Fig 2: Multiple signals contribute to the R-point transition. a HEK293 cells were treated with MEK1 inhibitor (U0126, 1 μM). Time-dependent interactions of BRD2–RUNX3 and p300–RUNX3, as well as phosphorylation of ERK1/2, were monitored by IP and/or IB. b HEK293 cells were treated with control or CyclinD1-specific siRNA (si-con or si-CycD1). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 and pRB at Ser-795 were measured by IP and IB. c Time-dependent formation of the JNK-CDK4 complexes and phosphorylations of RUNX3 at Ser-356 and CDK4 at Thr-172 were measured by IP and IB. d HEK293 cells were treated with JNK inhibitor (JNK-IN-8, 1 μM). Time-dependent formation of the BRD2–RUNX3 and RUNX3–CDK4 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. e HEK293 cells were treated with control or JNK-specific siRNA (si-con or si-JNK). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. f HEK293 cells were transfected with Myc-RUNX3 and Flag-CDK4 WT or Flag-CDK4-T172A (CDK4 mutant defective in phosphorylation by JNK). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent phosphorylation of pRB and expression of ARF were measured by IB. g HEK293 cells were treated with control or PIK3CA-specific siRNA (si-con or si- PIK3CA). Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. h HEK293 cells were treated with control or mTORC1 inhibitor (Rapamycin, 100 nM). Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. Ribosomal protein S6 kinase beta-1 (S6K1), which is phosphorylated by mTOR signaling, was used for control

Fig 3: The RUNX3–BRD2–nucleosome complex recruits SWI/SNF and TFIID. a Schematic diagram of BRD2 structure and interacting proteins. BD1 interacts with RUNX3 acetylated at Lys-94 and Lys-171; BD2 interacts with acetylated histones H4K4-ac, H4K12-ac, and H3K14-ac; and the C-terminal region interacts with the TFIID and SWI/SNF complexes. b, c HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points, and the levels of the indicated proteins were measured by IP and IB. The time-dependent interactions were measured by IP and IB. d HEK293 cells were treated with control siRNA (si-con) or BRD2-specific siRNA (si-BRD2), serum-starved for 24 h, and then stimulated with 10% serum for the indicated durations. The time-dependent interactions between the proteins were measured by IP and IB. e HEK293 cells were transfected with Myc-RUNX3, Flag-BRD2-WT, Flag-BRD2-ΔCt (lacking C-terminal aa 633–802), Flag-BRD2-ΔBD1 (lacking BD1), or Flag-BRD2-ΔBD2 (lacking BD2). Cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested after 2 h, and the interactions of the proteins were measured by IP and IB. f The RUNX3-binding site (GACCGCA) in the ARF enhancer region (ntd –1466) was deleted in HEK293 cells by the CRISPR/Cas9 method to obtain the HEK293-ARF-RX-D cell line. Deletion of the RUNX3-binding site was confirmed by nucleotide sequencing. Wild-type HEK293 cells (HEK293-ARF-WT) and HEK293-ARF-RX-D cells were serum-starved for 24 h. The cells were then treated with 10% serum, and the binding of the indicated proteins to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. g Schematic illustration of sequential molecular events at RUNX3 target loci during R-point regulation. RUNX3 binds to condensed chromatin marked by H3K27-me3 (inhibitory mark). p300 recruited to the loci acetylates RUNX3 and histones. Then, BRD2 binds both acetylated RUNX3 and acetylated histone through its two bromodomains. At 1 h after serum stimulation, SWI/SNF and TFIID are recruited to the loci through the C-terminal region of BRD2 to form Rpa-RX3-AC, and H3K27-me3 is replaced by H3K4-me3 (activating mark)

Fig 4: CDK4 plays key roles in the R-point transition. a HEK293 cells were serum-starved for 24 h, stimulated with 10% serum, and harvested at the indicated time points. Time-dependent formation of the BRD2–RUNX3, E2F1–RUNX3, CDK4–RUNX3, Cyclin D1–RUNX3, HDAC4–RUNX3, p16INK4a–CDK4, p21–CDK4, Cyclin D1–CDK4, and HDAC4–CDK4 complexes was measured by IP and IB. Time-dependent phosphorylation of pRB (at Ser-795) and ERK1/2 was measured by IB. b PLA assay showing the RUNX3–CDK interaction 2 h after serum stimulation. c HEK293 cells were treated with control or CDK4-specific siRNA (si-con or si-CDK4), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3, CDK4–RUNX3, HDAC4–RUNX3, and Cyclin D1–RUNX3 complexes and phosphorylated RUNX3 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. d HEK293 cells were treated with CDK4 inhibitor (PD0332991, 500 nM), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. e Cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent RUNX3–CDK4 interaction and RUNX3 phosphorylation at Ser-356 were measured by IP and IB. f, g HEK293 cells were transfected with Myc-RUNX3, Myc-RUNX3-S356A, or Myc-RUNX3-S356E, serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent formation of the BRD2–RUNX3 complex, RUNX3 phosphorylation at Ser-356, and ARF expression were monitored by IP and IB. h Schematic illustration of the process of Rpa-RX3-AC → Rpa-RX3-TR transition. CDK4 of Rpa-RX3-AC and Cyclin D1 of PRC1–Cyclin D1–HDAC4 provide docking sites for the interaction of the two complexes, enabling formation of Rpa-RX3-TR

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