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

PLoS One. 2016 Nov 22;11(11):e0166438. doi: 10.1371/journal.pone.0166438. eCollection 2016.


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.

MeSH terms

  • Animals
  • Chromatin Immunoprecipitation
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster
  • Enhancer of Zeste Homolog 2 Protein / antagonists & inhibitors
  • Enhancer of Zeste Homolog 2 Protein / genetics
  • Enhancer of Zeste Homolog 2 Protein / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Genome-Wide Association Study
  • Histones / genetics
  • Histones / metabolism*
  • Humans
  • Methylation / drug effects
  • Sequence Analysis, DNA


  • Drosophila Proteins
  • Enzyme Inhibitors
  • Histones
  • Enhancer of Zeste Homolog 2 Protein

Grant support

No specific funding was obtained for this project. General funds from Constellation Pharmaceuticals, Genentech Inc and Active Motif Inc were used to support the work. The funders provided support in the form of salaries for authors [BE, CCY, MC, PL, GDG, DA, TMM JB, CH, TKK, 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.