Fig. 2
RUNX3 sequentially recruits TrxG and PcG complexes. a Yeast two-hybrid screening using Gal4-BRD2 (aa 450–802) as bait identified RNF2 and MLL5 as BRD2-binding proteins (see STAR methods). DDO = SD-Leu/-Trp, DDO/X/A = SD-Leu/-Trp/X-α-gal/ABA, QDO/X/A = SD-Leu/-Trp/-His/-Ade/X-α-gal/ ABA medium. Selective colonies were identified by DNA sequencing. b HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points, and the time-dependent interactions between RUNX3, BRD2, Cyclin D1, MLL1, MLL5, RNF2, BMI1, EZH2, EED, and HDAC4 were measured by IP and IB. c PLA showing RUNX3-MLL1 and RUNX3-MLL5 at the indicated time points after serum stimulation. Green fluorescence indicates association of the indicated proteins. F-actin was stained (red) to visualize the cytoplasmic compartment. d Microscopy images of transgenic fly eyes. Lozenge is a Drosophila homolog of the RUNX genes. Glass multimer reporter (GMR)-Gal4 promotes eye-specific expression of UAS-inserted genes. GMR-driven Lozenge overexpression (GMR-Gal4/ + ;UAS-Lozenge (lz) → GMR > Lz) or GMR-driven Trithorax (Trx) overexpression (GMR-Gal4/ + ; TrxG14137 → GMR > Trx) conferred weak rough phenotypes. However, GMR-driven overexpression of both Lz and Trx (GMR > Lz + Trx) resulted in a severe defective eye phenotype with loss of external ommatidial facets. e HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. The binding of RUNX3, BRD2, MLL1, MLL5, CDK4, RNF2, Cyclin D1, HDAC4, EZH2, H2A-K119-Ub, 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. f Schematic illustration of the R-point transition. At 1 h after serum stimulation, RUNX3 associates with various proteins, including p300, BRD2, H4K12-ac, SWI/SNF, TFIID, and MLL1/5, to form Rpa-RX3-AC. Between 2 and 4 h after serum stimulation, Rpa-RX3-AC interacts with PRC1–CyclinD1–HDAC4 to from a transient complex, Rpa-RX3-TR. Subsequently, Rpa-RX3-TR is destroyed (at 4 h) to form Rpa-RX3-RE (at 8 h)
