Cullin4B Promotes Tumorigenesis By Coordinating With SUV39H1/HP1/DNMT3A In DNA Methylation-based Epigenetic Silencing

Cullin4B (CUL4B) is a component of the Cullin4B-Ring E3ligase complex (CRL4B) that mainly functions in proteolysis. Ubiquitin-mediated proteolytic pathway is the majority of protein degradation in cells, participates in a broad variety of physiologically and developmentally-controlled processes such as cell proliferation, cell cycle progression and neuronal functions. Dysregulated ubiquitin-dependent proteolysis has been implicated as a causative factor in cancer and several inherited diseases. Human cells express eight different cullins(CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7, and PARC). Among the eight Cullins identified, Cu14A and Cu14B are highly conserved. There is only one Cu14homologue in Fission yeast, plant, C. elegans and Drosophila. Further research indicates that Cu14A and Cu14B are not functionally redundant. Cu14b knockout mice are embryonically lethal (Liu et al.,2012), indicating a unique function of Cul4b that cannot be compensated by Cul4a.It is suggested that Mutations in Cu14could result in heterochromatin formation defect in fission yeast. In mammals, CUL4B targeted WDR5, a core subunit of histone H3lysine4(H3K4) methyltransferase complexes, for ubiquitylation and degradation in the nucleus. In addition, CUL4B is physically associated with Polycomb-repressive complex2(PRC2), and possesses transcription repressive activity by promoting H2AK119monoubiquitination. Interestingly, CUL4B, unlike CUL4A and other Cullins, carries a nuclear localization signal (NLS) in its N terminus and is also localized in the nucleus, suggesting that CUL4B might be involved in the nucleus-based functions.We report that CRL4B is associated with histone methyltransferase SUV39H1, heterochromatin protein1(HP1) and DNA methyltransferases3A (DNMT3A), which involved in DNA methylation-based gene silencing. Depletion of CUL4B resulted in loss of not only H2AK119monoubiquitination but also H3K9trimethylation and DNA methylation, leading to derepression of a collection of genes including the tumor suppressor IGFBP3. We demonstrated that CUL4B promotes cell proliferation, invasion and tumorigenesis, at least partially by repressing IGFBP3. We found that the expression of CUL4B is markedly up-regulated 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.PART ONE Identification of the CUL4B Interacting ProteinsIn an effort to better understand the mechanistic roles of CUL4B protein in the nucleus, we employed affinity purification and mass spectrometry to identify the proteins that are associated with CUL4B in vivo.1、We recently isolated CUL4B associated proteins with affinity purification and mass spectrometry assay. In these proteins, we also found that CUL4B could co-purified with DNMT3A, SUV39H1, and HP1α/β/γ, all of which are functionally linked to DNA methylation-based transcriptional repression. We employed affinity purification to identify the proteins that are associated with CUL4B in vivo. The presence of DNMT3A, SUV39H1, PRMT5and HP1in the CUL4B-interacting complex was detected by Western blotting analysis.2、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(FPLC). Nuclear extracts derived from HEK293T cells were fractionatedby DEAE sepharose, followed by superpose6gel filtration chromatography.Western blotting revealed a major peak at about669-1000kDa for CUL4B, DDB1, and ROC1, and also for DNMT3A, SUV39H1, HP1β,and HP1β. Significantly, the chromatographic profiles of CUL4B, DDB1, and ROC1were largely overlapped with SUV39H1/HP1/DNMT3A, substantiating the argument that these proteins are associated in vivo.3、Total proteins from HEK293T cells and HeLa cells (a human cervical carcinoma cell line) were extracted, and co-immunoprecipitation experiments were performed with antibodies against the endogenous proteins. Immunoprecipitation (IP) with antibodies against CUL4B and immunoblotting (IB) with antibodies against SUV39H1, HP1α, HP1β, HP1γ, DNMT1, DNMT3A or DNMT3B demonstrated that CUL4B was co-immunoprecipitated with all these proteins. Reciprocally, IP with antibodies against SUV39H1, HP1α, HP1β,HP1γ, DNMT1, DNMT3A or DNMT3B followed by IB with antibodies against CUL4B also revealed that all these proteins were co-immunoprecipitated with CUL4B. Moreover, SUV39H1, HP1and DNMT3A could also be co-immunoprecipitated with DDB1and ROC1, the other two main components of the CRL4B complex, in HEK293T or HeLa cells.4、In order to determine the molecular basis for the interaction of CRL4B with the SUV39H1/HP1/DNMTs complex, GST pull-down assays were performed using GST-fused CUL4B, DDB1or ROC1proteins and in vitro transcribed/translated individual components of the SUV39H1/HP1/DNMTs complex including SUV39H1, HP1α, HP1β, HP1γ, DNMT1, DNMT3A and DNMT3B. These experiments revealed that both CUL4B and DDB1could interact directly with SUV39H1, DNMT1, DNMT3and DNMT3B.The results showed that the CUL4B could associate with SUV39H1/HP1/DNMT3A complex, which involved in DNA methylation-based gene silencing. IP and GST pull-down assays revealed that CRL4B could interact directly with SUV39H1/HP1/DNMTs. Thus provides a new molecular basis for CUL4B in transcriptional regulation.PART TWO Genome-wide Transcriptional Targets for the CRL4B/SUV39H1/HP1/DNMT3A ComplexSince it is well established that SUV39H1/HP1/DNMT3A complex mainly functions in DNA methylation-based transcriptional repression, 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.1、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 against5-methylcytosine (5-mC). Specifically, following MeDIP, methylated DNAs with5-mC were amplified using nonbiased conditions, labeled, and hybridized to an oligonucleotide array covering over22,532human promoters in the NCBI database with a range from-800bp to+200bp relative to the transcription start site with a false recovery rate less than0.05. We found that a total of4,045genomic regions (1,878genes) had significantly changes in methylation patterns between CUL4B-depleted cells and control cells, including1,965hypermethylation sites (953genes) and2,080hypomethylation sites (925genes). 