Internal Ribosome Entry Site-Based Bicistronic In Situ Reporter Assays for Discovery of Transcription-Targeted Lead Compounds

Abstract

Although transgene-based reporter gene assays have been used to discover small molecules targeting expression of cancer-driving genes, the success is limited due to the fact that reporter gene expression regulated by incomplete cis-acting elements and foreign epigenetic environments does not faithfully reproduce chemical responses of endogenous genes. Here, we present an internal ribosome entry site-based strategy for bicistronically co-expressing reporter genes with an endogenous gene in the native gene locus, yielding an in situ reporter assay closely mimicking endogenous gene expression without disintegrating its function. This strategy combines the CRISPR-Cas9-mediated genome-editing tool with the recombinase-mediated cassette-exchange technology, and allows for rapid development of orthogonal assays for excluding false hits generated from primary screens. We validated this strategy by developing a screening platform for identifying compounds targeting oncogenic eIF4E, and demonstrated that the novel reporter assays are powerful in searching for transcription-targeted lead compounds with high confidence.

Figure 1. The schematic shows co-expression of a bicistronic in-situ reporter with an endogenous gene under the control of the native transcriptional regulatory machinery

IRES, internal ribosome entry site; Luc, luciferase gene; TF, transcription factors; GTF, general transcription factors; PolII, RNA polymerase II.

Figure 2. Genome editing by CRISPR-Cas9 followed by RMCE generates cells harboring a reporter gene in the endogenous EIF4E locus

(A) The schematic showing the two-step strategy for inserting the IRES-FLuc cassette into the EIF4E gene locus through CRISPR-Cas9 and RMCE. DSB, DNA double-strand break; LA, left homology arm; RA, right homology arm; PAM, protospacer adjacent motif; F and F3, wild-type and mutant FRT sites; sgEIF4E, EIF4E-specific sgRNA; pA, polyadenylation signal; TAA, the stop codon. (B) Identification of Zeocin-resistant clones carrying the tk-ble gene in the targeted EIF4E locus. Genomic DNAs from resistant clones were subjected to PCR using a primer pair RA-F and RA-R indicated in (B). Positive clones were expected to generate a ~1.5-kb fragment. (C) The tk-ble gene was replaced by the IRES-FLuc cassette in E8-FLuc clones confirmed by PCR. The primers are indicated in (B). The E8 clone carrying the tk-ble gene in the targeted EIF4E locus was chosen for RMCE. (D) Northern blotting confirmed co-expression of the FLuc gene and the endogenous EIF4E gene as single transcripts. Arrows indicate the fused, bicistronic transcripts. (E) Immunoblotting detected the eIF4E protein. No fusion of eIF4E to FLuc was found in F8-FLuc cells. (F) FLuc was highly expressed in recombinant cells. Clones were lysed for firefly luciferase activity assays. The FLuc expression level was comparable among clones. (G) E8-Fluc clones exhibited similar responses to chemical treatments. The indicated clones were treated with 2.5 μM of NSC607097 for 16 h for luciferase activity assays. See also Figure S1.

Figure 3. A drug screen using the bicistronic in-situ reporter assays identifies EIF4E inhibitors with a significantly reduced false positive rate

(A) The schematic showing the screening strategy. A random E8-FLuc clone and a clone (F55-pEIF4E-luc) carrying a ~ 1.5 kb EIF4E promoter in a permissive genomic site were treated with library chemicals in microplates for luciferase activity assays. (B) The results of E8-FLuc-based screen (red) and F55-pEIF4E-luc-based screen (blue). Recombinant cells in 96-well plates were treated with ~ 4,800 compounds (2.5 μM) for 16 h, and then lysed for firefly luciferase activity assays. The relative luciferase activities were converted into logarithm values (binary logarithm, i.e., log2) and plotted for each compound. The cutoff values set for positives are ±0.5849, i.e., either decrease to at least 66.7% or increase to at least 150% of the DMSO group, and indicated by the dotted lines. Chemicals that decreased the luciferase activity due to cytotoxicity were identified by MTT assays, and excluded from further investigation in this study. These chemicals were not shown in this graph. (C) Venn Diagram showing the numbers of hits (i.e., decreasing the FLuc activity) from the screens using E8-FLuc or F55-pEIF4E-luc cells. (D) qRT-PCR validation of the hits from two screens. The dotted lines indicate the cutoff (<0.667) for positives. Error bars represent SD for three replicate measurements. (E) Venn Diagram showing the validated EIF4E inhibitors. See also Figure S2 and Figure S3.

Figure 4. An orthogonal screening assay based on RLuc inserted in the same EIF4E locus identifies a majority of false hits

(A) Diagram showing that RMCE mediates rapid insertion of the RLuc gene into the same genomic location as FLuc. (B) PCR results confirmed the replacement of the tk-ble gene with RLuc in E8-RLuc cells. (C) E8-RLuc cells responded to chemical treatments as same as E8-FLuc cells. Cells were treated with NSC607097 for 16 h for luciferase activity assays. (D) Effects of primary hits (including those compounds that increased the FLuc activity by at least 1 fold) on EIF4E mRNA level and reporter gene activity. The only false hit (NSC321239) that could not be identified by the orthogonal assay is indicated by the red arrow. (E) NSC321239 is an inhibitor of both FLuc and RLuc. Lysates from cells expressing FLuc or RLuc were incubated with 2.5 μM of NSC321239, or Pifithrin-α (a known FLuc inhibitor), on ice before assaying for FLuc or RLuc activity. Error bars represent SD for three replicate measurements.