title: Screening for inhibitors of an essential chromatin remodeler in mouse embryonic stem cells by monitoring transcriptional regulation.
abstract: The SWI/SNF-like adenosine triphosphate (ATP)-dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression-based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.
authors.affiliation: Department of Pathology and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. dykhuize@stanford.edu
authors.forename: Emily C
authors.initials: EC
authors.lastname: Dykhuizen
authors.forename: Leigh C
authors.initials: LC
authors.lastname: Carmody
authors.forename: Nicola
authors.initials: N
authors.lastname: Tolliday
authors.forename: Gerald R
authors.initials: GR
authors.lastname: Crabtree
authors.forename: Michelle A J
authors.initials: MA
authors.lastname: Palmer
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects*; D002868:Chromosomal Proteins, Non-Histone / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D063150:Polycomb Repressive Complex 1 / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C489421:Bmi1 protein, mouse; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C445179:SWI-SNF-B chromatin-remodeling complex; D054852:Small Molecule Libraries; D014157:Transcription Factors; D008156:Luciferases; D063150:Polycomb Repressive Complex 1