2、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 of10-3. Ten pathways enriched with the most genes with hypermethylation or hypomethylation in CUL4B-depleted cells were plotted against that in control cells. Eleven hypomethylated gene including RPS6KA6, IGFBP3, AXIN1, SFRP5, FOXO3, WNT2B, IQGAP2, NKX3.1, RELN, KLF3and PER2, which represent each of the classified pathways, were selected for further quantitative ChIP (qChIP) analysis. The results validated our MeDIP-chip data.3、Among the target genes identified above, insulin-like growth factor binding protein (IGFBP3) is a well-established antiproliferative, proapoptotic and invasion suppressor protein. Aberrant promoter hypermethylation of IGFBP3and gene silencing have been observed in numerous cancers, including lung, hepatocellular, gastric, colorectal, breast, and ovarian cancers. We thus investigated the transcriptional regulation of IGFBP3by CRL4B/SUV39H1/HP1/DNMT3A complex in details. Quantitative ChIP (qChIP) using antibodies against CUL4B, DDB1, ROC1, SUV39H1, HP1α, HP1β or DNMT3A demonstrated that all these proteins could bind to IGFBP3promoter region。4、The histone modification marks H2AK119ubl and H3K9me2/3, which are added by CRL4B and SUV39H1, respectively, and DNA methylation, which is catalyzed by DNMT3A, were enriched in the same region of IGFBP3promoter.5、ChIP assays were performed in HeLa cells using antibodies against CUL4B, DDB1, SUV39H1, HP1α, DNMT3A,5-mC or control IgG. The results showed that both CRL4B complex and SUV39H1/HP1/DNMT3A complex occupied IGFBP3promoter, and that5’-methylated cytosine was enrich in the same region. To further test our proposition that CRL4B and SUV39H1/HP1/DNMT3A function in the same protein complex at IGFBP3promoter, sequential ChIP or ChIP/Re-ChIP experiments were performed. In these experiments, soluble chromatins were first immunoprecipitated with antibodies against CUL4B, DDB1, SUV39H1, HP1α, DNMT3A or5-mC. The immunoprecipitates were subsequently re-immunoprecipitated with appropriate antibodies. The results showed that in precipitates, the IGFBP3promoter that were immunoprecipitated with antibodies against CUL4B could be re-immunoprecipitated with antibodies against DDB1, SUV39H1, HP1α, DNMT3A or5-mC.6、We next investigated the effect of CUL4B depletion on IGFBP3expression by quantitative RT-PCR (qRT-PCR) and Western blotting. As expected, CUL4B depletion led to an increased expression of IGFBP3at both transcription (Fig.5A, upper panel) and protein levels in HeLa, SiHa and Ca Ski cells. Interestingly, treatment of HeLa cells with a deoxycytidine analog5-aza-deoxycytidine (5-aza-dC), which is widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation, restored IGFBP3expression; however, in CUL4B-depleted cells,5-aza-dC treatment had little effect on IGFBP3expression, suggesting that CUL4B is involved in DNA methylation-based epigenetic silencing of IGFBP3in human cervical carcinoma cells and supporting a functional connection between the CRL4B complex and SUV39H1/HP1/DNMT3A complex.7、qChIP assays were performed in CUL4B-depleted 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, HP1α, HP1β and DNMT3A. Consistent with this,’the levels of H2AK119ub1, H3K9me2, H3K9me3and5-mC were markedly decreased at IGFBP3promoter.PART THREE The Role of CUL4B in the Tumorigenesis of Cervical CarcinomaAs stated before, IGFBP3has been demonstrated to exert biological functions related to anti-proliferation, pro-apoptosis and anti-invasion and is considered to be a tumor suppressor. 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.1、HeLa cells with stable CUL4B knockdown showed a severe growth inhibition, which could be partially alleviated by co-knockdown of IGFBP3. 2、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.3、TUNEL assays revealed a striking increase in apoptosis cells,25.82%in CUL4B-RNAi group compared with only1.42%in control group (p<0.001), which could be partially rescued through IGFBP3knockdown (from25.82%to11.21%).4、Soft agar colony assays revealed that loss of CUL4B could significantly reduce both the clone size and clone number. Moreover, the results from trans-well invasion assays showed that knockdown of CUL4B resulted in about10-fold decrease in cell invasion. In addition, the decreased invasiveness upon CUL4B depletion was partially alleviated when IGFBP3was concomitantly knocked down.5、We collected64cervical carcinoma samples,30of them with paired adjacent normal tissues, from cervical cancer patients and performed tissue arrays by immunohistochemical staining. CUL4B was found to be significantly up-regulated in tumors and its expression appeared to be positively correlated with histological grades. There was a significant negative correlation between the expression of CUL4B and IGFBP3in these samples.Taken together, this study suggested that CRL4B could associate with SUV39H1/HP1/DNMT3A complex. These results showed that CRL4B, through catalyzing H2AK119, facilitated H3K9tri-methylation and DNA methylation, two key epigenetic modifications involved in DNA methylation-based gene silencing. We found that CUL4B could promote cell proliferation, invasion and tumorigenesis, at least in part, through repression of the tumor suppressor IGFBP3. The Expression of CUL4B is up-regulated in cervical carcinomas and negatively correlated with that of IGFBP3. Our findings advanced our understanding of the role of CUL4B in epigenetic transcriptional regulation and tumorigenesis.