SCFβ-TRCP promotes cell growth by targeting PR-Set7/Set8 for degradation

The Set8/PR-Set7/KMT5a methyltransferase plays critical roles in governing transcriptional regulation, cell cycle progression and tumorigenesis. Although CRL4Cdt2 was reported to regulate Set8 stability, deleting the PIP motif only led to partial resistance to ultraviolet-induced degradation of Set8, indicating the existence of additional E3 ligase(s) controlling Set8 stability. Furthermore, it remains largely undefined how DNA damage-induced kinase cascades trigger the timely destruction of Set8 to govern tumorigenesis. Here, we report that SCFβ-TRCP earmarks Set8 for ubiquitination and degradation in a casein kinase I-dependent manner, which is activated by DNA-damaging agents. Biologically, both CRL4Cdt2 and SCFβ-TRCP-mediated pathways contribute to ultraviolet-induced Set8 degradation to control cell cycle progression, governing the onset of DNA damage-induced checkpoints. Therefore, like many critical cell cycle regulators including p21 and Cdt1, we uncover a tight regulatory network to accurately control Set8 abundance. Our studies further suggest that aberrancies in this delicate degradation pathway might contribute to aberrant elevation of Set8 in human tumours.

a. Mass spectrometry analysis of β-TRCP1-associated proteins. b. Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitation (IP) derived from 293T cells transfected with HA-Set8 or empty vector (EV) as a negative control. c. U2OS cells harboring inducible Flag-tagged Set8 WT were treated with 0.1 µg ml -1 doxycline (Dox) to induce the ectopic expression of Flag-WT-Set8. The cells were then treated with 15 µM MG132 for 12 hr and harvested for anti-Flag immunoprecipitation (IP). Mouse normal IgG was used as a control. d. IB analysis of WCL and IP derived from 293T cells transfected with HA-Set8 and Flag-tagged wildtype (WT) or R474A mutant β-TRCP1 constructs, or EV. e. IB analysis of WCL and IP derived from 293T cells transfected with Flag-Set8 and Myc-Cullin 1, 2, 3, 4A or 5 expressing plasmids.   (c) infected with shRNA constructs specific for GFP, β-TRCP1 (three independent shRNAs, namely -A, -B and -C), or β-TRCP1+2, followed by selection with 1 µg ml -1 puromycin for three days to eliminate non-infected cells. d. IB analysis of WCL from T98G cells infected with shRNA specific for GFP, or several shRNA constructs against Cullin 1 (three independent lentiviral Cullin 1-targeting shRNA constructs namely, -A, -B, -C) followed by selection with 1 µg ml -1 puromycin for three days to eliminate non-infected cells. e. IB analysis of WCL from HeLa cells infected with shRNA construct specific for GFP, or several shRNA constructs against Cullin 4 (two independent lentiviral Cullin 4A-targeting shRNA constructs namely, -A, -B, and two independent lentiviral Cullin 4B-targeting shRNA constructs namely, -A, -B) followed by selection with 1 µg ml -1 puromycin for three days to eliminate non-infected cells. f. IB analysis of WCL and IP derived from 293T cells transfected with HA-Set8 and Myc-tagged Cullin 4A and Cullin 4B. g. IB analysis of WCL and IP derived from 293T cells transfected with Flag-Set8 and Myc-tagged Cullin 4A and Cullin 4B. h. IB analysis of WCL derived from Cullin 4A fl/fl and Cullin 4B fl/fl MEFs that are retrovirally infected with the Cre-recombinase encoding vectors to delete endogenous Cullin 4A or Cullin 4B, respectively. i. Quantification of the Set8 band intensities in (h). Set8 band intensity was normalized to Vinculin.

