Fig. 7
R-point surveils aberrant oncogene activation. a HEK293 cells were transfected with empty vector (Vec) or Myc-K-RASG12V. The time-dependent interactions among the components of Rpa-RX3-AC, Rpa-RX3-TR, and Rpa-RX3-RE were measured by IP and IB. Expression levels of ARF, p53, p21, and Myc-K-RasG12V were measured by IB. b Binding of the components of Rpa-RX3-AC, Rpa-RX3-TR, and Rpa-RX3-RE to the p14ARF promoter and histone marks (H4K12-ac, H3K27-me3, H3K4-me3, and H2A-K119-Ubi) at the locus were measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. c Wild-type HEK293 cells (HEK293-ARF-WT) and HEK293-ARF-RX-D cells were transfected with empty vector (Vec) or Myc-K-RasG12V. The binding of RUNX3, BRD2, H4K12-ac, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. d HEK293 cells were transfected with empty vector (Vec) or B-RAFV600E. Time-dependent formation of the RUNX3–p300 and BRD2–RUNX3 complexes was measured by IP and IB. Time-dependent expression of ARF and p53 was measured by IB. e HEK293 cells were transfected with empty vector (Vec) or Flag-MEK1-CA. Time-dependent interactions of CyclinD1–HDAC4 and CyclinD1–RNF2 were monitored by IP and IB. f Schematic illustration of differential regulation of the R-point in response to normal and oncogenic RAS. The RAS–RAF–MEK pathway inhibits formation of the PRC1/CyclinD1/HDAC4 complex, and thus inhibits the Rpa-RX3-AC → Rpa-RX3-TR transition. When the RAS–RAF–MEK pathway is activated by mitogenic stimulation, the activated pathway is downregulated after 4 h, allowing PRC1/CyclinD1/HDAC4 complex formation, which is followed by the Rpa-RX3-AC → Rpa-RX3-TR transition. If the RAF–MEK pathway is activated by oncogenic RAS, the constitutively activated signal inhibits formation of the PRC1/CyclinD1/HDAC4 complex for a long period of time, thereby inhibiting the Rpa-RX3-AC → Rpa-RX3-TR transition
