title: Preparation and Analysis of Native Chromatin-Modifying Complexes.
abstract: Nucleosomes, the basic units of chromatin, are decorated with a myriad of posttranslational modifications (PTMs) by the action of chromatin modifiers. These enzymes function almost exclusively as part of stable protein complexes that assist their recruitment to specific genomic loci, specify their substrate, and provide allosteric control. By altering the interactions within nucleosomes or with neighboring nucleosomes and serving as a platform to engage effector proteins, PTMs deposited by histone-modifying complexes influence virtually every nuclear process and are at the heart of the epigenetic mechanisms. Hence, it is critical to identify their components, define their structures, and characterize their biochemical activities. Here we describe protocols for tandem affinity purification (TAP) of native histone acetyltransferase (HAT) and methyltransferase (HMT) complexes from human cells engineered to express bait proteins from a genomic safe harbor or their endogenous chromosomal genes, using zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems. The approaches presented aim to preserve natural transcriptional and posttranscriptional regulation and minimize biochemical artifacts due to ectopic expression. Near homogenous preparations of native complexes are obtained in sufficient amounts to perform biochemical assays and characterize their components.
authors.affiliation: Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC, Canada. Electronic address: yannick.doyon@crchudequebec.ulaval.ca.
authors.forename: Y
authors.initials: Y
authors.lastname: Doyon
authors.affiliation: Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC, Canada; St-Patrick Research Group in Basic Oncology and Laval University Cancer Research Center, Quebec City, QC, Canada. Electronic address: jacques.cote@crhdq.ulaval.ca.
authors.forename: J
authors.initials: J
authors.lastname: Côté
mesh_terms: D064113:CRISPR-Cas Systems; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D000071221:Enhancer of Zeste Homolog 2 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D051548:Histone Acetyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; C485469:EPC1 protein, human; D012097:Repressor Proteins; D000076983:Histone Methyltransferases; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D011495:Histone-Lysine N-Methyltransferase; D051548:Histone Acetyltransferases; D004720:Endonucleases; D000069896:Transcription Activator-Like Effector Nucleases
keywords: CRISPR/Cas9; EZH2; Genome editing; Histone acetyltransferase; Histone methyltransferase; NuA4; PRC2; Protein complexes; TAL effector nucleases; TIP60; Tandem affinity purification; Zinc-finger nucleases