Oncogene (2015) 34, 104–118 & 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

ORIGINAL ARTICLE

CRL4B promotes tumorigenesis by coordinating with SUV39H1/HP1/DNMT3A in DNA methylation-based epigenetic silencing Y Yang1, R Liu1, R Qiu2, Y Zheng2, W Huang2, H Hu1, Q Ji1, H He3, Y Shang2, Y Gong1 and Y Wang1,2 Cullin 4B (CUL4B) is a component of the Cullin4B-Ring E3 ligase complex (CRL4B) that functions in proteolysis and is implicated in tumorigenesis. Here, we report that CRL4B is associated with histone methyltransferase SUV39H1, heterochromatin protein 1 (HP1) and DNA methyltransferases 3A (DNMT3A). We showed that CRL4B, through catalyzing H2AK119 monoubiquitination, facilitates H3K9 tri-methylation and DNA methylation, two key epigenetic modifications involved in DNA methylation-based gene silencing. Depletion of CUL4B resulted in loss of not only H2AK119 monoubiquitination but also H3K9 trimethylation and DNA methylation, leading to derepression of a collection of genes, including the tumor suppressor IGFBP3. We demonstrated that CUL4B promotes cell proliferation and invasion, which are consistent with a tumorigenic phenotype, at least partially by repressing IGFBP3. We found that the expression of CUL4B is markedly upregulated in samples of human cervical carcinoma and is negatively correlated with the expression of IGFBP3. Our experiments unveiled a coordinated action between histone ubiquitination/methylation and DNA methylation in transcription repression, providing a mechanism for CUL4B in tumorigenesis. Oncogene (2015) 34, 104–118; doi:10.1038/onc.2013.522; published online 2 December 2013 Keywords: CRL4B; SUV39H1; DNMT3A; HP1; transcriptional repression

INTRODUCTION Histones are subjected to a variety of post-translational modifications including acetylation, methylation, phosphorylation, sumoylation and ubiquitination1–3 that impact on chromatin configuration, transcription process, and other chromatin-based events. Although identified 38 years ago,4 histone ubiquitination remains one of the least understood histone modifications and is mainly found on H2A lysine 119 (K119) and H2B (K120 in human and K123 in yeast).5 It has been reported that H2BK120 ubiquitination is mediated by human RNF20/RNF40 and UbcH6 and is functionally associated with transcriptional activation,6,7 while H2AK119 ubiquitination is catalyzed by the Polycombrepressive complex 1 and Cullin4B-Ring E3 ligase complex (CRL4B) that are linked to transcriptional repression.8–10 Cullin 4-Ring E3 ligases (CRL4), assembled with CUL4, DDB1 and ROC1 as the core components, participate in a broad variety of physiologically and developmentally-controlled processes such as cell cycle progression, replication and DNA damage response.11 In mammals, there are two Cullin 4 members, CUL4A and Cullin 4B (CUL4B), which share 82% sequence identity. Although Cul4a null mice do not exhibit evident developmental defects,12–14 CUL4A has been shown to target p53 and several cyclindependent kinase inhibitors, such as p21 and p27 for proteolysis in cultured cells15–17 and was found to be highly expressed in breast and hepatocellular carcinomas.18,19 Loss-of-function mutation in the X-linked CUL4B, on the other hand, causes mental retardation, short statue, absence of speech and other

phenotypes in humans,20,21 and Cul4b knockout mice are embryonically lethal,10 indicating a unique function of Cul4b that cannot be compensated by Cul4a. Interestingly, CUL4B, unlike CUL4A and other Cullins, carries a nuclear localization signal in its N terminus and is also localized in the nucleus,22,23 suggesting that CUL4B might be involved in the nucleus-based functions. It has been reported that CRL4 promotes ubiquitination of histones H2A,24 H3 and H425 to facilitate cellular response to DNA damage. Interestingly, CUL4B, but not its paralog CUL4A, exhibited, through catalyzing H2AK119 ubiquitination (H2AK119ub1), a transcription repression activity10 and targeted WDR5, a core subunit of histone H3 lysine 4 (H3K4) methyltransferase complexes,22 for ubiquitination and degradation in the nucleus. Methylation of cytosine within the context of a simple dinucleotide site, CpG, is an important epigenetic process that is implicated in mammalian development. Specifically, DNA methylation plays a key role in controlling several biological processes, such as X chromosome inactivation, genomic imprinting, genomic stability and chromatin structure, most likely as a result of its well-documented repressive effect on gene transcription.26 Methylation of CpG dinucleotides in mammals is carried out by three active DNA methyltransferases, DNMT1, DNMT3A and DNMT3B.27 The mechanisms by which DNA methylation brings about transcriptional repression have been the subject of intense research over the last decade. One well-documented mechanism is that DNA methylation is intimately linked to histone trimethylation at Lys9 of H3 (H3K9me3), which is known to be

1 Key Laboratory of Experimental Teratology, Ministry of Education, Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong, China; 22011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China and 3Ontario Cancer Institute, Princess Margaret Cancer Center/University Health Network, Toronto, Ontario, Canada. Correspondence: Dr Y Wang, 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, 22 Qi Xiang Tai Road, Tianjin 300070, China. E-mail: [email protected] Received 19 June 2013; revised 16 October 2013; accepted 18 October 2013; published online 2 December 2013

CRL4B modulates DNA methylation-based gene silencing Y Yang et al

105 involved in gene silencing.28,29 It was found that mutations of the gene dim-5 in Neurospora crassa or kryptonite in Arabidopsis thaliana, which encodes for H3K9 methyltransferases in the corresponding organisms, results in a loss of DNA methylation in these species.29,30 In Fission yeast, methylation of histone H3K9 within heterochromatin by the Clr4 histone methyltransferase produces a requisite recognition mark for the heterochromatin protein 1 (HP1) proteins Swi631 and Chp2,32 thus DNMT recruitment. These data indicate that, in Neurospora, Arabidopsis and Fission yeast, histone methylation can direct DNA methylation. In mammals, DNMT3A is associated with SUV39H1, the enzyme responsible for H3K9 di- and trimethylation, and HP1a/b/g.33 It is believed that methylation at H3K9 by SUV39H1 creates a binding site for HP1 that recognizes the H3K9 methyl epitope to facilitate DNMT recruitment.34,35 It becomes increasingly clear that DNA methylation and histone methylation are interdependent events to maintain a repressed chromatin state. Although exepriments showed that CRL4B possesses an intrinsic transcription repressive activity,10,22 the exact role of CRL4B in epigenetic transcription regulation is still poorly understood. Here, we report that CRL4B is associated with the SUV39H1, HP1 and DNMT3A. We showed that CRL4B facilitate H3K9me3 and DNA methylation through catalyzing H2AK119ub1. We demonstrated that CUL4B promotes cell proliferation, invasion and tumorigenesis via transcription repression of genes including IGFBP3. We found that the expression of CUL4B is upregulated in samples of human cervical carcinoma and is negatively correlated with the expression of IGFBP3. These results add to the understanding of the role of CUL4B in epigenetic regulation and tumorigenesis. RESULTS Cullin 4B-RING E3 ligase (CRL4B) is physically associated with DNMT/SUV39H1/HP1 complex in vivo In an effort to better understand the mechanistic roles of CUL4B protein in tumorigenesis, we employed affinity purification and mass spectrometry to identify the proteins that are associated with CUL4B in vivo. In these experiments, HA-tagged vector (Control) and HA-tagged CUL4B (HA-CUL4B) were stably expressed in HEK293T cells (a human embryonic kidney cell line). Cellular extracts were prepared and subjected to affinity purification using anti-HA affinity gels. After extensive washing, the bound protein complex was eluted with excess HA peptide and the proteins in the complex were resolved on SDS–PAGE and then visualized by silver staining. The protein bands on the gel were retrieved and analyzed by mass spectrometry. The results indicate that CUL4B co-purified with DDB1, EZH2 and COPS4, as reported previously.10,36,37 Interestingly, we found that CUL4B also copurified with DNMT3A, SUV39H1 and HP1a/b/g (Figure 1a), all of which are functionally linked to DNA methylation-based transcriptional repression. In addition, NSD1 and SMYD2, two histone methyltransferases for H3 Lys36 (H3K36),38–40 and PRMT5, the histone methyltransferase for H3 Arg2 (H3R2), Arg8 (H3R8) and H4 Arg3 (H4R3)41–43 were also detected in the immunocomplex. The detailed results of the mass spectrometric analysis are provided in Supplementary File 1. The presence of DNMT3A, SUV39H1 and HP1 in the CUL4B-interacting complex was confirmed by western blotting analysis (Figure 1b). To further support the observation that CUL4B complex is associated with SUV39H1/HP1/ DNMT3A complex in vivo, protein fractionation experiments were carried out with nuclear proteins by fast protein liquid chromatography. Nuclear extracts derived from HEK293T cells were fractionated by DEAE sepharose, followed by superpose 6 gel filtration chromatography. Western blotting revealed a major peak at about 669–1000 kDa for CUL4B, DDB1 and ROC1, and also for DNMT3A, SUV39H1, HP1a, and HP1b & 2015 Macmillan Publishers Limited

