Figure 1.
Differential expression analysis between hPSCs and hFs and the in silico screening of transcription factor (TF) binding sites in gene promoters, returned a gene list with corresponding scores. In screening for TFA, the enrichment of high target propensity genes in differentially-expressed genes was assessed. Significant TFs were further assessed for their transcript and protein levels. Subsequently, functional gene groups were evaluated for regulation by the validated TFs (target-cohort analysis). hPSC properties were then elucidated in terms of the functions of regulated gene groups as well as biological information from databases and literature. Trans-activation of interesting target-cohorts by the TF was also validated using luciferase assay.
Figure 2.
Significant E2F activity and E2F1 differential expression between hPSCs and hFs.
(A) Box plots depicting statistical significance of TFA for all 22 TRANFAC PWMs, based on 7 hPSC-hF comparisons. (B) Box plots summarizing statistical significance of TFA for 7 hPSC- hF comparisons, based on 22 PWMs. Median −log10(P-value) is greater than 10 for most hPSC-hF comparisons. The whiskers represent 10th and 90th percentiles while the circles outside them are outliers. (C) Quantitative Real-time PCR analysis of gene expression in hPSCs [iPS(IMR90), iPS(foreskin) and HES-3] and hFs [IMR-90, Hs27 and Hs68]. Gene expression was normalized to that of GAPDH and expressed as fold change relative to HES-3. The values shown are mean ± SD of a representative experiment performed in triplicate and repeated twice for each biological replicate (cell line). (D) Verification of up-regulation of the TF in hPSCs compared to hFs. Actin served as loading control.
Figure 3.
Identification of target-cohorts in WNT and FGF self-renewal pathways.
(A) Target propensity separation threshold used to separate genes above and below E2F target propensity thresholds. The P-values at each target propensity threshold for various measures of differential expression profiles is computed during target-cohort analysis. The average P-value at each target propensity threshold is used to determine the optimal target propensity threshold to separate high and low target propensity genes. As target propensity threshold decreased to 4.9, statistical significance increases sharply before plateau-ing off. This is called ‘target propensity separation threshold’. (B) Differential expression similarity with E2F1 (covariance measure) is plotted against the differential expression scores for genes below (pink squares) and above (blue triangles) separation threshold. For most genes below separation threshold (with the exception of one outlier), |covariance|<0.135 while differential expression <0.9. For genes above separation threshold, those with |covariance|>0.135 and differential expression >0.9 (outside red box), are called target-cohorts, and likely to be enriched in target genes. (C) E2F target-cohorts identified in WNT pathway using separation threshold = 4.9 (red circle) and 4.0 (black circle). For separation threshold = 4.9, target-cohorts are genes marked with an ‘*’ and have differential expression >0.9 and either |dot product|>3.2 or |covariance|>0.135. To explore more genes for trans-activation by E2F, target propensity separation threshold is lowered to 4.0 with the resulting criteria to identify target-cohorts being differential expression >0.55 and either |dot product|>2.0 or |covariance|>0.1. (D) Similar to the procedure highlighted in (A) and (B), target-cohorts in FGF pathways were identified using separation threshold = 5.1, with differential expression >0.617 and either |covariance|>0.126 or |dot product|>1.87.
Figure 4.
Validation and increased differential expression scores of target-cohorts in various new and canonical E2F functions.
Luciferase activity of wild-type promoter-reporter constructs (WNT components- FRZB, SMAD1 and WNT5A) in (A) hESCs, (B) in hESCs cultured with differentiation medium, and (C) in hESCs with E2F binding sites mutated. (a–c) Fold inductions using E2F expression vectors are in comparison to that of empty vector. All luciferase activities were measured relative to the renilla luciferase internal control. Data are illustrated as mean ± SD of a representative experiment performed in triplicate and repeated twice. (D) Representative gene groups in functional categories identified by target-cohort analysis. The X-axis represents differential expression score while the Y-axis presents the similarity with E2F1 (covariance measure). Overall, high target propensity genes (triangles) have values more dispersed from the origin, compared to low target propensity genes (squares), implying higher differential expression scores and similarities with E2F1. Corresponding P-values were displayed as a column in the order [differential expression, dot Product, covariance].
Figure 5.
Interplay between E2F and other cellular regulators in the direct regulation of self-renewal associated functions.
(A) Co-regulated functions of E2F [14–16, this work], HCF1 [46], [47], NRF1/2 [48], [49] are associated with cell proliferation (B) E2F, WNT and FGF activities are deeply integrated in a self-renewal module of interplay between gene regulation and signal transduction. Besides directly targeting genes with the canonical functions of proliferation, differentiation and apoptosis, E2F transcriptionally regulate the component genes of these signaling pathways with the same functions. Remarkably, WNT pathway is engaged in a positive feedback with cell cycle progression [51]. With mitogens and intracellular regulators (such as E2F) driving cell cycle progression in hPSCs, a self-feeding state of high cell-proliferation may be programmed into hPSCs via the WNT pathway. (C) Key components of WNT and FGF pathway are regulated by E2F. The ovals and blocks represent key components of the pathways while the black arrows depict direction of information flow. E2F target-cohorts (red) and known target genes (black) encoding signaling components, are listed beside them. If a gene is both a known target and a target-cohort, it is colored red. Known targets include WNT2B, SMARCA3, SMARCA5, RRAS, MAP2K7, YHWAE (BIND database), FGF1 [54], FGF2 [53], FGF7, PRKCL2, MAP3K7, MAPK3 (ERK1), [39], [58], SOX2, OCT4, NANOG [55]. Genes denoted with ‘**’ are experimentally verified in this study to be regulated by E2F (Luciferase-based assay) while those with ‘*’ are experimentally shown to be differentially expressed (RT-QPCR).