Detection and quantification of histone methyltransferase activity in vitro

Nwamaka J Idigo; Philipp Voigt

doi:10.1007/978-1-0716-2481-4_2

Detection and quantification of histone methyltransferase activity in vitro

Nwamaka J Idigo, Philipp Voigt

School of Biological Sciences

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

Abstract

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

Original language	English
Title of host publication	Histone methyltransferases
Subtitle of host publication	Methods and protocols
Pages	43-61
Number of pages	19
Volume	2529
ISBN (Electronic)	978-1-0716-2481-4
DOIs	https://doi.org/10.1007/978-1-0716-2481-4_2
Publication status	E-pub ahead of print - 23 Jun 2022

Publication series

Name	Methods in molecular biology (Clifton, N.J.)
Publisher	Humana Press
ISSN (Print)	1064-3745

Keywords

histone methyltransferases
histone-lysine N-methyltransferase
histones
methylation
protein processing
S-Adenosylmethionine
post-translational

Access to Document

10.1007/978-1-0716-2481-4_2

3 Finished

Wellcome Centre for Cell Biology
Tollervey, D.
UK-based charities
1/12/16 → 1/12/21
Project: Research
PRCTOERC: Novel Regulatory Principles of Polycomb Repressive Complex 2
Voigt, P.
EU government bodies
1/06/15 → 31/05/22
Project: Research
Roles of symmetric and asymmetric histone H3 lysine 27 trimethylation in gene repression and epigenetic inheritance
Voigt, P. & Finnegan, D.
UK-based charities
1/11/14 → 31/12/21
Project: Research

Cite this

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title = "Detection and quantification of histone methyltransferase activity in vitro",

abstract = "Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH). ",

keywords = "histone methyltransferases, histone-lysine N-methyltransferase, histones, methylation, protein processing, S-Adenosylmethionine, post-translational",

author = "Idigo, {Nwamaka J} and Philipp Voigt",

note = "Funding Information: We thank Pablo De Ioannes and Karim-Jean Armache for helpful discussions and advice on SAH quantification methods. Work in the Voigt lab is supported by the Wellcome Trust ([104175/Z/14/ Z], Sir Henry Dale Fellowship to P.V.) and through funding from the European Research Council (ERC) under the European Union{\textquoteright}s Horizon 2020 research and innovation programme (ERC-STG grant agreement No. 639253 to P.V.). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust [203149]. Publisher Copyright: {\textcopyright} 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.",

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doi = "10.1007/978-1-0716-2481-4_2",

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AU - Idigo, Nwamaka J

AU - Voigt, Philipp

N1 - Funding Information: We thank Pablo De Ioannes and Karim-Jean Armache for helpful discussions and advice on SAH quantification methods. Work in the Voigt lab is supported by the Wellcome Trust ([104175/Z/14/ Z], Sir Henry Dale Fellowship to P.V.) and through funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-STG grant agreement No. 639253 to P.V.). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust [203149]. Publisher Copyright: © 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2022/6/23

Y1 - 2022/6/23

N2 - Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

AB - Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

KW - histone methyltransferases

KW - histone-lysine N-methyltransferase

KW - histones

KW - methylation

KW - protein processing

KW - S-Adenosylmethionine

KW - post-translational

U2 - 10.1007/978-1-0716-2481-4_2

DO - 10.1007/978-1-0716-2481-4_2

M3 - Chapter (peer-reviewed)

C2 - 35733009

SN - 978-1-0716-2480-7

VL - 2529

T3 - Methods in molecular biology (Clifton, N.J.)

SP - 43

EP - 61

BT - Histone methyltransferases

ER -

Detection and quantification of histone methyltransferase activity in vitro

Abstract

Publication series

Keywords

Access to Document

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Projects

Wellcome Centre for Cell Biology

PRCTOERC: Novel Regulatory Principles of Polycomb Repressive Complex 2

Roles of symmetric and asymmetric histone H3 lysine 27 trimethylation in gene repression and epigenetic inheritance

Cite this