A}MOP grants [T{\'A}MOP 4.2.2-08/1-2008-0015], [T{\'A}MOP 4.2.1/B-09/1/KONV-2010-0007], [T{\'A}MOP 4.2.2.A-11/1/KONV-2012-0023 “V{\'E}D-ELEM”], and [T{\'A}MOP 4.2.4. A/2-11-1-2012-0001, GINOP-2.3.2-15-2016-00044, National Excellence Program]´, by JSPS KAKENHI, JP 25116005, JP26291071 (to HK) and by Natural Sciences and Engeneering Research Council of Canada (NSERC) 46399-2012 grant (to JA). Publisher Copyright: {\textcopyright} 2017 The Author(s).",

year = "2017",

month = dec,

day = "1",

doi = "10.1038/s41598-017-12608-9",

language = "English (US)",

volume = "7",

journal = "Scientific reports",

issn = "2045-2322",

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TY - JOUR

T1 - Nucleosome stability measured in situ by automated quantitative imaging

AU - Imre, László

AU - Simándi, Zoltán

AU - Horváth, Attila

AU - Fenyofalvi, György

AU - Nánási, Péter

AU - Niaki, Erfaneh Firouzi

AU - Hegedüs, Éva

AU - Bacsó, Zsolt

AU - Weyemi, Urbain

AU - Mauser, Rebekka

AU - Ausio, Juan

AU - Jeltsch, Albert

AU - Bonner, William

AU - Nagy, László

AU - Kimura, Hiroshi

AU - Szabó, Gábor

N1 - Funding Information: The authors thank Adel Nagy Vezendine for conscientious technical assistance and Katrina Good for a careful proofreading of the manuscript. We thank Tom Misteli, NIH, for providing the LacO array system. This work was supported by Hungarian National Science and Research Foundation (OTKA) grants [K72762]; [NK101337]; by TÁMOP grants [TÁMOP 4.2.2-08/1-2008-0015], [TÁMOP 4.2.1/B-09/1/KONV-2010-0007], [TÁMOP 4.2.2.A-11/1/KONV-2012-0023 “VÉD-ELEM”], and [TÁMOP 4.2.4. A/2-11-1-2012-0001, GINOP-2.3.2-15-2016-00044, National Excellence Program]´, by JSPS KAKENHI, JP 25116005, JP26291071 (to HK) and by Natural Sciences and Engeneering Research Council of Canada (NSERC) 46399-2012 grant (to JA). Publisher Copyright: © 2017 The Author(s).

PY - 2017/12/1

Y1 - 2017/12/1

N2 - 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.

AB - 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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DO - 10.1038/s41598-017-12608-9

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SN - 2045-2322

VL - 7

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JF - Scientific reports

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ER -

Nucleosome stability measured in situ by automated quantitative imaging

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