An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition.

Brian Egan,Paul Labhart,Jennifer Busby,Patrick Trojer,Chisato Henry,Marie Classon,Theresa K Kelly,Tobias M Maile,Suchit Jhunjhunwala,Madeleine Lisa Craske,David Arnott,Charles A Tindell,Gulfem D Guler,Feng Zhao,Barbara M Bryant,Chih-Chi Yuan,Charlie Hatton

doi:10.1371/journal.pone.0166438

Abstract

Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.

Highlights

ChIP-seq is a powerful and commonly used technique for the detection of transcription factor binding patterns and histone post-translational modification (PTM) occupancy profiles acrossPLOS ONE | DOI:10.1371/journal.pone.0166438 November 22, 2016ChIP-Seq Normalization to Detect Global Histone Modification ChangesCAT, SJ, FZ, CH, BMB, MC, PT], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript
The primary method of mapping D. melanogaster tags used a partial dm3 reference genome containing only H2Av bound regions that we identified in H2Av ChIP-seq experiments using only D. melanogaster chromatin from S2 or OSS cells
To better understand how EZH2 inhibitor-mediated reduction in H3K27me3 levels affects the distribution of this modification across the genome, we performed H3K27me3 ChIP-seq in cells treated with EZH2 inhibitors as compared to untreated cells

Summary

Introduction

ChIP-seq is a powerful and commonly used technique for the detection of transcription factor binding patterns and histone post-translational modification (PTM) occupancy profiles acrossPLOS ONE | DOI:10.1371/journal.pone.0166438 November 22, 2016ChIP-Seq Normalization to Detect Global Histone Modification ChangesCAT, SJ, FZ, CH, BMB, MC, PT], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. ChIP-seq is a powerful and commonly used technique for the detection of transcription factor binding patterns and histone post-translational modification (PTM) occupancy profiles across. ChIP-Seq Normalization to Detect Global Histone Modification Changes. CAT, SJ, FZ, CH, BMB, MC, PT], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section

Methods

Results

Conclusion