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J Biomol Screen DOI:10.1177/1087057112455060

Screening for inhibitors of an essential chromatin remodeler in mouse embryonic stem cells by monitoring transcriptional regulation.

Publication TypeJournal Article
Year of Publication2012
AuthorsDykhuizen, EC, Carmody, LC, Tolliday, N, Crabtree, GR, Palmer, MAJ
JournalJ Biomol Screen
Date Published2012 Oct
KeywordsAnimals, Cell Line, Chromatin, Chromatin Assembly and Disassembly, Chromosomal Proteins, Non-Histone, DNA-Binding Proteins, Embryonic Stem Cells, Gene Expression Regulation, Developmental, Genes, Reporter, High-Throughput Screening Assays, Luciferases, Mice, Polycomb Repressive Complex 1, Proto-Oncogene Proteins, Reverse Transcriptase Polymerase Chain Reaction, Small Molecule Libraries, Transcription Factors

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.


Alternate JournalJ Biomol Screen
PubMed ID22853929
PubMed Central IDPMC4097195
Grant ListR37 GM038627 / GM / NIGMS NIH HHS / United States
NS046789 / NS / NINDS NIH HHS / United States
R01 NS046789 / NS / NINDS NIH HHS / United States
R37 NS046789 / NS / NINDS NIH HHS / United States
R01 GM038627 / GM / NIGMS NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 CA163915 / CA / NCI NIH HHS / United States
GM38627 / GM / NIGMS NIH HHS / United States
N01CO12400 / CA / NCI NIH HHS / United States
N01-CO-12400 / CO / NCI NIH HHS / United States