(Figure 1c). Significantly, the chromatographic profiles of CUL4B, DDB1 and ROC1 were largely overlapped with SUV39H1/HP1/ DNMT3A, substantiating the argument that these proteins are associated in vivo. To further validate the in vivo interaction between CRL4B and SUV39H1/HP1/DNMTs, total proteins from HEK293T cells and HeLa cells (a human cervical carcinoma cell line) were extracted, and coimmunoprecipitation experiments were performed with antibodies detecting the endogenous proteins. Immunoprecipitation (IP) with antibodies against CUL4B and immunoblotting with antibodies against SUV39H1, HP1a, HP1b, HP1g, DNMT1, DNMT3A or DNMT3B demonstrated that CUL4B was co-immunoprecipitated with all these proteins (Figures 2a and b, left panels). Reciprocally, IP with antibodies against SUV39H1, HP1a, HP1b, HP1g, DNMT1, DNMT3A or DNMT3B followed by immunoblotting with antibodies against CUL4B also revealed that all these proteins were coimmunoprecipitated with CUL4B (Figures 2a and b, right panels). Furthermore, using CUL4B-depleted cells as negative control, we also confirmed the interaction between SUV39H1/HP1a and CUL4B in control shRNA (shSCR)-transfected cells but not in the CUL4B-depleted cells (Figure 2c). Moreover, SUV39H1, HP1a and DNMT3A could also be co-immunoprecipitated with DDB1 and ROC1, the other two main components of the CRL4B complex, in HEK293T or HeLa cells (Figures 2d and e). Taken together, these data strongly support the hypothesis that CRL4B is physically associated with SUV39H1/HP1/DNMT3A complex in vivo. CRL4B complex is associated with DNA/histone methyltransferase activities in vivo In order to determine the molecular basis for the interaction of CRL4B with the SUV39H1/HP1/DNMTs complex, GST pulldown assays were performed using GST-fused CUL4B, DDB1 or ROC1 proteins and in vitro transcribed/translated individual components of the SUV39H1/HP1/DNMTs complex, including SUV39H1, HP1a, HP1b, HP1g, DNMT1, DNMT3A and DNMT3B. These experiments revealed that both CUL4B and DDB1 could interact directly with SUV39H1, DNMT1, DNMT3A and DNMT3B (Figure 3a). The direct interaction between CRL4B and SUV39H1 prompted us to investigate whether CUL4B could be associated with an HMT enzymatic activity. To this end, CUL4B-containing protein complex was immunoprecipitated from HEK293T cells stably expressing HA-CUL4B with the anti-HA affinity gel and analyzed for enzymatic activities. The immunoprecipitates (IPs) were first incubated with unmodified histone H3 tail (residues 1–21), and the levels of methylated histones in the reactions were then analyzed by western blotting. Notably, the CUL4B-containing complex indeed possessed an enzymatic activity that led to a significant increase in the tri-methyl H3K9 and an evident methyl-transferase activity for di-methyl H3K9 (Figure 3b). The results showed that CRL4B is associated clearly with an H3K9 methyl-transferase activity, both for trimethylation and dimethylation. However, the H3K4 methyltransferase activity was not dose-dependent; the weak and unspecific histone H3K4 methyl-transferase activity might be conducted by WDR5-containing H3K4 methylation complex pulldowned by HA-CUL4B as reported previously.22 It is believed that the methylation at H3K9 by the SUV39H1 enzyme creates a binding site for HP1 that recognizes the H3K9 methyl moiety to facilitate DNMT recruitment.33 The physical association of CRL4B, SUV39H1 and DNMTs prompted us to investigate whether CUL4B could be associated with a DNMT enzymatic activity. For this purpose, we first examined, by GST pulldown assays, if GST-tagged CUL4B or DDB1 could purify (pulldown) DNA methyltransferase activity from HeLa nuclear extracts. Enzymatic activity was measured as radioactivity of S-adenosyl-L-[methyl-3H]methionine incorporated into an oligonucleotide substrate. The results indicated that both Oncogene (2015) 104 – 118

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Figure 1. Identification of the CUL4B Interacting Proteins. (a) Immunoaffinity purification of CUL4B-containing protein complexes. Cellular extracts from HEK293T cells stably expressing HA vector or HA-CUL4B were immunopurified with anti-HA affinity columns and eluted with HA peptide. The eluates were resolved by SDS–PAGE and silver-stained. The proteins bands were retrieved and analyzed by mass spectrometry. Detailed results from the mass spectrometric analysis are provided as Supplementary File 1. (b) Western blotting analysis of the identified proteins in the purified fractions using antibodies against the indicated proteins. (c) Co-fractionation of CRL4B and SUV39H1/HP1/DNMT3A complex by fast protein liquid chromatography. Nuclear extracts of HEK293T cells were fractionated on a DEAE sepharose column followed by a Superose 6 gel filtration column. The fractions were analyzed by western blotting. Molecular weight standards are shown on top. The predicted molecular size of each protein is summarized on the right. The elution positions of calibration proteins with known molecular masses (kDa) are indicated, and an equal volume from each fraction was analyzed.

