A small-molecule inducer of PDX1 expression identified by high-throughput screening

Abstract

Pancreatic and duodenal homeobox 1 (PDX1), a member of the homeodomain-containing transcription factor family, is a key transcription factor important for both pancreas development and mature β cell function. The ectopic overexpression of Pdx1, Neurog3, and MafA in mice reprograms acinar cells to insulin-producing cells. We developed a quantitative PCR-based gene expression assay to screen more than 60,000 compounds for expression of each of these genes in the human PANC-1 ductal carcinoma cell line. We identified BRD7552, which upregulated PDX1 expression in both primary human islets and ductal cells, and induced epigenetic changes in the PDX1 promoter consistent with transcriptional activation. Prolonged compound treatment induced both insulin mRNA and protein and also enhanced insulin expression induced by the three-gene combination. These results provide a proof of principle for identifying small molecules that induce expression of transcription factors to control cellular reprogramming.

Figure 1. see also Figure S1, Table S1. Three transcription factors induce beta cell-like characteristics in human PANC-1 cells

Immunofluorescent analysis of control and triply overexpressed (TOE) PANC-1 cells after two weeks in cell culture. (A) C-peptide, (B) NEUROG3, (C) PDX1, (D) MAFA, and (E) cytokeratin-19 (CK19). Nuclei were stained with Hoechst. Scale bars = 100 µm. (F) qPCR analysis of insulin, MAFA, NEUROG3, PDX1, PAX4, GCG0, SST, and PPY gene expression in TOE cells after three-day and two-week culture. Gene expression was normalized by GAPDH expression, and scored relative to control vector-transfected cells. “Ct (Ctrl)” indicates basal Ct value of each gene in DMSO-treated PANC-1 cells. “ΔCt” refers to the GAPDH-normalized levels of expression of these genes in control PANC-1 cells. Data represent the mean ± SDs of three independent experiments. (G) Immunofluorescent analysis of Ki67 (green) performed on TOE cells two weeks after transfection and selection. Nuclei were stained with Hoechst. (H) Total nuclei of TOE cells in each well were detected by Hoechst fluorescence and counted by automated microscopy. Data represent the mean and standard error of eight independent replicates. * p < 0.0001 (t-test). (I) Quantification of PANC-1 colony formation in soft agar by nucleic acid detection. * p < 0.01 (t-test).

Figure 2. see also Figures S2, S3, Table S1. BRD7552 induces PDX1 gene and protein expression

(A) Assay development of gene expression in 384-well plates. Triply overexpressed (TOE) cells were compared to DMSO-treated PANC-1 cells for expression of indicated genes. Z’ factors for each assay are listed below each gene name. (B) Chemical structure of BRD7552. (C) qPCR analysis of PDX1 expression in PANC-1 cells following 3-, 5- and 9-day treatment with the indicated concentrations of BRD7552. “ΔCt (DMSO)” indicates the relative expression level of the target genes compared to control gene GAPDH, as an indication of the basal levels of expression in PANC-1 cells. The fold change was scored relative to control cells treated with DMSO. Data represent the mean ± SDs of three independent experiments. (D) qPCR analysis of PDX1 expression induced by 5 µM BRD7552 at the indicated time points. * p < 0.001 (t-test). (E) Assessment of protein levels of PDX1 and cytokeratin-19 in PANC-1 cells treated with the indicated concentrations of BRD7552 for 5 days. Actin was used as an internal loading control. (F) Quantification of PDX1 protein levels induced by three-day BRD755 treatment, as measured by ELISA. Data represent the mean and standard deviation of three independent experiments.

Figure 3. see also Figure S4, Table S1. BRD7552 induces insulin expression in PANC-1 cells

(A) qPCR analysis of insulin expression on PANC-1 cells following 3-, 5- and 9-day treatment with the indicated concentrations of BRD7552. Gene expression was normalized by GAPDH and actin expression, and scored relative to the control cells treated with DMSO. Data represent the mean ± SDs of three independent experiments. (B) qPCR analysis of PDX1 and (C) insulin expression in dissociated human islet cells from donor 3 (Figure S3), treated with the indicated concentrations BRD7552 for three or five days. Gene expression was normalized by GAPDH and actin expression, and scored relative to the control vector-transfected cells. Data represent the mean ± SDs of three independent experiments. (D) qPCR analysis of PDX1 expression in HDDCs at passage 3 or 4 after incubation with BRD7552 at the indicated concentrations for three days. Gene expression was normalized by TBP expression, and scored as a fold change relative to 0.1% DMSO-treated cells. P: passage.

Figure 4. see also Figures S5, S6, S7, Table S2. Chromatin modifications induced by BRD7552 at the PDX1 promoter are consistent with transcriptional activation

(A) Location of the primer pairs used for ChIP-qPCR for human PDX1 gene. PANC-1 cells were treated with DMSO (light gray bars) and 5 µM BRD7552 (dark gray bars) for three days. Modifications analyzed by ChIP-qPCR: (B) acetylated H3, (C) H3K4me3, (D) H3K9me3, (E) H3K27me3. Data represent the mean ± SDs of four independent qPCR experiments.

Figure 5. see also Figure S8, Table S1. FOXA2-targeted gene set is linked to BRD755 treatment in PANC-1 cells by gene-set enrichment analysis (GSEA)

(A) GSEA enrichment plot showing expression enrichment of a set of FOXA2-downregulated genes. Bars represent individual genes in a ranked data set list. Expression of the majority of these genes was enriched in the DMSO-treated PANC-1 cells compared to BRD7552-treated cells. Heat map of the 27 genes in the leading edge of the gene set. (B) qPCR analysis of PDX1 expression induced by 5 µM BRD7552 and FOXA2 knockdown in PANC-1 cells. Data represent the mean ± SD. * p < 0.001 (t-test).

Figure 6. Most structural features of BRD7552 are required for activity

The common chemical structure of all analogs is shown at the top. Alterations of the stereochemistry at the C2 position, as well as the identities of R (red) and R’ (blue) groups, are indicated below. PDX1 induction of each small molecule was tested at 10 µM after three- and five-day treatments. The fold change was normalized by GAPDH expression, and scored relative to DMSO-treated control cells. Data represent the mean of two independent experiments.

Figure 7. see also Table S1. BRD7552 can partially complement PDX1 in PANC-1 cells

(A) qPCR analysis of insulin expression of transfected PANC-1 cells, in the presence or absence of 5 µM BRD7552, after two weeks in culture. Gene expression was normalized by GAPDH and actin expression, and scored relative to the control vector-transfected cells. Data represent the mean ± SDs of three independent experiments. (B) Immunofluorescence analysis of C-peptide expression (red) in transfected PANC-1 cells in the absence or presence of 5 µM BRD755 after two weeks. Nuclei were stained blue with Hoechst. Scale bars = 100 µm.