title: A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.
abstract: Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin regulation. As a proof of principle, we used CROSS to identify chromatin factors involved in histone H3 methylation on either lysine-4 or lysine-27. Furthermore, we show that CROSS can be used to identify chromatin factors that affect growth in cancer cell lines. Taken together, CROSS is a powerful method to identify the writers and erasers of novel and known chromatin marks and facilitates the identification of drugs targeting epigenetic modifications.
authors: 1Department of Molecular Cancer Research, University Medical Centre, Utrecht, The Netherlands.
authors: Roy
authors: R
authors: Baas
authors: Daphne
authors: D
authors: Lelieveld
authors: Hetty
authors: H
authors: van Teeffelen
authors: Philip
authors: P
authors: Lijnzaad
authors: Bas
authors: B
authors: Castelijns
authors: F M
authors: FM
authors: van Schaik
authors: Michiel
authors: M
authors: Vermeulen
authors: David A
authors: DA
authors: Egan
authors: H Th Marc
authors: HT
authors: Timmers
authors: Petra
authors: P
authors: de Graaf
mesh_terms: D002460:Cell Line; D002843:Chromatin / Q000235:genetics; D044127:Epigenesis, Genetic; D057166:High-Throughput Screening Assays; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics; D008745:Methylation; D008853:Microscopy; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D002843:Chromatin; D006657:Histones; D011506:Proteins; D008239:Lysine
keywords: RNA interference; RNAi; cancer and cancer drugs; cell-based assays; epigenetics; microscopy; shRNA