title: An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition.
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.
authors.affiliation: Active Motif Inc., Carlsbad, California, United States of America.
authors.forename: Brian
authors.identifier: http://orcid.org/0000-0002-6354-6575
authors.initials: B
authors.lastname: Egan
authors.affiliation: Constellation Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Chih-Chi
authors.initials: CC
authors.lastname: Yuan
authors.affiliation: Active Motif Inc., Carlsbad, California, United States of America.
authors.forename: Madeleine Lisa
authors.initials: ML
authors.lastname: Craske
authors.affiliation: Active Motif Inc., Carlsbad, California, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: Labhart
authors.affiliation: Department of Molecular Oncology, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Gulfem D
authors.initials: GD
authors.lastname: Guler
authors.affiliation: Department of Protein Chemistry, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Arnott
authors.affiliation: Department of Protein Chemistry, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Maile
authors.affiliation: Constellation Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Busby
authors.affiliation: Active Motif Inc., Carlsbad, California, United States of America.
authors.forename: Chisato
authors.initials: C
authors.lastname: Henry
authors.affiliation: Active Motif Inc., Carlsbad, California, United States of America.
authors.forename: Theresa K
authors.initials: TK
authors.lastname: Kelly
authors.affiliation: Department of Molecular Oncology, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Tindell
authors.affiliation: Department of Bioinformatics, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Suchit
authors.initials: S
authors.lastname: Jhunjhunwala
authors.affiliation: Constellation Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhao
authors.affiliation: Constellation Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Charlie
authors.initials: C
authors.lastname: Hatton
authors.affiliation: Constellation Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Barbara M
authors.initials: BM
authors.lastname: Bryant
authors.affiliation: Department of Molecular Oncology, Genentech Inc., South San Francisco, California, United States of America.
authors.forename: Marie
authors.initials: M
authors.lastname: Classon
authors.affiliation: Constellation Pharmaceuticals Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Patrick
authors.initials: P
authors.lastname: Trojer
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D000071221:Enhancer of Zeste Homolog 2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D055106:Genome-Wide Association Study; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008745:Methylation / Q000187:drug effects; D017422:Sequence Analysis, DNA
chemical_list: D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D006657:Histones; D000071221:Enhancer of Zeste Homolog 2 Protein