Fig 1.
Visualization of transcripts oscillating with the cell cycle reveals expression patterns.
(A) The scheme of the experimental and analytical workflow. (B) HeLa-Fucci cells were sorted into three restricted cell cycle phases, G1, S, and G2/M, according to the expression of Fucci markers. (C) A heat map analysis shows differential gene expression patterns in sorted HeLa-Fucci cells. (D) For each individual transcript in the HeLa-Fucci transcriptome, the TriComp algorithm was used to calculate polar coordinates (θ, r). θ denotes the relationship between gene expression levels in the three cell cycle phases, and r denotes the intensity of this relationship. Genes with similar expression patterns were divided into six categories (6x60°) based on their θ-value. (E) Polar coordinates for all transcripts found to oscillate significantly in synchrony with the cell cycle (FDR≤0.001, logCPM ≥1, FC ≥1.5) in sorted HeLa-Fucci cells show an unequal distribution. (F-H) θ-values describing the distribution of relationship patterns of oscillation for all significantly oscillating transcripts in HeLa cells (FDR≤0.001) (F), transcripts belonging to GO terms associated with the cell cycle (H), and common cell cycle markers (G). Significance stars denote the probability of arising from the same distribution as the full set of significantly oscillating transcripts using log-likelihood-ratio statistical comparison with a six-group binning (***: p < 0.001).
Fig 2.
Comparisons of different cell cycle data sets show consistent expression patterns.
(A) Comparative analysis of HeLa-Fucci and U2OS-Fucci cell cycle-dependent transcriptomes after plotting their respective θ-value for each shared transcript (stringency level: FDR≤0.001). The green area denotes a correlation of less than 60 degrees in the θ-value. (B, C) Number of transcripts in each of the six TriComp θ-categories (FDR≤0.001). (D) Plot of the θ-value for HeLa-Fucci transcripts versus the reported phase peak value for each probe reported by Whitfield et al.[19]. (E) Category distribution plots for the core set of 67 genes reported by [18] versus the θ-value for genes significantly oscillating (FDR≤0.001) in HeLa-Fucci cells.
Fig 3.
Large TF families oscillate and show differential distribution during the cell cycle.
(A-D) θ-value plots for all TFs and TF families with a significantly differing pattern in (A, C) HeLa-Fucci cells and (B, D) U2OS-Fucci cells (FDR ≤0.001; logCPM ≥1; FC ≥1.5). (E) θ-value plot of cell-cycle-synchronized TFs in HeLa-Fucci cells versus U2OS-Fucci cells, with suggested phase-specific expression groups denoted I-VI and the main TFs involved in G1 restriction and onset of S phase indicated in red. (F) Schematic showing how repressor activity may shape transcriptional boundaries during the cell cycle, exemplified by E2F7/8 repression of E2F1/2 expression.
Fig 4.
E2F1-controlled gene expression is compartmentalized.
(A-D) Comparative analysis of E2F (A, C) and non-E2F targets (B, D) was performed using both the HeLa-Fucci and U2OS-Fucci cell cycle-dependent transcriptomes by plotting θ-values, with expression groups 2–5 highlighted by a pink box (A, B), and a distribution analysis with a log-likelihood ratio statistical comparison using the HeLa-Fucci cell θ-values (C, D). (E-H) mRNA expression in the HeLa-Fucci data set (RPKM), and protein levels as Tukey boxplots of average antibody intensities per cell nucleus, subdivided by cell cycle phase according to the quantification of Fucci reporters using fluorescent imaging. Error bars denote SEM. (I) An example of known temporal relationships among cell cycle-controlled TFs, verified by our data.
Fig 5.
Transcriptional networks in G2/M-G1 phases are associated with developmental programmes.
(A, B) Circular plot of θ and r coordinates for selected cell type-specific TF subfamilies expressed in (A) Hela-Fucci cells or (B) U2OS-Fucci cells (FDR ≤0.001, logCPM ≥1, FC ≥1.5). (C, D) GO enrichment analysis of the identified oscillating TFs against a background of all TFs. (E) NOTCH mRNA expression levels (RPKM) in HeLa-Fucci and U2OS-Fucci cells. (F) Representative western blot of total full-length (FL) NOTCH2 and the transmembrane/intracellular region (NTM/ICD) in sorted HeLa-Fucci cells. (G) Quantitation of western blot data for total NOTCH2 protein expression relative to the unsorted sample (Student’s t-test; * p<0.05). (H, J, K) mRNA expression of Notch signalling target genes HES7, NRARP and LFNG in the HeLa-Fucci data set (RPKM). (I) Map of a Notch-dependent oscillator as expressed during embryonic somitogenesis, indicating the key oscillators HES7, NRARP and LFNG. (L-M) Examples of known relationships between TFs identified to oscillate in synchrony with the cell cycle in the (L) HeLa-Fucci and (M) U2OS-Fucci data sets. Error bars denote SEM.
Fig 6.
The circadian clock transcriptional network is synchronized with the cell cycle.
(A) Schematic illustrating the feedback loops of the circadian clock oscillators. (B) Plot of the θ-value for core circadian genes in HeLa-Fucci cells (p-value≤0.001). (C) Protein expression levels of core components of the circadian clock in HeLa-Fucci cells analysed by correlating fluorescent immunostaining intensity to cell cycle phase determined by Fucci reporters or DNA content (DAPI); bars represent the mean of the logarithmic intensity of cells relative the average mean logarithmic intensity of all cells. Error bars denote SEM. (D) Comparison between cell cycle oscillating transcripts in HeLa-Fucci cells (FDR≤0.001) and a published circadian clock transcriptome in non-proliferating liver cells[55]. (E) Plot of the θ-values for core circadian genes in HeLa-Fucci cells (FDR≤0.001) versus the circadian peak values in liver cells reported by Yoshitane H, et al. 2014. (F) Proposed model of the integration of cell cycle, circadian clock and genes associated with development found to be synchronized with the cell cycle.