Fig 2: A schematic representation of the E2F1 transcription level regulated by the interaction between HRP‐3 and HDAC 1/2 and the associated cell cycle changes

Fig 3: Interaction between HRP‐3 and HDAC1/2 on the E2F1 promoter of lung cancer cells. A‐B, Expression levels of HRP‐3 (red/green) and HDAC1 (green)/HDAC2 (red) proteins were determined using confocal microscopy. C, A549 cells were fixed and incubated with mouse or rabbit anti‐HRP‐3 together with mouse anti‐HDAC1 or rabbit‐HDAC2, followed by in situ proximity ligation assay (PLA) analysis. Confocal images of cells with PLA‐positive signals are shown (red dots). Interaction between anti‐HRP‐3 and anti‐α‐tubulin was used as a negative control. *P < .05; **P < .01. D, reChIP assay was performed after an initial immunoprecipitation with anti‐HRP‐3 and anti‐HDAC1/2 antibodies in A549 cells. A protein‐DNA complex was recovered after a second immunoprecipitation with HRP‐3 and HDAC1/2 antibodies. E, A549 cells were transfected with 50 nmol/L scrambled or siHRP‐3 and cultured for 48 h. ChIP assay was performed using sheared chromatin from formaldehyde–cross‐linked A549 cells using anti‐HRP‐3, anti‐HDAC1, anti‐HDAC2, and anti‐E2F1 as the immunoprecipitating antibodies

Fig 4: Depletion of HRP‐3 promotes accumulation of abnormal S‐phase via the E2F1Cyclin E axis. A, Expression levels of HRP‐3 (red) and α‐tubulin (green) protein were determined using confocal microscopy. Cells were identified by DAPI (blue) staining of nuclei. B, A549 cells were synchronized by a double thymidine block released into fresh medium at the indicated time points and analyzed by immunoblotting with antibodies against the indicated proteins (left). Levels of HRP‐3, E2F1, and Cyclin E detected by Western blotting. E2F1 and Cyclin E were used as positive controls for the interphase. Graphs representing mean percentages of G0/G1‐, S, G2/M‐phase, and >4N cell populations synchronized using a double thymidine block are obtained from cell cycle analysis using a FACScan flow cytometer (right). C, S‐phase accumulation analysis of A549 cells (transfected with 50 nmol/L scrambled or siHRP‐3) using a FACScan flow cytometer. *P < .05. D‐E, A549 cells were transfected with 50 nmol/L scrambled and siHRP‐3 and harvested. The mRNA levels and protein levels of HRP‐3, E2F1, and Cyclin E were determined by qRT‐PCR and Western blotting. **P < .01. F, A549 cells were cotransfected with 50 nmol/L siHRP‐3 and 1 µg control constructs or constructs containing the human E2F1 promoter for 24 h. E2F1 promoter transcriptional activity was assessed using a luciferase reporter gene assay. **P < .01. G‐H, A549 cells were transfected with either 50 nmol/L of siHRP‐3, 50 nmol/L of siE2F1, or both. The mRNA levels and protein levels of HRP‐3, E2F1, and Cyclin E were determined by qRT‐PCR and Western blotting. **P < .01. I, S‐phase accumulation analysis in A549 cells transfected with either 50 nmol/L siHRP‐3, 50 nmol/L siE2F1, 50 nmol/L siCyclin E, or all three of them using a FACScan flow cytometer. *P < .05

Fig 5: The sequential molecular events for the R-point decision. a, b Upon mitogenic stimulation, RUNX3 binds to inactive chromatin marked by H3K27-me3. pRB–E2F1 and p300 associate with RUNX3. p300 acetylates RUNX3 and histones. BRD2 binds to acetylated RUNX3 through its first bromodomain (BD1). c One hour after mitogenic stimulation, the second bromodomain (BD2) of BRD2 binds to H4K12-ac: BRD2 binds both acetylated RUNX3 and acetylated histone through its bromodomains. Subsequently, SWI/SNF, MLL1/5, and TFIID bind to the C-terminal region of BRD2. At this point, inhibitory histone marks (H3K27-me3) are erased, and activatory marks (H3K4-me3) are enriched at the locus. Soon thereafter, TAF7 (inhibitory TAF) is released from the large complex, and expression of ARF, p53, and p21 is induced. The large complex, of which RUNX3 is the core, was named as Rpa-RX3-AC. d Two hours after mitogenic stimulation, CDK4 (associated with p21) binds to RUNX3 and becomes an additional component of Rpa-RX3-AC. At this point, the Cyclin D1–PRC1 complex forms separately from Rpa-RX3-AC. e When the RAS–MEK signal is downregulated, the Cyclin D1–PRC1 complex matures into the Cyclin D1–HDAC4–PRC1 complex, which in turn binds to Rpa-RX3-AC through the interaction between Cyclin D1 and CDK4 (a component of Rpa-RX3-AC), yielding Rpa-RX3-TR. Activation of CDK4 through the association with Cyclin D1 is critical for the inactivation of the chromatin loci and the dissociation of the entire complex. If the RAS signal is constitutively activated, the Cyclin D1–PRC1 complex fails to mature into the Cyclin D1–HDAC4–PRC1 complex, and consequently cannot form Rpa-RX3-TR. Therefore, if R-point commitment is normal, cells expressing constitutively active RAS cannot progress through the R-point into S-phase. f If the mitogenic signal is downregulated in a normal manner, Rpa-RX3-TR dissociates (4 h after stimulation) into two pieces, RUNX3–Cyclin D1–HDAC4 and BRD2–PRC1–SWI/SNF–TFIID, which remain associated with chromatin. g Soon thereafter, EZH2 associates with RUNX3–Cyclin D1–HDAC4 to form Rpa-RX3-RE, which remains on the chromatin. EZH2 contributes to the enrichment of an inactive chromatin mark (H3K27-me3) at the locus