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Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

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A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone

Roy Baas¹, Daphne Lelieveld, Hetty van Teeffelen

¹1Department of Molecular Cancer Research, University Medical Centre, Utrecht, The Netherlands.

Journal of Biomolecular Screening

|December 17, 2013

Summary

We developed a new screening method, CROSS, to find proteins that control histone modifications. This powerful tool helps identify epigenetic regulators and potential drug targets for cancer.

Keywords:

RNA interference RNAi cancer and cancer drugs cell-based assays epigenetics microscopy shRNA

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Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

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Published on: January 12, 2024

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Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2)

Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2)

Published on: December 19, 2025

Area of Science:

Epigenetics and Molecular Biology
Chromatin Biology
Gene Regulation

Background:

Posttranslational modifications of histones are crucial for regulating gene expression and chromatin structure in eukaryotes.
The dynamic balance of histone mark deposition (writers) and removal (erasers) dictates chromatin modification levels.
Identifying proteins that modulate these histone marks is essential for understanding epigenetic regulation.

Purpose of the Study:

To introduce a novel, high-throughput microscopy-based screening method for identifying proteins that regulate histone modification levels.
To validate the method's efficacy in discovering chromatin factors involved in specific histone methylation marks.
To demonstrate the method's utility in identifying factors affecting cancer cell growth.

Main Methods:

Developed a screening method named CROSS (Chromatin Regulation Ontology SiRNA Screening).
Utilized a siRNA library targeting 529 proteins involved in chromatin regulation.
Employed microscopy to assess changes in histone modification levels after protein depletion.

Main Results:

Successfully identified chromatin factors regulating histone H3 methylation at lysine-4 and lysine-27.
Demonstrated the method's capability to identify proteins influencing cancer cell line growth.
Validated CROSS as an effective tool for high-throughput screening of epigenetic regulators.

Conclusions:

CROSS is a powerful and versatile method for identifying novel and known histone 'writers' and 'erasers'.
This screening approach facilitates the discovery of potential drug targets for modulating epigenetic modifications.
The method has broad applications in understanding chromatin regulation and developing epigenetic therapies.