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, 10 (1), 1897

RUNX3 Regulates Cell Cycle-Dependent Chromatin Dynamics by Functioning as a Pioneer Factor of the Restriction-Point

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RUNX3 Regulates Cell Cycle-Dependent Chromatin Dynamics by Functioning as a Pioneer Factor of the Restriction-Point

Jung-Won Lee et al. Nat Commun.

Abstract

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
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. 2
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)
Fig. 3
Fig. 3
Formation of the PRC1–CyclinD1–HDAC4 complex. a HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent formation of the RNF2–Cyclin D1, EZH2–Cyclin D1, HDAC4–Cyclin D1, and RNF2–HDAC4 complexes was measured by IP and IB. b HEK293 cells were transfected with HA-Cyclin D1, Myc-HDAC4, and Flag-RNF2, and the interactions between the proteins were measured by IP and IB. c, d HA-Cyclin D1, Myc-RNF2, and Myc-HDAC4 were translated in vitro and the interactions among the proteins were measured by IP and IB. e Regions of Cyclin D1 required for the interaction with RUNX3, RNF2, and HDAC4 are summarized. Cyclin D1 regions known to interact with pRB and CDK4/6 are also indicated. Cyc Box = Cyclin Box. f HEK293 cells were treated with control or HDAC4-specific siRNA (si-con or si-HDAC4), 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. g HEK293 cells were treated with control, RNF2-specific, or Cyclin D1-specific siRNA (si-con, si-RNF2, or si-CycD1), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3, Cyclin D1–RUNX3, HDAC4–RUNX3, and RNF2–BRD2 complexes was measured by IP and IB. h Schematic illustration of the process of Rpa-RX3-TR formation. Cyclin D1, which is induced 2 h after serum stimulation, interacts with PRC1 (containing RNF2) and matures into the PRC1–CyclinD1–HDAC4 complex. The PRC1–CyclinD1–HDAC4 complex then interacts with Rpa-RX3-AC to form Rpa-RX3-TR
Fig. 4
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
Fig. 5
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. 6
Fig. 6
The roles of the RAS pathway in regulating the R-point transition. The RAS–RAF–MEK pathway facilitates formation of Rpa-RX3-AC and inhibits formation of the PRC1–Cyclin D1–HDAC4 complex. The RAS–RAC–JNK pathway activates CDK4 within Rpa-RX3-AC. The RAS–PI3K pathway facilitates formation of Rpa-RX3-TR by contributing to translation of Cyclin D1
Fig. 7
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
Fig. 8
Fig. 8
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
Fig. 9
Fig. 9
RUNX3 defends against endogenous oncogenic K-Ras. a H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were synchronized by serum deprivation and stimulated with 10% serum and 1 μM 4-OHT for the indicated durations (0, 4, and 8 h). Time-dependent subcellular localization of the expressed proteins was analyzed by double immunofluorescence staining (green = RUNX3; red = F-actin). b, c H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were serum-starved for 24 h, stimulated with 10% serum or 10% serum + 1 μM 4-OHT. Cells were harvested at the indicated time points, and the time-dependent interactions of RUNX3 with BRD2, p300, H4K12-ac, TFIID complex (TAF1, TAF7, and TBP), SWI/SNF complex (BRG-1 and BAF155), and MLL1/5 were measured by IP and IB. Expression of p14ARF, p53, and p21 was measured by IB. The binding of the proteins and 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 H460-vec, H460-ERT2-RUNX3, and H460-ERT2-RUNX3-K94/171R cells were treated with indicated si-RNA, serum-starved for 24 h, and then stimulated with 10% serum or 10% serum + 1 μM 4-OHT for the indicated durations. Apoptotic cells were detected by flow cytometry after Annexin V–FITC/PI staining. The levels of p53 were measured by IB
Fig. 10
Fig. 10
The R-point governs multiple programs of tumor suppression. a H460-vec, H460-ERT2-RUNX3, and H460-ERT2-RUNX3-K94/171R cells were serum-starved for 24 h, and then stimulated with 10% serum or 10% serum + 1 μM 4-OHT for 0, 8, or 16 h. RNA was extracted from the cells, and gene expression patterns were analyzed by mRNA sequencing. Expression of genes 8 or 16 h after serum stimulation was quantified as log2(fold change) relative to the average of control reactions (i.e., before serum stimulation, 0 h) for each cell line. Differential gene expression in response to expression of wild-type RUNX3 was analyzed by plotting log2(ERx3 + 16 h/ERx3 + 0 h) and log2(Evec + 16 h/Evec + 0 h); the results are shown on the left. Differential gene expression changes in response to expression of RUNX3-K94/171R were analyzed by plotting log2(ERx3KR + 16 h/ERx3KR + 0 h) and log2(Evec + 16 h/Evec + 0 h); the results are shown on the right. ERx3 + 16 h and ERx3 + 0 h, ERx3KR + 16 h and ERx3KR + 0 h, and Evec – 16 h and Evec – 0 h are the average expression levels of genes 0 or 16 h after 4-OHT stimulation in H460-ERT2-RUNX3, H460-ERT2-RUNX3-K94/171R, and H460-vec cells, respectively. Yellow spots indicate genes regulated in a RUNX3-independent manner. Red and blue spots indicate genes regulated in a RUNX3-dependent manner 8 and 16 h after 4-OHT stimulation, respectively (FDR < 0.001, p < 0.05). b RUNX3-dependent genes involved in various signaling pathways were analyzed using the DAVID Bioinformatics Resources 6.8. Gene expression levels 16 h after 4-OHT stimulation in the indicated cells were quantified as the fold change relative to the average of un-stimulated levels. Z-scores of log2(ERx3 + 16 h/ERx3 + 0 h) and log2(ERx3KR + 16h/ERx3KR + 0 h) for major signaling pathways are shown for H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells. Numbers at the top or bottom of the bars indicate the numbers of genes significantly up- or downregulated by inducer treatment. All categories were enriched with p < 0.05

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