Supplementary
Data are shown as mean ± s.d. of three independent experiments. *p<0.05, Student's t-test j. IB analysis of WCL derived from HeLa cells with or without MG132 treatment. k. HeLa cells were infected with the shGFP or shβ-TRCP1 followed by selection with 1 µg ml -1 puromycin for three days to eliminate non-infected cells. The generated stable cell lines were then treated with nocodazole. At the indicated time points, WCL were prepared and immunoblots were probed with the indicated antibodies. l. mRNA levels of β-TRCP2 in HeLa cells infected with the indicated shRNA constructs. β-TRCP2 mRNA levels were normalized to GAPDH, and then normalized to the control cells (shGFP), n=3.

Supplementary Figure 3. Casein Kinase Iδ (CKIδ) controls Set8 stability.
a-b. Immunoblot (IB) analysis of WCL from HeLa cells treated with the CKI inhibitor D4476 (a) or IC261 (b) at the indicated concentrations for 12 hours. c-d. IB analysis of WCL from 293T cells treated with the CKI inhibitor D4476 (c) or IC261 (d) at the indicated concentrations for 12 hours. e. 293T cells were transfected with EV or CKIδ expression plasmid. The transfected cells were then split into 60-mm dishes. Cells were treated with 20 µg ml -1 CHX after 20 hours. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. f. Quantification of the Set8 band intensities in (e). Set8 band intensity was normalized to tubulin, and then normalized to the t=0 controls, n=3.   Data are shown as mean ± s.d. of three independent experiments. *p<0.05, Student's t-test b. HeLa cells were transfected with the indicated plasmids. The transfected cells were treated with 20 µg ml -1 CHX after 20 hours. At indicated time points, whole cell lysates (WCL) were prepared and the immunoblots were probed with the indicated antibodies. c. Quantification of the Set8 band intensities in Fig. 4h. Set8 band intensity was normalized to Set8 in WCL, d-e. IB analysis of WCL and immunoprecipitation (IP) derived from 293T cells transfected with Flag-Set8 and Myc-tagged Rbx1 or Skp1. f. Purified CKIδ protein was incubated with 5 µg of the indicated GST-Set8 (173-267aa) proteins in the presence of ATP. The kinase reaction products were incubated with purified E1, E2, ubiquitin, and the SCF β-TRCP complex for 45 min. Afterwards, the products were analyzed by IB with anti-Set8 antibody. g. In vivo ubiquitination assays to demonstrate that SCF β-TRCP promotes Set8 ubiquitination using the WT and K-0 mutant ubiquitin as indicated. His-pull-down products of ubiquitinated Set8 and WCL were subjected to IB analysis with indicated antibodies. h. Mass spectrometry analysis to identify the Ser253 phosphorylation event in Set8.

Supplementary Figure 5. Effects of β-TRCP-mediated degradation of Set8 on cell growth.
a. U2OS cells were treated with doxycycline to induce the Set8 WT or S253A expression and then treated with UVC and 20 µg ml -1 CHX for different time periods. At the indicated time points, WCL were prepared, and immunoblots were probed with the indicated antibodies. b. U2OS cells with induced Flag-Set WT , Set8 ∆PIP , Set8 S253A or Set8 ∆PIP/S253A were treated with UV (50 J m -2 ), IB analysis was performed to analyze the levels of Flag-Set8 and tubulin. c. Quantification of the Set8 band intensities in (b). Set8 band intensity was normalized to tubulin.  , h) were cultured in the absence or presence of 0.1 µg ml -1 doxycycline (Dox). Cell cycle was analyzed by PI staining using FACS. i-j. Quantification of the H4K20me1 (i) and H4K20me2 (j) bands intensities in Fig. 6b. The band intensity was normalized to Vinculin. k.
U2OS cells harboring inducible Flag-Set8 WT , Set8 S253A , Set8 ΔPIP , or Set8 ΔPIP/S253A were synchronized in M phase with nocodazole for 18 hours. During the last 4 hours in nocodazole, expression of Set8 was induced by doxycycline (Dox). Then, cells with or without MG132 treatment were released from nocodazole in the presence of Dox, and IB analysis was performed to detect the indicated proteins.     Flag-Set8 CDC20 Vinculin