CRL4(Cdt2) regulates cell proliferation and histone gene expression by targeting PR-Set7/Set8 for degradation

Abstract

PR-Set7/Set8 is a cell-cycle-regulated enzyme that monomethylates lysine 20 of histone H4 (H4K20). Set8 and monomethylated H4K20 are virtually undetectable during G1 and S phases of the cell cycle but increase in late S and in G2. We identify CRL4(Cdt2) as the principal E3 ubiquitin ligase responsible for Set8 proteolytic degradation in the S phase of the cell cycle, which requires Set8-PCNA interaction. Inactivation of the CRL4-Cdt2-PCNA-Set8 degradation axis results in (1) DNA damage and the induction of tumor suppressor p53 and p53-transactivated proapoptotic genes, (2) delayed progression through G2 phase of the cell cycle due to activation of the G2/M checkpoint, (3) specific repression of histone gene transcription and depletion of the histone proteins, and (4) repression of E2F1-dependent gene transcription. These results demonstrate a central role of CRL4(Cdt2)-dependent cell-cycle regulation of Set8 for the maintenance of a stable epigenetic state essential for cell viability.

Figure 1

Association of Set8 with PCNA via a CRL4^Cdt2 substrate-specific PIP box triggers its degradation. (A) Sequence alignment of the PIP box of various substrates of CRL4^Cdt2 Red, canonical residues essential for PCNA interaction; blue, residues common to CRL4^Cdt2 substrates, and green, frequent residues in these substrates. (B) Sequence alignment of Set8 PIP box from different species. (C) Schematic of Set8 protein with its two PIP boxes. (D) GST pull-down experiment showing PIP box 2 of Set8b, but not PIP box 1, is essential for direct Set8-PCNA interaction. Coomassie stained gel of the purified Gst and Gst-Set8b proteins shown on left for loading control. (E) Immuno-fluorescence images of U2OS showing that endogenous Set8 is detected in PCNA-foci positive (S-phase) cells only when proteasomes are inactivated by MG132. Red arrows indicate the cell magnified below. (F) Quantitation of data in (E). (G) Similar to E, except that Myc-Set8b or Myc-Set8b ^ΔPIP2 were ectopically expressed and detected by the anti-Myc epitope antibody in the absence of MG132. (H) Half-life measurement of Set8b or Set8b^ΔPIP2 proteins in U2OS cells following cycloheximide (CHX) treatment. Immunoblots for Myc epitope. β-actin shows equal protein loading. (I) Similar to (H), but measures the half-life of endogenous Set8 protein in U2OS cells depleted of PCNA by si-RNA. Immunoblots for endogenous Set8 and PCNA. si-RNA targeting luciferase (si-GL2) is a negative control.

Figure 2

Low Set8 levels in the cell-cycle coincides with high CRL4 activity and PCNA mediates Set8-Cdt2 co-localization in vivo. (A and B) Top: FACS analysis of U2OS cells showing the synchronization of cells by nocodazole or double thymidine block (DTB), respectively, and subsequent release. Bottom: proteins extracted from the synchronized cells shown above were immunoblotted with antibodies specific for the indicated proteins. *: cross-reacting unrelated band. (C and D) Immuno-fluorescence images of U2OS showing the localization of endogenous Cdt2 and myc-tagged wt Set8b (C) or Set8b^ΔPIP2 (D) in cells treated without or with MG132. White arrow: a representative cell, which is magnified below.

Figure 3

Set8 is a direct substrate for CRL4^Cdt2. (A) Western blotting of Set8 protein in cells depleted of the indicated proteins by siRNA. (B) Quantitative RT-PCR of Set8 mRNA in control (si-GL2) or cells depleted of the indicated proteins by siRNA, expressed relative to β-actin mRNA. Average ± standard deviation of three separate PCR reactions. (C) Set8 protein half-life is increased only in cells depleted of various components of the CRL4^Cdt2 complex but not other related proteins. Half-life measurement of Set8 protein in control cells and in cells depleted of various proteins as indicated. 50 ug of protein from si-GL2 transfected cells or 20 ug of other samples were loaded to normalize for the starting point of Set8 expression. β-actin is loading control. The ratio of Set8 protein normalized to actin relative to the starting point (0 hr of cycloheximide treatment) is indicated below each lane. The efficiency of knockdown is shown in figure S3A. The rate of Set8 degradation is also displayed in figure S3B, C. (D) Left; Cdt2 stimulates Set8b ubiquitylation in vivo. Western blotting of Set8 expressing the indicated proteins in 293T cells. Immunoprecipitated ubiquitylated proteins (anti-HA) from MG132-treated cells were Western-blotted for Myc-Set8. Expression of the overexpressed proteins is shown below. Right, similar to the panel on left without overexpressing HA-ubiquitin. Western blot of immunoprecipitated Set8 proteins (anti-myc) with anti-K-48 ubiquitin-specific antibody. (E) Destabilization of Set8b by Cdt2 requires Cul4A/B. Western blotting of endogenous Set8 shows that ectopic Cdt2 decreases co-transfected Set8b only in control 293T but not in cells depleted of Cul4A/B by siRNA. The upper band in the anti-Set8 immunoblot, which appears after Cul4 si-RNA, represents either the stabilized Set8a isoform of Set8 protein or a modified form of Set8b, because it disappears upon co-silencing endogenous Set8 with siRNA (data not shown). (F) Endogenous Set8 interacts with endogenous Cdt2 in vivo in cells treated with MG132. Western blot analysis of Cdt2 and Set8 in control (IgG) or anti-Cdt2 immunoprecipitates. (G) Wt-Set8b but not Set8b^ΔPIP2 interacts with endogenous PCNA and endogenous Cdt2. Western blot of the anti-myc immunoprecipitates from cells expressing the indicated proteins. *: cross-reacting unrelated band. (H) DDB1 interacts with Set8. Western blot of the anti-Set8 immunoprecipitate from 293T cells expressing the indicated proteins. (I) CRL4^Cdt2 promotes Set8 ubiquitylation in vitro. Incubation of immuno-purified CRL4^Cdt2 (shown by Western in top two panels) with Set8b in an in vitro ubiquitin ligase assay increased the formation of Set8 ubiquitylated species (bottom panel). Auto-ubiquitylation of Cdt2 is also observed (middle panel).

