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Helmholtz Munich | Amelie J. Kraus

A beginner’s guide to catching the readers – Till Bartke publishes protocols to identify modification-specific nucleosome interactors


As part of the book series ‘Methods in Molecular Biology’, Springer Nature has released the methods and protocols collection ‘Histone Methyltransferases’. The chapter contributed by Till Bartke and colleagues from the Institute of Functional Epigenetics includes detailed descriptions of how to identify methylation-specific nucleosome interactors via mass-spectrometry.

To fit the expansive length of DNA into the tiny nucleus of a eukaryotic cell, it is densely folded into a higher-order structure called chromatin. Chromatin, however, is not a rigid structure and can be dynamically altered to provide DNA accessibility for the DNA-templated processes within the nucleus. The smallest structural unit of chromatin is nucleosomes, which is composed of DNA wrapped around a histone octamer. Changes in the chromatin structure involve changes to the nucleosomes, making the DNA more or less tightly bound to histone proteins by adding distinct chemical modifications, such as methylation, for example

Histone Methylation – a versatile epigenetic player in regulating DNA activity

Histone methylation can alter the nucleosomal properties, thereby changing the interaction of nucleosomes with other proteins. Most notably, it can recruit so-called ‘epigenetic readers’ that recognize specific methylations and stimulate additional changes in the chromatin, ultimately altering the activity of the underlying DNA. New tools and methods developed over the past 20 years have led to remarkable findings that have proven the importance of histone methylation for regulating gene transcription and other DNA-templated processes. The book ‘Histone Methyltransferases’, edited by Daniel Holoch and Raphael Margueron and now published by Springer Nature, is an overarching collection of best practice approaches for studying the functions of histone methylation. In one chapter, Till Bartke and colleagues from the Institute of Functional Epigenetics at Helmholtz Munich provide a series of detailed protocols for the methods they have developed to identify nucleosome-interacting proteins.

How is the message of modified nucleosomes processed and spread in the nucleus?

Since starting his postdoc in the laboratory of Tony Kouzarides at the Gurdon Institute in Cambridge, Till Bartke has aimed at answering this question. To this end, he developed SILAC nucleosome affinity purifications (SNAP) as a tool for studying histone methylation reader proteins (Bartke et al.Cell, 2010). Over the past decade, he and his team have continuously refined and optimized their procedures (Makowski et al.Nat. Commun., 2018Nakamura et al.Nat. Cell Biol., 2019), which they now provide as streamlined protocols to the scientific community. The result is a comprehensive workflow that includes optimized protocols for purifying recombinant (unmodified) human histones and preparing semi-synthetic methylated histones, nucleosome reconstitution using biotinylated DNA, and descriptions of how to use the reconstituted nucleosomes as baits in affinity pull-downs, as well as a fast and reliable approach to analyze the pull-down samples by mass spectrometry in order to identify modification-specific nucleosome interacting proteins. Till Bartke says: “Our aim was to compile a collection of the main procedures that we use in the lab and to provide them in a standardized format for everyone to follow. Our protocols are versatile and easy to customize so that they can be applied to different biological questions.” In the notes section the chapter also sums up the lessons learned during the past years while developing the protocols, including valuable tips, tricks, and background information, making it a useful resource for other researchers even if they are not biochemists or proteomics specialists.

For more detailed information, please contact Till Bartke.

Reference: Tvardovskiy A, Nguyen N, and Bartke T. (2022). Identifying specific protein interactors of nucleosomes carrying methylated histones using quantitative mass spectrometryMethods Mol. Biol. 2529, 327-403. DOI: 10.1007/978-1-0716-2481-4_16