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.

Figure 1

The esBAF complex associated with chromatin. The specificity imparted by the particular composition of BAF subunits in embryonic stem (ES) cells is in part through the combinatorial assembly of chromatin targeting motifs, including DNA-binding domains, bromodomains, chromodomains, and plant homeodomains.

Figure 2

Depletion of BRG1 results in increased Bmi1 expression. (A) BRG1 protein expression in embryonic stem (ES) cells homozygous for a floxed Brg1 allele with an actin-CreER transgene after 72 h of EtOH or tamoxifen treatment. (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) of Brg1 and Bmi1 mRNA expression following tamoxifen treatment in the Brg1f/f-actin-CreER ES cell line. Fold change is calculated using Gapdh as an internal control. (C) BRG1 protein expression in E14 ES cells 72 h after treatment with an empty lentiviral construct or a lentiviral construct containing shBrg1. (D) Induction of Bmi1 transcript levels in E14 ES cells 72 h after treatment with shBrg1.

Figure 3

C_T value as a function of number of cells plated per well of a 384-well plate. The direct relationship between Bmi1 and actin C_T values means that the ΔΔC_T value provides an accurate normalization of Bmi1 transcript levels by actin, even at low densities of cells.

Figure 4

(A) Scatter plot of the quantitative polymerase chain reaction (qPCR) primary screen. Broad ID refers to a unique compound identifier assigned to each compound screened. The activity score is calculated as −ΔΔC_T. An activity score of –1.33 was set as the cutoff for hits (green line), which correlates to a 2.5-fold increase in Bmi1 transcript levels. Solid gray line represents no change in Bmi1 transcript levels (mean value for DMSO-treated wells); dotted gray lines represent three standard deviations from this mean. Active: both replicates meet the hit calling threshold; inconclusive: only one replicate meets the hit calling threshold; inactive: neither replicate meets the hit calling threshold. (B) Dose response of Bmi1 induction by a representative hit (compound 63), as determined by quantitative reverse transcriptase PCR (qRT-PCR). (C) Dose response of Bmi1 induction by compound 63, as determined by luciferase levels using the reporter embryonic stem (ES) cell line.

Figure 5

Increase in Bmi1 levels upon BRG1 depletion as indicated by the luciferase reporter. Data are shown at 72 h after viral infection with shRNA against Brg1.

Figure 6

Transcriptional profiles of E14 ES cells treated with shBrg1 (Brg1 KD) and three representative hits. The fold change was calculated as comparison to DMSO-treated embryonic stem (ES) cells using Gapdh as the housekeeping gene. Twenty compounds out of ∼30 000 screened produced a transcriptional profile similar to Brg1-deficient cells.