GST-CUL4B and GST-DDB1 were able to pull down DNA methyltransferase activity from nuclear extracts (Figure 3c). Next, we asked whether endogenous CRL4B complex could purify DNA methyltransferase activity from nuclear extracts. To test this, we immunoprecipitated native CUL4B and DDB1 with their corresponding specific antibodies, followed by DNMT enzymatic assay. As shown in Figure 3d, a significant amount of DNA methyltransferase activity was purified using endogenous antibodies for CUL4B, DDB1 and DNMT3A, whereas control immunoprecipitation with normal IgG showed background activity. Collectively, these results support the argument that CRL4B complex is associated with DNA methyltransferase activity in vivo. Genome-wide transcriptional targets for the CRL4B/SUV39H1/HP1/ DNMT3A complex Since it is well established that SUV39H1/HP1/DNMT3A complex mainly functions in DNA methylation-based transcriptional Oncogene (2015) 104 – 118

repression,33 the physical association between CRL4B and SUV39H1/HP1/DNMT3A prompted us to investigate the hypothesis that CRL4B might also be functionally linked to DNA methylation-based transcriptional repression. To this end, we established HeLa cells in which CUL4B expression was stably knocked-down by its shRNA. The alteration of genome-wide DNA methylation in these cells was then analyzed using methyl-DNA immunoprecipitation (MeDIP)-chip approach (NimbleGen) with an antibody against 5-methylcytosine (5-mC). Specifically, following MeDIP, methylated DNAs with 5-mC were amplified using nonbiased conditions, labeled and hybridized to an oligonucleotide array covering over 22 532 human promoters in the NCBI database with a range from  800 to þ 200 bp relative to the transcription start site with a false recovery rate o0.05. We found that a total of 4045 genomic regions (1878 genes) had significant changes in methylation patterns between CUL4Bdepleted cells and control cells, including 1965 hypermethylation sites ( 953 genes) and 2080 hypomethylation sites (925 genes) & 2015 Macmillan Publishers Limited

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Figure 2. Physical Interaction between CRL4B and the DNMT/SUV39H1/HP1 Complex. (a, b) Association of CUL4B with the SUV39H1/HP1/ DNMT complex. Whole cell lysates from HEK293T (a) and HeLa (b) were immunoprecipitated (IP) with the antibodies against the indicated proteins. Immunocomplexes were resolved on SDS–PAGE and analyzed by immunoblotting (IB) using antibodies against the indicated proteins. (c) Protein extracts from the control scrambled shRNA (shSCR) or CUL4B shRNA transcfected HEK293T or HeLa cells were immunoprecipitated with an anti-CUL4B antibody and immunoblotted with the indicated antibodies. (d, e) Association of DDB1 or ROC1 with the SUV39H1/HP1/DNMT complex. Whole cell lysates from HEK293T (d) and HeLa (e) were immunoprecipitated (IP) with the antibodies against the indicated proteins. Immunocomplexes were resolved on SDS–PAGE and analyzed by immunoblotting (IB) using antibodies against the indicated proteins.

(Figure 4a). These genes were then classified into various cellular signaling pathways using Molecule Annotation System software (http://www.capitalbio.com/support/mas) with a P value cutoff of 10  3 (Figure 4b, right panel). Ten pathways enriched with the most genes with hypermethylation or hypomethylation in CUL4Bdepleted cells were plotted against that in control cells. Eleven hypomethylated genes and two hypermethylated genes, including RPS6KA6, IGFBP3, AXIN1, SFRP5, FOXO3, WNT2B, IQGAP2, NKX3.1, & 2015 Macmillan Publishers Limited

RELN, KLF3, PER2, USF2 and SLBP, which represent each of the classified pathways, were selected for further quantitative MeDIP analysis. The results validated our MeDIP-chip data (Figure 4c). Besides the hypomethylatied regions/genes, a significant number of regions/genes were hypermethyalted following the depletion of CUL4B. Based on our previous work that CUL4B possesses intrinsic transcription repressive activity,10 these hypermethylation sites might be due to secondary effects induced by CUL4B Oncogene (2015) 104 – 118

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Figure 3. CRL4B interacts directly with DNMTs and SUV39H1 and exhibits DNA Histone methyltransferase activity. (a) GST pulldown experiments with bacterially expressed GST fusion proteins and the in vitro transcribed/translated indicated proteins. In vitro transcribed and translated full-length SUV39H1, HP1a, HP1b, HP1g, DNMT1, DNMT3A or DNMT3B was incubated with the indicated GST fusions of CUL4B, DDB1 or ROC1. GST was used as a negative control for association with in vitro translated proteins. (b) The CUL4B-containing protein complex possesses histone methyl-transferase activity. Cellular extracts were obtained from HeLa cells stably expressing HA-CUL4B and were immunoprecipitated by anti-HA affinity gel histone with methyl-transferation (HMT) buffer. The immunoprecipitates (IPs) were incubated with unmodified histone H3 N terminal tail (residues 1–21). The reaction mixtures were analyzed by western blotting using antibodies against the indicated histone marks or proteins. (c) GST-fused CUL4B or DDB1 protein was used to purify DNA methyltransferase activity from nuclear extracts. GST–DNMT3A and GST were also used as a positive and a negative control, respectively. After incubation, the beads were washed and assayed for DNA methyltransferase activity read as c.p.m. of S-adenosyl-L[methyl-3H]methionine incorporated into an oligonucleotide substrate. (d) Endogenous CRL4B complex associates with DNA methyltransferase activity. HeLa nuclear extracts were immunoprecipitated (IP) with specific antibodies against CUL4B, DDB1 or DNMT3A, along with control IgG, and tested for associated DNA methyltransferase activity. Error bars represent mean±s.d. of three independent experiments.

depletion. The detailed results of the MeDIP chip analysis are provided in Supplementary File 2. Among the target genes identified above, IGFBP3 is a wellestablished antiproliferative, proapoptotic and invasion suppressor protein.44 Aberrant promoter hypermethylation of insulin-like growth factor binding protein (IGFBP3) and gene silencing have been observed in numerous cancers, including lung, hepatocellular, gastric, colorectal, breast and ovarian cancers.45–48 We thus investigated the transcriptional regulation of insulin-like growth factor binding protein (IGFBP3) by CRL4B/SUV39H1/HP1/ DNMT3A complex in detail. Quantitative ChIP using antibodies against CUL4B, DDB1, ROC1, SUV39H1, HP1a, HP1b or DNMT3A demonstrated that all these proteins could bind to IGFBP3 promoter region, whereas CUL4A, another Cullin 4 member, or G9A, a histone lysine methyltransferase catalyzing H3K9me2, were not detected in this region (Figure 4d). Moreover, the histone modification marks H2AK119ub1 and H3K9me2/3, which are added by CRL4B and SUV39H1, respectively, and DNA Oncogene (2015) 104 – 118

methylation, which is catalyzed by DNMT3A, were enriched in the same region of IGFBP3 promoter (Figure 4e, left panel). These results suggest that the CRL4B complex and SUV39H1/HP1/ DNMT3A complex are functionally connected in IGFBP3 silencing. To further understand the regulation of IGFBP3 by the CLR4B/ SUV39H1/HP1/DNMT3A complex, ChIP assays were performed in HeLa cells using antibodies against CUL4B, DDB1, SUV39H1, HP1a, DNMT3A, 5-mC or control IgG. The results showed that both CRL4B complex and SUV39H1/HP1/DNMT3A complex occupied IGFBP3 promoter, and that 50 -methylated cytosine was enriched in the same region (Figure 4f, upper panels). To further test our proposition that CRL4B and SUV39H1/HP1/DNMT3A function in the same protein complex at the IGFBP3 promoter, sequential ChIP or ChIP/Re-ChIP experiments49,50 were performed. In these experiments, soluble chromatins were first immunoprecipitated with antibodies against CUL4B, DDB1, SUV39H1, HP1a, DNMT3A or 5-mC (Figure 4f, lower panel). The immunoprecipitates were subsequently re-immunoprecipitated with appropriate antibodies. & 2015 Macmillan Publishers Limited