Figure 4

CRL4^Cdt2 promotes Set8 degradation after UV. (A) UV irradiation of U2OS cells decreases total and chromatin-bound Set8. Western blot of Set8 after treating U2OS with various doses of UV as indicated. Actin serves as a loading control. (B, C, D) Degradation of Set8 following UV requires Cul4 and DDB1 (B), Cdt2 (C) and PCNA (D). Western blots of Set8 and the indicated protein from untreated or UV-irradiated U2OS cells transfected with the indicated siRNAs. The level of Set8 protein relative to actin and normalized to the non-UV irradiated sample is displayed below each lane. (E) Set8b interaction with PCNA is required for its degradation following UV. Western blot of 293T cell lysates showing that ectopic wt Set8b, but not Set8b^ΔPIP2 is degraded via the proteasome.

Figure 5

Failure to degrade Set8 via the CRL4^Cdt2-PCNA pathway inhibits G2/M cell cycle progression and cell proliferation. (A) Expression of Set8b^ΔPIP2 but not a catalytically inactive Set8b^ΔPIP2 (Set8b^{ΔPIP2_R265G/D338A}) or wt Set8 inhibits the proliferation of U2OS cells. Day 0 post-selection corresponds to 48 hrs after retroviral transduction. (B) Phase contrast images of U2OS cells taken on day four post-transduction. Expression of Set8b^ΔPIP2 causes an increase in cellular and nuclear size with flattening of cells. (C) FACS analysis of U2OS cells shown in (B). Percentages of cells in the various cell cycle stages are indicated. Mean ± standard deviation of three independent transductions. (D) FACS analysis of U2OS cells transduced with retroviruses expressing the indicated proteins and synchronized (48 hrs post-transduction) by DTB block and release at various time points, in the presence of nocodazole. (E) Western blotting of lysates of U2OS shown in (D). Set8b^ΔPIP2 expression delays progression into mitosis as indicated by the delayed appearance of H3S10 phosphorylation. The immunoblots of protein lysates from the various cells for a given antibody were exposed for the same time to demonstrate differences in the abundance of the various proteins.

Figure 6

Deregulated Set8 expression increases H4K20me2 and H4K20me3, induces DNA damage and activates the p53 tumor suppressor pathway. (A) H4K20me1 is increased initially but is converted to H4K20me2 and H4K20me3 in cells containing stable Set8. Western blot analysis of U2OS lysates, 24 and 48 hours following transduction with mock, wt Set8b, or Set8b^ΔPIP2-expressing retroviruses. (B) DNA damage-induced accumulation of phosphorylated p53 (Ser-15) and γH2AX and activated checkpoint pathways in cells expressing stable Set8. Rest as in (A). (C) RNA level of various p53-regulated genes in U2OS cells transduced with the indicated retroviruses as determined by quantitative RT-PCR. Data are represented relative to the expression of β-actin mRNA. (D) Similar to (C), but shows the induction of p53-regulated genes p21, FAS and PUMA in Cdt2-depleted (si-Cdt2) U2OS cells. (C, D) show the average ± standard deviation of three separate PCR reactions.

Figure 7

Inactivation of the CRL4-Cdt2-PCNA-Set8 pathway represses transcription of E2F1-regulated genes and histone genes and causes chromatin decompaction. (A and B) RNA level of some E2F1-regulated genes (A) and the indicated histone genes (B) expressed relative to β-actin mRNA as determined by quantitative RT-PCR. (C) Similar to (B), and represents the relative expression of histone genes in U2OS cells depleted of the indicated proteins by siRNA. (D) Chromatin immunoprecipitation (ChIP) analysis of the indicated histone promoters shows the enrichment of the H4K20me3 chromatin mark at various promoters in cells transduced with viruses expressing the indicated Set8 proteins or control pMSCV vector. The data represent the PCR-amplification from the H4K20me3 immunoprecipitates normalized to those from total histone H4 immunoprecipitates. (A, B, C &D) show the average ± standard deviation of three separate PCR reactions. (E) Ponceau-S staining of U2OS cell lysates after SDS-PAGE, showing the reduction of histone proteins upon the expression of Set8b^ΔPIP2. Also shown is a Western blot that shows that reduction of individual histone proteins in Set8b^ΔPIP2-expressing cells. (F) Cells expressing Set8b^ΔPIP2 exhibit decondensed chromatin. Micrococcal nuclease digestion of nuclei of cells expressing the indicated proteins shows the loss of nucleosomal pattern in nuclei from Set8b^ΔPIP2 -expressing cells. (G) Summary model depicting the deregulated gene expression and biological consequences of the failure to degrade Set8 via the CRL4^Cdt2-PCNA during the S-phase of the cell cycle.