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Characterization of SET-Domain Histone Lysine Methyltransferase Substrates Using a Cofactor S-Adenosyl-l-Methionine Surrogate

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Histone Methyltransferases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2529))

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Abstract

Identification of histone lysine methyltransferase (HKMT) substrates has recently benefited from chemical-biology-based strategies in which artificial S-adenosyl-l-methionine (SAM) cofactors are engineered to allow substrate labeling using either the wild-type target enzyme or designed mutants. Once labeled, substrates can be selectively functionalized with an affinity tag, using a bioorthogonal ligation reaction, to allow their recovery from cell extracts and subsequent identification. In this chapter, we describe steps on how to proceed to set up such an approach to characterize substrates of specific HKMTs of the SET domain superfamily, from the characterization of the HKMT able to accommodate a SAM surrogate containing a bioorthogonal moiety, to the proteomic analysis conducted on a cell extract. We focus in particular on the controls that are necessary to ensure reliable proteomic data analysis. The example of PR-Set7 on which we have implemented this approach is shown.

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Abbreviations

ACN:

Acetonitrile

BSA:

Bovine serum albumin

CuAAC:

Cu(I)-catalyzed alkyne-azide cycloaddition

DTT :

Dithiothreitol

EDTA :

Ethylenediamine tetraacetic acid

HKMT:

Histone lysine methyltransferase

LC:

Liquid chromatography

MALDI TOF:

Matrix assisted laser desorption ionization time-of-flight

MS:

Mass spectrometry

PEG:

Polyethylene glycol

PTM:

Post-translational modification

SAM:

S-Adenosyl-l-methionine

SDS-PAGE :

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SET:

Su(var)3–9, Enhancer-of-zeste and Trithorax

TFA :

Trifluoroacetic acid

THPTA:

Tris(3-hydroxypropyltriazolylmethyl)amine

XIC:

Extracted ion chromatogram

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Acknowledgments

We thank the Ministère de la Recherche for a grant to A.D. We also thank Damarys Loew and Berangere Lombard from the Institut Curie Mass Spectrometry and Proteomics facility for help with proteomic study. The present work was supported by l’Agence Nationale de la Recherche (AMetHist, ANR-17-CE12-0028).

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Authors and Affiliations

  1. Sorbonne Université, École normale supérieure, PSL University, CNRS, Laboratoire des Biomolécules, LBM, Paris, France

    Alexandre Désert & Fabienne Burlina

  2. Université Paris-Saclay, CNRS, Institut de Chimie Moléculaire et des Matériaux d’Orsay (ICMMO), UMR 8182, Orsay, France

    Karine Guitot & Dominique Guianvarc’h

  3. Institut Curie, Paris Sciences et Lettres Research University, Sorbonne University, Paris, France

    Audrey Michaud, Daniel Holoch & Raphaël Margueron

  4. INSERM U934/CNRS UMR3215, Paris, France

    Audrey Michaud, Daniel Holoch & Raphaël Margueron

Authors
  1. Alexandre Désert
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  2. Karine Guitot
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  3. Audrey Michaud
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  4. Daniel Holoch
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  5. Raphaël Margueron
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  6. Fabienne Burlina
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  7. Dominique Guianvarc’h
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Corresponding author

Correspondence to Dominique Guianvarc’h .

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Editors and Affiliations

  1. Institute Curie, INSERM U934/CNRS UMR 3215, Paris, France

    Raphaël Margueron

  2. Institute Curie, INSERM U934/CNRS UMR 3215, Paris, France

    Daniel Holoch

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Désert, A. et al. (2022). Characterization of SET-Domain Histone Lysine Methyltransferase Substrates Using a Cofactor S-Adenosyl-l-Methionine Surrogate. In: Margueron, R., Holoch, D. (eds) Histone Methyltransferases. Methods in Molecular Biology, vol 2529. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2481-4_14

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  • DOI: https://doi.org/10.1007/978-1-0716-2481-4_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2480-7

  • Online ISBN: 978-1-0716-2481-4

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