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Figure 4. Genome-wide identification of transcription targets for the CRL4B/DNMT/SUV39H1/HP1 Complex. (a) MeDIP-chip analysis of the changed DNA methylation patterns. Hypermethylation and hypomethylation: the status of DNA methylation in CUL4B depleted HeLa cells compared to control shSCR transfected cells. The detailed results of the MeDIP experiments are provided as Supplementary File 2. (b) Pathway analysis of the identified genes with changed promoter DNA methylation status. Ten of the most significantly enriched pathways were plotted for genes of hypermethylation (upper panel) or hypomethylation (lower panel) in CUL4B depleted HeLa cells compared to control. (c) Quantitative MeDIP analysis of selected promoters of the hypomethylated and hypermethylated genes using antibody against 5-mC. Results are represented as fold change over control. ACTB served as a negative control. Error bars represent mean±s.d. of three independent experiments. (d) Quantitative ChIP (qChIP) of IGFBP3 promoter in HeLa cells using antibodies specifically against CUL4B, DDB1, ROC1, SUV39H1, HP1a, HP1b, DNMT3A, CUL4A or G9A. (e) qChIP analysis of IGFBP3 and ACTB promoters in HeLa cells using epigenetic modification antibodies specific against H2AK119ub1, H3K9me2, H3K9me3 and 5-mC. The constantly transcriptional activated housekeeping gene ACTB served as a negative control. (d, e) Results are represented as fold change over control. Error bars represent mean±s.d. of three independent experiments. (f ) The CRL4B and SUV39H1/HP1/DNMT3A complexes exist in the same protein complex on the IGFBP3 promoters. ChIP and ReChIP experiments were performed in HeLa cells with the indicated antibodies.

The results showed that in precipitates, the IGFBP3 promoter that were immunoprecipitated with antibodies against CUL4B could be re-immunoprecipitated with antibodies against DDB1, SUV39H1, HP1a, DNMT3A or 5-mC. Similar results were obtained when initial ChIP was done with antibodies against DDB1, SUV39H1, HP1a and DNMT3A. Taken together, these results support the idea that CRL4B and SUV39H1/HP1/DNMT3A occupy IGFBP3 promoter in one multiunit complex. & 2015 Macmillan Publishers Limited

Coordinated action of CRL4B and SUV39H1/HP1/DNMT3A in transcriptional silencing of IGFBP3 We next investigated the effect of CUL4B depletion on IGFBP3 expression by quantitative RT–PCR (qRT–PCR) and western blotting. As expected, CUL4B depletion led to an increased expression of IGFBP3 at both transcription (Figure 5a, upper panel) and protein levels (Figure 5a, lower panel) in HeLa, SiHa (a human cervical carcinoma cell line) and Ca Ski (a human Oncogene (2015) 104 – 118

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110 cervical carcinoma cell line from a metastasis in the small bowel mesentery) cells. Interestingly, treatment of HeLa cells with a deoxycytidine analog 5-aza-deoxycytidine (5-aza-dC), which is widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation,51 restored IGFBP3 expression (Figure 5b); however, in CUL4Bdepleted cells, 5-aza-dC treatment had little effect on IGFBP3 expression (Figure 5b), suggesting that CUL4B is involved in DNA methylation-based epigenetic silencing of IGFBP3 in human cervical carcinoma cells and supports a functional connection between the CRL4B complex and SUV39H1/HP1/DNMT3A complex. In order to further understand the functional connection between these two complexes, we next investigated the molecular details involved in the recruitment of the CRL4B/ SUV39H1/HP1/DNMT3A complex on IGFBP3 promoter. For this purpose, quantitative ChIP assays were performed in CUL4Bdepleted HeLa cells. The results showed that depletion of CUL4B led to an evident decrease in the recruitment of CRL4B/SUV39H1/ HP1/DNMT3A proteins, including DDB1, ROC1, SUV39H1, HP1a, HP1b and DNMT3A (Figure 5c). Consistent with this, the levels of H2AK119ub1, H3K9me2, H3K9me3 and 5-mC were markedly decreased at the IGFBP3 promoter (Figure 5d), and the CpGs residing within the IGFBP3 gene were heavily hypomethylated in CUL4B-depleted cells (Figures 5e and f). Collectively, these data suggest that CRL4B, through its enzymatic activity responsible for H2AK119ub1, might function to promote the recruitment or/and the stabilization of the SUV39H1/HP1/DNMT3A complex onto the promoter of target genes such as IGFBP3, supporting a molecular mechanism in which the CRL4B and SUV39H1/HP1/DNMT3A complexes are functionally coordinated in epigenetic gene silencing. CUL4B promotes the proliferation and invasion of cervical carcinoma cells As stated before, IGFBP3 has been demonstrated to exert biological functions related to anti-proliferation, pro-apoptosis and anti-invasion and is considered to be a tumor suppressor.52 Based on our observation that CUL4B is functionally involved in promoter methylation and transcriptional silencing of IGFBP3, we next investigated what role, if any, CUL4B plays in the tumorigenesis of cervical carcinoma. For this purpose, HeLa cells were transfected with shRNAs specifically against CUL4B and/or IGFBP3. Each of these shRNA led to a significant reduction in the expression of its target gene without causing detectable changes of the non-targeted genes (Figure 6a). We first preformed growth assays in cells with loss-of-function of CUL4B. HeLa cells with stable CUL4B knockdown showed a severe growth inhibition, which could be partially alleviated by co-knockdown of IGFBP3 (Figure 6b). Colony formation assays further showed that CUL4B depletion was associated with a significant decrease in colony numbers which could be partially ascribed to IGFBP3’s anti-proliferation effect (Figure 6c). In addition, TUNEL assays revealed a striking increase in apoptosis

cells, 25.82% in CUL4B-RNAi group compared with only 1.42% in control group (Po0.001), which could be partially rescued through IGFBP3 knockdown (from 25.82–11.21%, Figure 6d), in agreement with the functional link between CUL4B and IGFBP3 described above. Next, we investigated whether or not CUL4B has a role in anchorage-independent growth and tumor metastasis. Soft agar colony assays revealed that loss of CUL4B could significantly reduce both the clone size and clone number (Figure 6e). Moreover, the results from trans-well invasion assays showed that knockdown of CUL4B resulted in about 10-fold decrease in cell invasion (Figure 6f). In addition, the decreased invasiveness upon CUL4B depletion was partially alleviated when IGFBP3 was concomitantly knocked-down (Figures 6e and f). Taken together, these experiments support a notion that CUL4B promotes cell survival, proliferation, anchorage-independent growth and invasive potential of cervical carcinoma, and it does so, at least in part, through repression of the tumor suppressor IGFBP3. In addition, CUL4B knockdown showed similar effects, including decreased colony formation and invasion, but increased apoptosis on the SiHa and Ca Ski cervical carcinoma cell lines, suggesting CUL4B is associated with a tumorigenic phenotype (Figure 6g). Furthermore, we investigated the role of CUL4B in tumor development and progression in vivo by implanting HeLa, SiHa and Ca Ski cells that had been engineered to stably express CUL4B shRNA or control scrambled shRNA onto the subcutaneous sites of athymic BALB/c mice. Growth of the implanted tumors was monitored by measuring the tumor sizes every 4 days over a period of 4 weeks. The results showed that tumor growth was greatly suppressed upon CUL4B knockdown (Figure 6h). Western blotting analysis confirmed the increases in the levels of IGFBP3 proteins in the tumors with CUL4B knockdown (Figure 6h). Taken together, these experiments indicate that CUL4B promotes cervical carcinoma cell proliferation, invasion and tumorigenesis in vitro and in vivo. The expression of CUL4B is upregulated in cervical carcinomas and negatively correlated with that of IGFBP3 To further define the role of CUL4B in tumorigenesis, we collected 64 cervical carcinoma samples, 30 of them with paired adjacent normal tissues, from cervical cancer patients and performed tissue arrays by immunohistochemical staining. CUL4B was found to be significantly upregulated in tumors, and its expression appeared to be positively correlated with histological grades (Figures 7a and b). Moreover, the results also revealed a statistically significant increase in CUL4B expression in tumors compared to the adjacent normal cervical tissue (Figure 7c). In 12 of 15 selected paired samples of each grade cancers, the level of CUL4B mRNA was found to be higher in tumor tissue than in adjacent tissue (Figure 7d), while in 13 of 15 paired samples the level of IGFBP3 mRNA was found to be lower in tumor tissue than in adjacent tissue (Figure 7e), as measured by qPCR. In addition, statistical analysis found a Spearman correlation coefficient of  0.6699

Figure 5. CUL4B targeted IGFBP3 for epigenetic silencing. (a) Clones in which CUL4B was stably knocked-down were compared with the parental cell line with respect to the levels of mRNA (upper panel) and protein (lower panel) of IGFBP3 in HeLa, SiHa and Ca Ski cells. The mRNA levels were normalized to those of GADPH (upper panel) and b-actin served as a loading control for the western blotting (lower panel). (b) Upper panel, real-time PCR analysis of IGFBP-3 mRNA showing restoration of IGFBP-3 expression in HeLa-shSCR. HeLa-shSCR and HeLashCUL4B cells were treated with 0, 0.1, 0.5 or 2 mM of 5-aza-dC for 8 days. Culture media was changed to serum free media for 24 h before IGFBP-3 analysis. Lower panel shows western blotting corresponding to IGFBP-3 expressions HeLa-shSCR and HeLa-shCUL4B culture media after 5-aza-dC treatment. (c, d) qChIP analysis of IGFBP3 promoter in the HeLa-shSCR and HeLa-shCUL4B cells using indicated antibodies. Results are represented as fold change over control. Error bars represent mean±s.d. of three independent experiments. (e) Genomic sequence analysis of IGFBP3 promoter (Accession no. M35878), note the 20 CpG island were in capital letters. Nomenclature of promoter is relative to the mRNA cap site designated þ 1. (f ) Bisulphite sequencing shows that depletion of CUL4B (shCUL4B) leads to decreased methylation at a number of CpG sites within the IGFBP3 promoter. Each row represents a single sequence analysed, and each square is a single CpG site. White and black squares represent unmethylated and methylated CpGs, respectively. Oncogene (2015) 104 – 118

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111 (Po0.0001) and a Pearson correlation coefficient of  0.6279 (Po0.0001) when the relative level of IGFBP3 expression was plotted against the relative level of CUL4B expression in 30 paired samples (Figure 7f), indicating a significant negative correlation

& 2015 Macmillan Publishers Limited

between the expression of CUL4B and IGFBP3 in these samples. These data are consistent with a role of CUL4B in promoting carcinogenesis and support the observation that IGFBP3 is a downstream target of CUL4B.

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112

Figure 6. CUL4B Promotes Cervical Carcinogenesis. (a) The knockdown efficiencies of CUL4B and IGFBP3 were confirmed by western blotting. (b) CUL4B promotes cellular proliferation. HeLa cells transfected with shSCR, shCUL4B or/and shIGFBP3 were subjected to growth curve analysis. (c) CUL4B enhances the colony-forming efficiency of cervical cancer cells. HeLa cells transfected with shSCR, shCUL4B or/and shIGFBP3 were maintained in culture media for 14 days under the presence of 1 mg/ml G418 prior to being stained with crystal violet. Representative photos were showed on the left and statistically analyzed on the right. (d) HeLa cells were transfected with indicated shRNAs. After 48 h of the plasmid transfection, TUNEL assay were performed with a fluorescence method. For each group, 6 different fields under fluorescence microscopy with 10-fold magnifications were randomly chosen and counted. Representative photos were showed on the left and statistically analyzed on the right. (e) CUL4B enhances the anchorage-independent growth of cervical cancer cells. HeLa cells transfected with shSCR, shCUL4B or/and shIGFBP3 were used for growth in soft agar. After 14 days of incubation, shCUL4B-transfected cells show growth reduction in size and number of colonies (10  magnification). The bar graph represents average number of colonies from 3 plates. In each experiment, at least 6 randomly selected view fields were scored. The number of colonies in each condition was counted and expressed as mean±s.d. from triplicate experiments. (f ) CUL4B enhances the invasivemess of cervical cancer cells. HeLa cells transfected with shSCR, shCUL4B or/and shIGFBP3 were starved for 18 h before cell invasion assays were performed using Matrigel transwell filters. The invaded cells were stained and counted. The images represent one field under microscopy in control (shSCR) and CUL4B knockdown (shCUL4B) groups (10  magnification), respectively. Each bar represents the mean±s.d. for triplicate measurements. *Po0.05 and **Po0.01 (two-tailed unpaired t-test). (g) CUL4B is associated with a tumorigenic phenotype, which includes increased colony formation and invasion, but reduced apoptosis in SiHa and Ca Ski cell lines. For each group, 6 different fields under fluorescence microscopy with 10-fold magnifications were randomly chosen and counted. Representative photos were showed on the left and statistically analyzed on the right. Each bar represents the mean±s.d. for triplicate measurements. *Po0.05 and **Po0.01 (two-tailed unpaired t-test). (h) CUL4B promotes cervical carcinogenesis. HeLa, SiHa and Ca Ski cells stably expressing CUL4B shRNA or shSCR were transplanted into athymic mice. Tumors were measured every 4 days using a vernier calliper and the volume was calculated according to the formula: p/6  length  width2. The growth curves of tumors, representative images of tumor-bearing mice and their tumors and the average tumor mass of each group are shown. Each point represents the mean±s.e.m. *Po0.05 and **Po0.01 (two-tailed unpaired t-test). Western blotting analysis showed the increase of IGFBP3 proteins in the CUL4B knockdown tumors.

DISCUSSION The Cullin 4-RING E3 ligase complex participates in a broad variety of biological processes, such as cell cycle progression, replication and DNA damage response.11 In mammalian cells, the levels of H3K27me3, H3K4me3 and H3K9me3 were all affected in CUL4depleted HeLa cells.36 However, the molecular mechanisms underlying the role of CRL4B in epigenetic regulation are not fully understood. We report in this study that CUL4B interacts with SUV39H1/HP1/DNMTs complex for DNA methylation-based Oncogene (2015) 104 – 118

epigenetic transcriptional silencing. Through physical interaction, CRL4B and SUV39H1/HP1/DNMT3A form a larger complex, which can coordinate in catalysing H2AK119 mono-ubiquitination, H3K9 di-/tri-methylation and DNA methylation at target genes such as IGFBP3 (Figure 7g). We showed that the abundance of SUV39H1, HP1a, HP1b and DNMT3A at IGFBP3 promoter greatly decreased after CUL4B knockdown, supporting the notion that CRL4B contributes to SUV39H1/HP1/DNMT3A recruitment or/and the stabilization onto target promoters. In addition, we also found that & 2015 Macmillan Publishers Limited

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113

Figure 7. CUL4B is highly expressed in cervical carcinomas and negatively correlated with IGFBP3. (a) Immunohistochemical staining of CUL4B in normal cervical tissue and cervical carcinomas (histological grades I, II & III). For each grade, the representive photos of two specimens were shown. (b, c) The positively stained nuclei (%) in grouped samples (b) or 30 paired samples (c) were analyzed by two-tailed unpaired t-test (*Po0.05; **Po0.01; ***Po0.001). (d) The expression of CUL4B mRNA is upregulated in cervical carcinomas. (e) The expression of IGFBP3 mRNA is downregulated in cervical carcinomas. (d, e) Total RNAs in paired samples of cervical carcinomas versus adjacent normal cervical tissues were extracted and the expression of each gene was measured by qPCR. mRNA levels were normalized to those of GADPH. Each bar represents the mean±s.d. for triplicate experiments (*Po0.05; **Po0.01; ***Po0.001). (f ) CUL4B mRNA level is negatively correlated with the level of IGFBP3 mRNA. The relative level of CUL4B expression was plotted against the relative level of IGFBP3 expression (***Po0.001). (g) Graphic model as discussed in the text. DNA (black line); nucleosomes with single N terminus of H3 and C terminus of H2A (blue ball). & 2015 Macmillan Publishers Limited

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114 a number of CpG islands located within the IGFBP3 promoter region were significantly hypomethylated in CUL4B-depleted cells through BSP assay, indicating that CUL4B is required for the maintenance of epigenetic silencing of target genes. The CRL4B subunits do not contain chromatin-binding domains, which could contribute to SUV39H1/HP1/DNMT3A recruitment, and none of the SUV39H1/HP1/DNMT3A contains documented conserved domains that might contribute to CRL4B interaction, such as WD40 domain.36,37 Clearly, future investigations are needed to explore the scope and the exact molecular mechanisms of the formation of the CRL4B/SUV39H1/HP1/DNMT3A complex, and to determine whether or not this functionality involves additional elements. In addition, the functional association of CRL4B with SUV39H1/ HP1/DNMT3A during normal development and physiology remains to be investigated. The crucial role of DNMT3A during development is highlighted by early embryonic lethality in knockout mice.53–55 Remarkably, evidence showed that Cul4b or Ddb1 ablation also causes early embryonic lethality,10,56 whereas Cul4a null mice do not exhibit severe developmental defects.12–14 It is worth noting that the phenotype of Cul4b null embryos is very similar to that in Dnmt3a null embryos, clearly pointing to a concerted role of CUL4B and DNMT3A in embryogenesis, supporting our current report of a physical association and thus a functional connection between CRL4B and SUV39H1/HP1/ DNMT3A. Moreover, HP1, DNMT3A and CUL4B are all essential for neural development.57–60 For example, depletion of Dnmt3a was associated with severe neurogenic defects and CNS abnormalities.61,62 Notably, HP1b, Dnmt3a and Cul4b are highly expressed in the cerebral cortex of mouse brain,60,63–65 indicating that they are functionally connected. Further research will need to be concentrated on the functional interplay between the CRL4B and SUV39H1/HP1/DNMT3A in neural development, which will be expected to shed new light on the pathogenesis of X-linked mental retardation. CUL4 is one of the three founding members of the Cullin family that is conserved from yeast to humans. Compared with other cullin members, CUL4B is the only one that harbors a nuclear localization signal. We showed that the ability of catalyzing H2AK119 mono-ubiquitination is essential for the transcription repressive function of CUL4B,10 and is required for SUV39H1/HP1/ DNMT3A to fulfill the mission of epigenetic silencing. It is known that the CRL4 complex also functions as a ubiquitin E3 ligase in poly-ubiquitinating histones H2A, H2B, H3 and H4 to facilitate cellular response to UV damage by affecting nucleosome stability.25,66 However, the mono-ubiquitination of lysine 119 of histone H2A does not constitute a degradation signal but may represent a bulky structural alteration that may impact the binding by other transcriptional factors, cofactors, and histone H1 to chromatin.67 In mammalian cells, it was shown recently that H2AK119ub1 marks certain inactive chromatin regions, possibly playing a role in maintaining the inactive chromatin state.68 This raises the question of the actual role of H2AK119ub1, the product of CRL4B enzymatic activity, in epigenetic transcriptional regulation. One possibility is that H2AK119ub1 could create a repressive environment that may promote the binding of other repressor complexes such as PRC2 or SUV39H1/HP1/ DNMT3A complex. Indeed, de-ubiquitination of H2AK119 is reported to promote histone H1 phosphorylation and H3K4 di- and trimethylation, which may further result in H1 release from chromatin and gene activation.69,70 In addition, as a very large modification, H2AK119ub1 could possibly prevent H2BK120ub1, thus inhibiting H3K79 methylation, a pivotal activation signal.71,72 Furthermore, H2AK119ub1 is reported to be refractory to histone disassembly to inhibit polymerase activity.73,74 It is also possible that CUL4-mediated ubiquitination may be involved in other types of histone modifications and epigenetic mechanisms, such as heterochromatin formation, parental Oncogene (2015) 104 – 118

imprinting or X-chromosome inactivation.75–77 Moreover, in Neurospora crassa, the CRL4B complex was also found to interact with DNMT3A/B that is responsible for de novo DNA methylation.78 Interestingly, Pcu4-Rik1-Rbx1 complex, the homolog of CRL4 in fission yeast Schizosacchromyces pombe was shown to be involved in the regulation of noncoding RNA transcription, an emerging powerful element engaged in epigenetic regulation and heterochromatin formation.79,80 It is not expected that the CUL4B-DDB1 complex has relationships with all types of epigenetic regulators, but it will not be surprising if future investigations uncover additional epigenetic elements that are associated with this complex. IGFBPs are circulating transport proteins for IGF, with IGFBP3 being the predominant IGFBP in circulation.81 Contrary to the original understanding based on the somatomedin hypothesis, IGFBP3 is not just a passive carrier of IGF-I. It is reported that IGFBP3 can regulate cell growth and death either dependent or independent of its interaction with IGF, and inhibit tumor invasion and angiogenesis through blocking the PI3K/AKT1 signalling pathway.81,82 Aberrant promoter hypermethylation and gene silencing of IGFBP3 are observed in many cancers, such as lung, hepatocellular, gastric, colorectal, breast and ovarian cancer, and associated with poor clinical outcome.45–48,52 Therefore, the regulation of IGFBP3 by CRL4B/SUV39H1/ HP1/DNMT3A may have significant physiological implications. In agreement with this notion, we demonstrated that CUL4B promotes cervical carcinoma cell survival, proliferation, anchorage independent growth and invasive potential and it does so, at least in part, through repression of the powerful tumor suppressor IGFBP3. We also showed that CUL4B expression was significantly higher in tumor samples compared to adjacent normal tissue and that the level of CUL4B expression was negatively correlated with the level of IGFBP3 expression. Moreover, IGFBP3 is induced by wild-type p53,52 and enhances the p53-dependent apoptotic response of tumor cells to DNA damage.83 Aberrant promoter methylation of IGFBP3 at the p53 regulatory element causes gene silencing resistant to p53.84 Interestingly, it is reported that CRL4A could degrade p53 to promote cell cycle progression and immortalization.15,85 So, it is reasonable to speculate that CRL4 negatively controls the p53-IGFBP3 axis. Collectively, in addition to the propositions that CRL4B possibly promotes tumorigenesis through degradation of several cyclin-dependent kinase inhibitors,16,17 and coordinates with PRC2 in H3K27me3mediated transcriptional silencing,10 our findings that CUL4B controls DNA methylation-based transcriptional repression add a new element to the understanding of the oncogenic potential of CRL4B. In summary, our study revealed that CRL4B regulates transcription by mono-ubiquitinating H2AK119 and by coordinating/ facilitating the function of SUV39H1/HP1/DNMT3A catalyzing H3K9me2/3 and DNA methylation, thus providing a new molecular basis for the interplay between histone ubiquitination/methylation and DNA methylation in chromatin remodeling. Our data indicate that CUL4B is required for aberrant hyermethylation of the promoter region, thus contributes to epigenetic silencing of tumor suppressors, and promotes cervical carcinogenesis and progression, supporting the pursuit of CUL4B as a target for cancer therapy.

MATERIALS AND METHODS Antibodies and reagents The sources of the antibodies were: anti-HA, anti-CUL4B and anti-CUL4B (mouse-Sigma-Aldrich, St Louis, MO, USA; rabbit-BD, Franklin Lakes, NJ, USA); anti-DDB1 and anti-IGFBP3 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); anti-G9A, anti-SUV39H1, anti-DNMT1, anti-DNMT3A, antiDNMT3B, anti-H3K9me2, anti-H3K9me3, anti-H3 and anti-ROC1 (Abcam, & 2015 Macmillan Publishers Limited

CRL4B modulates DNA methylation-based gene silencing Y Yang et al

115 Hong Kong, China); anti-H2AK119ub1, and anti-H3 (Millipore, Billerica, MD, USA). Protein A/G Sepharose CL-4B beads were from Amersham Biosciences (Indianapolis, IN, USA), and protease inhibitor mixture cocktail was from Roche Applied Science, Indianapolis, IN, USA. The siRNAs were purchased from Sigma-Aldrich. The shRNAs were obtained from GenePharma Co Ltd (Shanghai, China) and the targeted sequences were (sense sequences): CUL4B: 50 -CCGGGCCATGAAAGAAGCATTTGAACTCGA GTTCAAATGCTTCTTTCATGGCTTTTT-30 . IGFBP3: 50 -CCGGGCCTCGATTTATATT TCTGTTCTCGAGAACAGAAATATAAATCGAGGCTTTTTG-30 .

Cell culture and stable cell line generation HeLa and HEK293T cell lines were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen, Grand Island, NY, USA). SiHa and Ca Ski human cervical cancer cell lines were from Chinese Academy of Medical Sciences. All media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (Gibco BRL, Gaithersburg, MD, USA). Cells were maintained in a humidified incubator equilibrated with 5% CO2 at 37 1C. Stable cell lines expressing the CUL4B or shCUL4B were generated by transfection of pSG5HA-CUL4B or pGPU6-GFP-shCUL4B into HEK293T, HeLa, SiHa cells using lipofectamin 2000 and screened for expression in single colonies under the presence of 1 mg/ml G418.

Immunopurification and mass spectrometry A stable HeLa cell line expressing the HA-CUL4B was produced by transfection of the cells with HA-CUL4B and selection in medium containing 1 mg/ml of G418. Anti-HA immunoaffinity columns were prepared using anti-HA affinity gel (Sigma) following the manufacturer’s suggestions. Cell lysates were obtained from about 5  108 cells and applied to an equilibrated HA column of 1-ml bed volume to allow for adsorption of the protein complex to the column resin. After binding, the column was washed with cold BC500 buffer containing 50 mM Tris, 2 mM EDTA, 500mM KCl, 10% glycerol and protease inhibitors. HA peptide (0.2 mg/ml, SigmaAldrich) was applied to the column to elute the HA protein complex as described by the vendor. Fractions of the bed volume were collected and resolved on SDS-polyacrylamide gel and silver stained; bands were excised and subjected to LC-MS/MS sequencing and data analysis.

Fast protein liquid chromatography HeLa nuclear extracts were prepared and dialyzed against buffer D (20 mM HEPES, pH 8.0, 10% glycerol, 0.1 mM EDTA, 300 mM NaCl) (Applygen Technologies, Beijing, China). Approximately 6 mg of nuclear protein was concentrated to 1 ml using a Millipore Ultrafree centrifugal filter apparatus (10 kDa nominal molecular mass limit), and then applied to an 850  20 mm Superose 6 size exclusion column (Amersham Biosciences, Salt Lake City, UT, USA) that had been equilibrated with buffer D containing 1 mM dithiothreitol and calibrated with protein standards (blue dextran, 2000 kDa; thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Ovalbumin, 43 kDa; all from Amersham Biosciences). The column was eluted at a flow rate of 0.5 ml/min and fractions were collected.

Histone methyltransferase assays and protein sequencing Immunopurified HA-CUL4B complex were incubated with 5 mg unmodified histones H3 peptides (residues 1–21, Millipore) and 2 ml S-adenosyl-Lmethionine (Sigma) in buffer MAB (50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl2, 10 mM 2-mercaptoethanol and 250 mM w/v sucrose) at 30 1C for 4 h. The reaction products were then resolved by SDS±PAGE and western blotted with indicated antibodies.

GST pull-downs and immunoprecipitation for DNA methyltransferase assays GST fusion proteins were produced in Escherichia coli (BL-21 strain) and purified with Glutathione Sepharose 4B beads according to the manufacturer’s instructions. For immunoprecipitations preceding the DNA methyltransferase assay, we used anti-CUL4B, anti-DDB1 and anti-DNMT3A. IgG served as a negative control. DNA methyltransferase assays were carried out as described.33 Briefly, equivalent amounts of GST fusion proteins prebound to glutathione beads were added to HeLa nuclear extract (100 ml) or antibodies immunoprecipitation from 107 cells in modified IPH buffer (250 ml; 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.1% NP-40, 0.1 mM PMSF) and incubated at 4 1C for 12 h. Beads were washed three times with IPH buffer and assayed for methyltransferase activity in a 100 ml reaction & 2015 Macmillan Publishers Limited

containing a 33-bp hemimethylated oligonucleotide substrate86 (500 ng), S-Adenosyl-L-[methyl-3H]-methionine (2 ml; 77 Ci/mmol; Amersham), TrisHCl (50 mM, pH 7.5), EDTA (5 mM), 50% glycerol, DTT (5 mM) and protease inhibitors. After incubation at 37 1C for 1 h, we removed unincorporated nuclides with Biospin chromatography columns (BioRad, Berkeley, CA, USA) and determined the incorporation of radioactivity by liquid scintillation counting.

Immunoprecipitation For immunoprecipitation assays, cells were washed with cold PBS and lysed with cold lysis buffer at 4 1C for 30 min. Whole cell lysates were incubated with appropriate primary antibodies or normal rabbit/mouse immunoglobin G (IgG) on a rotator overnight at 4 1C, followed by addition of protein A/G Sepharose CL-4B beads for 2h at 4 1C. Beads were then washed 5–8 times with lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% NP-40, 0.25% sodium deoxycholate and protease inhibitor mixture). The immune complexes were subjected to SDS–PAGE, followed by immunoblotting with secondary antibodies. Immunodetection was performed using enhanced chemiluminescence (ECL System, Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions.

ChIP and re-ChIP ChIPs and re-ChIPs were performed in HeLa cells as described previously.49,50 Briefly, 1  107 cells were cross-linked with 1% formaldehyde, sonicated, pre-cleared and incubated with 5–10 mg of antibody per reaction. Complexes were washed with low and high salt buffers, and the DNA was extracted and precipitated. For Re-ChIP assays, immune complexes were eluted from the beads with 20 mM dithiothreitol at 37 1C for 30 min. Eluates were then diluted 30-fold with ChIP dilution buffer and followed by reimmunoprecipitation with specific second antibodies. The final elution step was performed using 1% SDS solution in Tris-EDTA buffer, pH 8.0. The enrichment of the DNA template was analyzed by conventional PCR using primers specific for each target gene promoter. The primers used were listed as following:

RPS6KA6

F

50 -TGAAGGCAACCAGCAGGAG-30

RPS6KA6 IGFBP3 IGFBP3 AXIN1 AXIN1 FOXO3 FOXO3 WNT2B WNT2B IQGAP2 IQGAP2 NKX3.1 NKX3.1 RELN RELN SFRP5 SFRP5 PER2 PER2 KLF3 KLF3 USF2 USF2 SLBP SLBP ACTB ACTB

R F R F R F R F R F R F R F R F R F R F R F R F R F R

50 -TGACAAATACTAGGGCAGGGAT-30 50 -TTCAGCAGTGCCCAGTTTATT-30 50 -GCTACACCGCAAGTCTCCAA-30 50 -TTGGGACTCAGGAGGGTGGAG-30 50 -GTACATCGGAGGGCAGTCAGG-30 50 -GCGGGAGCAAGGAGGTGAT-30 50 -GGGAGCGAGTCGGAACATAAA-30 50 -CGCTCCTGTAGGCAGTGTTG-30 50 -GCACTCATTTCCTCCATCCTT-30 50 -GCTTCACAAAGTGCTGGGATT-30 50 -CGTACCTGTGCCCTCACCTATT-30 50 -ATTGCGGATAAAGGAACCACC-30 50 -TCTTCAGGACCGTCAGAGCC-30 50 -CCACAACCGAGCAGCACA-30 50 -GCAGCGACAGAGCCTCATCT-30 50 -CTCCTGGTTGTTTCTGGGCTCT-30 50 -AATAAGGCTAGATGGTGGGTAGGT-30 50 -CCGACGAGGTGAACATGGAG-30 50 -GCCGCTGTCACATAGTGGAAAA-30 50 -CCCCAGGGCACAAAGAAAGA-30 50 -CGCACTCAGCTCACTGCAAGA-30 50 -CACTGATGAAGCCCTGCCATAC-30 50 -CGGCGCTTTACAGACGCAT-30 50 -CACAGCTGAGTGGACCCCTG-30 50 -CAGAAACCCGCGTCCCC 50 -CCCTCCTCCTCTTCCTCAATCT-30 50 -AACGGCGCACGCTGATT-30

Real-time quantitative RT–PCR Total RNA was extracted with Trizol reagents following the manufacturer’s instructions (Invitrogen). Any potential DNA contamination was removed by RNase-free DNase treatment (Promega, Madison, WI, USA). cDNA was prepared with the MMLV Reverse Transcriptase (Promega). Relative Oncogene (2015) 104 – 118

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116 quantitation was determined using the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) that measures real-time SYBR green fluorescence and then calculated by means of the comparative Ct method (2  DDDCt) with the expression of GAPDH as an internal control. The experiment was performed in triplicate.

ACKNOWLEDGEMENTS This work was supported by grants (81322032, 31171240 and 90919053 to YW) from the National Natural Science Foundation of China, grant (NECT-12–1067 to YW) from the Ministry of Education of China, and grant (2013CB910900 to YG) from the National Basic Research Program of China.

Bisulphite genomic sequencing Methylation status of the IGFBP3 promoter in HeLa cells was assessed by bisulphite genomic sequencing. Genomic DNA was extracted using QIAamp DNA kit (Qiagen, Hilden, Germany) and subjected to EpiTect Bisulfite kit (Qiagen) according to the manufacture’s protocol. The amplified product was subcloned into the pGEM-T Easy vector by TA cloning (Promega) and sequenced via automated sequencing. Primers for IGFBP3 amplification are: F: 50 -TTTGAGAGTGGAAGGGGTAAGGG-30 ; R: 50 -CCCACTACATAACACCTACAACC-30 .

Soft agar colony assay HeLa cells transfected with indicated shRNAs (grown in G418 selection medium for 10 days) were trypsinized and suspended in DMEM medium containing 0.3% lukewarm agar at a cell concentration of 5  103 cells/ml. The suspension was spread on top of 0.5% solidified agar plates. Colony formation was observed after a 2-week culture at 37 1C in air containing 5% CO2. Colonies were counted and photographed using a microscope (Olympus, Tokyo, Japan).

Tunel assay HeLa cells were seeded onto 6-well plates for 24 h and transfected with indicated shRNAs using Lipofectamine 2000 (Invitrogen). After 48 h of the plasmid transfection, cells were harvested, and TUNEL assay were performed according to the manufacturer’s instructions (#TB235, Promega) with a fluorescence method. Positive and negative controls with the TUNEL assay were performed according to the instructions provided by the manufacturer.

Cell invasion assay Transwell chamber filters (Millipore) were coated with Matrigel. After infected with vectors or shRNAs, HeLa cells were suspended in serum-free DMEM media at a concentration of 5.0  105/ml, 300 ml suspensions were placed to the upper chamber of the transwell. The chamber was then transferred to a well containing 500 ml of media containing 10% fetal bovine serum. After incubation for 36 h, cells in the top well were removed by wiping the top of the membrane with cotton swabs. The membranes were then stained and the remaining cells were counted. Four highpowered fields were counted for each membrane.

Tumour xenografts HeLa, SiHa and Ca Ski cervical cancer cells stably transfected with a CUL4B shRNA or scrambled control shRNA were collected and 4  106 viable cells in 100 ml PBS were injected subcutaneously into the 6–8-week-old female BALB/c nude mice (Vital River, Beijing, China). Six animals per group were used in each experiment. Tumors were measured every 4 days using a vernier caliper and the volume was calculated according to the formula: p/6  length  width2. All studies were approved by the Animal Care Committee of Shandong University School of Medicine.

Tissue specimen and immunohistochemistry Cervical carcinoma tissues were obtained from Qilu Hospital of Shandong University. Samples were frozen in liquid nitrogen immediately after surgical removal and maintained at  80 1C until analyzed. Samples were fixed in 4% paraformaldehyde (Sigma-Aldrich) at 4 1C overnight, and then embedded in paraffin, sectioned at 8 mm onto Superfrost-Plus Slides and processed as per standard protocols using DAB staining, and monitored microscopically. All human tissue was collected using protocols approved by the Ethics Committee of Shandong University School of Medicine, and informed consent was obtained from all patients.

CONFLICT OF INTEREST The authors declare no conflict of interest.

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