Figure 3.
β-Catenin and FOXO3 mutually promote the expression of LIC-related genes. (A) Schematic overview of experimental approach. Inactivating DNA mutations in CTNNB1 and FOXO3 genes were serially induced by the direct delivery of ribonucleoprotein Cas9 complex. Positive clones were selected after serial dilutions into a 96-well plate and validated by DNA sequencing of target regions and western blot analysis for protein expression. Afterward, the FOXO3null CTNNB1null cells were cotransduced with an active FOXO3-TM alone or in combination with a stable isoform of β-catenin (ΔGSK) for performing RNA-seq and functional assays. (B) Heat map and hierarchical clustering of gene expression RNA-seq data. FOXO3null CTNNB1null PF382 cells were transduced as reported in panel A and sorted by FACS for RNA isolation and sequencing. Differentially expressed genes, scaled with mean is 0 and standard deviation is 1, are represented (adjusted P ≤ .1 and log fold change ≤ −0.5) by Morpheus-Broad Institute software (https://software.broadinstitute.org/morpheus). (C) Table of the hallmark gene sets significantly enriched in FOXO3null CTNNB1null PF382 cells, doubly transduced with β-catenin (ΔGSK) and FOXO3 € with respect to other cell conditions by gene set enrichment analysis of RNA-seq data. (D) Location of predicted FOXO3 and β-catenin sites relative to transcription start site and FOXO3 and β-catenin chromatin immunoprecipitation (ChIP-seq) signal intensity (ChIP-seq score in log2). In the plot, each dot represents overlapping FOXO3 and β-catenin peaks within 1 kilobase around the transcription start site. The ChIP-seq peaks over 140 genes highly expressed in the FOXO3null CTNNB1null PF382 cells cotransduced with FOXO3-TM and β-catenin (ΔGSK) are highlighted in red. (E) ChIP-seq analysis. ChIP-seq was performed with the anti–β-catenin, anti-FOXO3, anti-H3K27me3, and anti-H3K27Ac antibodies in PF382 cell line. Peaks of aligned reads over the CDK4 locus are shown along with MACS2 peak calls (P ≤ .05). The active genomic region identified as overlapping between FOXO3 and β-catenin ChIP-seq peaks in the CDK4 locus is highlighted in red. (F) CDK4 messenger RNA expression level of human FOXO3null CTNNB1null PF382 and RPMI-8402 cell lines with engineered levels of the active FOXO3-TM mutant alone or in combination with the stable ΔGSK isoform of β-catenin. Transduced cells were sorted by FACS sorting and RNA was isolated to perform the TaqMan reverse transcription droplet digital polymerase chain reaction assay. (G) Protein expression level of intracellular CDK4 by flow cytometric analysis in the transduced cells as reported in panel F. crRNA, crispr RNA; ES, enrichment score; FDR, false discovery rate; NES, normalized enrichment score; NOM, nominal P value.

β-Catenin and FOXO3 mutually promote the expression of LIC-related genes. (A) Schematic overview of experimental approach. Inactivating DNA mutations in CTNNB1 and FOXO3 genes were serially induced by the direct delivery of ribonucleoprotein Cas9 complex. Positive clones were selected after serial dilutions into a 96-well plate and validated by DNA sequencing of target regions and western blot analysis for protein expression. Afterward, the FOXO3null CTNNB1null cells were cotransduced with an active FOXO3-TM alone or in combination with a stable isoform of β-catenin (ΔGSK) for performing RNA-seq and functional assays. (B) Heat map and hierarchical clustering of gene expression RNA-seq data. FOXO3null CTNNB1null PF382 cells were transduced as reported in panel A and sorted by FACS for RNA isolation and sequencing. Differentially expressed genes, scaled with mean is 0 and standard deviation is 1, are represented (adjusted P ≤ .1 and log fold change ≤ −0.5) by Morpheus-Broad Institute software (https://software.broadinstitute.org/morpheus). (C) Table of the hallmark gene sets significantly enriched in FOXO3null CTNNB1null PF382 cells, doubly transduced with β-catenin (ΔGSK) and FOXO3 € with respect to other cell conditions by gene set enrichment analysis of RNA-seq data. (D) Location of predicted FOXO3 and β-catenin sites relative to transcription start site and FOXO3 and β-catenin chromatin immunoprecipitation (ChIP-seq) signal intensity (ChIP-seq score in log2). In the plot, each dot represents overlapping FOXO3 and β-catenin peaks within 1 kilobase around the transcription start site. The ChIP-seq peaks over 140 genes highly expressed in the FOXO3null CTNNB1null PF382 cells cotransduced with FOXO3-TM and β-catenin (ΔGSK) are highlighted in red. (E) ChIP-seq analysis. ChIP-seq was performed with the anti–β-catenin, anti-FOXO3, anti-H3K27me3, and anti-H3K27Ac antibodies in PF382 cell line. Peaks of aligned reads over the CDK4 locus are shown along with MACS2 peak calls (P ≤ .05). The active genomic region identified as overlapping between FOXO3 and β-catenin ChIP-seq peaks in the CDK4 locus is highlighted in red. (F) CDK4 messenger RNA expression level of human FOXO3null CTNNB1null PF382 and RPMI-8402 cell lines with engineered levels of the active FOXO3-TM mutant alone or in combination with the stable ΔGSK isoform of β-catenin. Transduced cells were sorted by FACS sorting and RNA was isolated to perform the TaqMan reverse transcription droplet digital polymerase chain reaction assay. (G) Protein expression level of intracellular CDK4 by flow cytometric analysis in the transduced cells as reported in panel F. crRNA, crispr RNA; ES, enrichment score; FDR, false discovery rate; NES, normalized enrichment score; NOM, nominal P value.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal