| Protein ubiquitination is involved in multiple biological progresses including development, cell cycle, signaling transduction, and viral infection. Cullin-Ring E3ligases (CRLs) represent the biggest E3ligase family. Cullin acts as the scaffold to assemble various E3ligase complexes that specifically add ubiquitins to various substrates and direct them for proteasome degradation. Cullin4-Ring E3ligases (CRL4) recognize specific substrates through the adaptor DDB1and a substrate receptor that are positioned at the N terminal of Cullin4. There are two closely related Cullin4members in human, namely CUL4A and CUL4B, which form CRL4A and CRL4B complexes, respectively. Unlike CUL4A, CUL4B has a unique nuclear location signal and is primarily located in nucleus. Mutations in CUL4B are associated with X-linked mental retardation. CUL4B was found uniquely to regulate microRNA expression to mediate CDK2-CDC6cascade in DNA replication. Cul4b-null embryos were impaired in development and died in E8.5.To gain insights into the molecular mechanisms by which CRL4B complex functions, we purified and characterized CUL4B-interacting proteins. Our data indicated that CRL4B is physically and functionally associated with PRC2complex via DDB1-RbAp46/48interaction and CRL4B acts as a novel chromatin modifier in transcription repression. CRL4B catalyzes H2AK119monoubiquitination and coordinates with PRC2to promote H3K27methylation. CUL4B was shown to promote cancer cell proliferation, metastasis and tumor progression. CUL4B is frequently upregulated in tumor samples and is positively correlated with tumor grade. Our findings establish CUL4B as a target for cancer therapy. PART ONECRL4B monoubiquitinates H2AK119and cooperates with PRC2complex to repress transcriptionWe purified CUL4B-interacting proteins using FLAG M2affinity gel in combination with mass spectrometric analysis. We utilized a Gal4-reporter system and fusion proteins to investigate the role of CUL4B in transcriptional regulation. We made the following findings:(1) CRL4B is physically associated with PRC2complex through DDB1-RbAp46/48:We employed affinity purification and mass spectrometry to identify the proteins that are associated with CUL4B in vivo. FLAG-tagged CUL4B (FLAG-CUL4B) was co-purified with the peptides of EZH2, EED, and RbAp48, which are the core components of PRC2. Physical association of CRL4B with PRC2complex but not with PRC1was confirmed by immunoprecipitation. To further support this notion, protein fractionation experiments were carried out with nuclear proteins from HeLa by fast protein liquid chromatography (FPLC). A major peak was revealed at about669kDa-1300kDa for CUL4B, DDB1, and ROC1, and also for PRC2core components EZH2, SUZ12, EED, and RbAp46/48, indicating the two complexes co-exist in a larger complex. GST pull down showed that DDB1and RbAp46/48serves as the bridging molecules between CRL4B and PRC2, for knockdown of DDB1or RbAp46/48disrupted the physical association between CUL4B and EZH2or SUZ12.(2) The physical association between CRL4B and PRC2, one transcriptional repressive complex, suggests that CRL4B may have a role in transcription regulation. We tested this possibility by utilizing Gal4reporter system and fusion protein. We found that the DDB1-interacting domain and Cullin domain of CUL4B are each required for transcriptional repression. Luciferase assay results revealed CUL4B as a moderate transcription corepressor. Knockdown of DDB1, EZH2and RbAp46/48each resulted in attenuated repressive function of CUL4B, but BM11and RING1B, the core components of PRC1, had no effect on the repressive function of CUL4B when knocked down, suggesting that the function of CRL4B is dependent on PRC2but not on PRC1.(3) Decrease in CUL4B level resulted in reduced H3K27me3and H2AK119ubl: Immunofluorescence staining and Western blot showed that CUL4B depletion would reduce the level of H2AK119ub1and H3K27me3but increase that of H3K4me3in vitro. Similar results were obtained with Cul4b constitutive or conditional knockout mice including Cul4b-null embryos at E7.5and liver-specific knockout tissues.(4) CRL4B catalyzes H2AK119monoubiquitination:Since CUL4B increases H2AK119ubl independently of PRC1complex, we performed in vitro ubiquitination assays to determine whether CRL4B could ubiquitinate H2A and repress transcription through monoubiquitinating H2AK119. The results showed that H2A is monoubiquitinated by CRL4B in an E1/E2and E3-dependent manner in the presence of ATP and ubiquitin, but depletion of Cullin domain impaired H2AK119monoubiquitination. Mutation of H2AK119(K to R), but not that of H2AK118(K to R), disabled CRL4B in H2A monoubiquitination, suggesting the specificity of CRL4B-catalyzed ubiquitination. Compared with RING1B, CRL4B is less efficient in histone ubiquitination. However ablation either of them led to a significant reduced level of H2AK119ubl, suggesting that CRL4B may function as substituted substitute enzyme forH2AK119ubl.(5) RbAp48as the substrate receptor of H2A:In vitro ubiquitination assay showed that RbAp48but not EED, the histone chaperone with WD repeat domain, could effectively increase the efficiency of CRL4B to monoubiquitinate H2AK119. Interference of RbAp46/48led to the decrease of H2AK119ub1.Together, the results in this part demonstrated a direct physical association between CRL4B and PRC2through DDB1-RbAps. CRL4B monoubiquitinated H2AK119and cooperated with PRC2complex to promote the trimethylation of H3K27and to repress gene expression. PART TWOCRL4B coordinates with PRC2to promote tumor progression by repressing tumor suppressors.EZH2, which catalyzes the methylation of H3K27, has been reported to be dysregulated in tumor tissues. EZH2promotes cancer cell proliferation, invasion and tumor progression by repressing the expression of many tumor suppressors including CDH1and p16INK. In order to study the mechanism by which CUL4B coordinates with PRC2in regulating tumor suppressors, we performed ChlP-on-chip to identify the target genes co-regulated by CRL4B and PRC2complexes. We determined whether CRL4B and PRC2could co-occupy the promoters at p16and PTEN loci. Also, clinical samples were analyzed to determine the role of CUL4B in tumor progression.(1) CRL4B cooperates with PRC2to repress genes involved in multiple pathways: In order to understand the mechanism by which PRC2and CUL4B complexes cooperate in transcriptional repression, we performed ChIP-on-chip using EZH2and CUL4B antibodies in KYSE410cells and analyzed their target genes.619genes were identified to be co-regulated by EZH2and CUL4B including some tumor suppressors such as p16and PTEN.18cancer-associated genes were identified to be upregulated after CUL4B or EZH2interference. qChIP analysis using antibodies against CUL4B, DDB1, EZH2, RING1B, H3K27me3and H2AK119ubl indicated that CUL4B and EZH2are required for H3K27me3and H2AK119ub1. Moreover, downregulation of CUL4B resulted in reduced EZH2binding to the promoters of their target genes together with significantly decreased H3K27me3, decreased H2AK119ubl and moderately increased H3K4me3while EZH2did not affect the binding of CUL4B to chromatin, suggesting that the recruitment of PRC2to the promoters of target genes depended on CRL4B.(2) CRL4B cooperates with PRC2to repress p16and PTEN:p16and PTEN were critical tumor suppressors. p16INK negatively regulates G1to S transition during cell cycle and PTEN deficiency is a poor prognosis marker in cancer patients. We first confirmed that CRL4B and PRC2also cooperate in repressing p16and PTEN in other types of cells. We found that knockdown of CUL4B invariably led to the upreguation of p16and PTEN. Similar results were obtained in Cul4b null mouse embryos. ChIP/Re-ChIP illustrated the co-occupacy of p16and PTEN promoters by CUL4B, DDB1, EZH2and SUZ12. qChIP assays using primers covering20kb flanking TSS revealed that the binding peaks of CRL4B and PRC2components overlapped with each, but not with that of PRC1component RING1B. While knockdown of CUL4B or DDB1displaced EZH2and SUZ12from the promoters of PTEN and p16, the binding of CUL4B and DDB1to these promoters was only slightly affected when EZH2or SUZ12was knocked down. Together, these data confirmed the co-occupacy of p16and PTEN promoters by CRL4B and PRC2and further supported the notion that CRL4B facilitated the recruitment of PRC2to target sites.(3) CUL4B promotes tumor cell proliferation, colony formation and migration:In order to determine the oncogenic role of CUL4B, we determined the effect of CUL4B expression on tumor cell proliferation and migration. We observed that the expression level of CUL4B was positively correlated with the percentage of cells in S phase. Colony formation assays further showed that overexpression of CUL4B was associated with a marked increase not only in colony numbers but also in colony diameters. Interference of p16partially rescued the proliferation defect caused by CUL4B knockdown, indicating that CUL4B regulates cell cycle via repressing p16. We also investigated whether CUL4B has a role in EMT and tumor metastasis using trans-well invasion assay. CUL4B overexpression in KYSE410cells via stable transfection could promote cell migration while ectopic expression of PTEN or dominant-negative CUL4Bâ–łCullin reduced it. Analysis of NCI60expression profiles showed that CUL4B is positively correlated with mesenchymal markers and negatively correlated with epithelial markers.(4) CUL4B promotes tumor growth:We next investigated the role of CUL4B in tumor formation and progression in vivo by implanting KYSE410and EC9706cells that had been engineered to stably express CUL4B shRNA or control scrambled shRNA onto the subcutaneous sites of athymic BALB/c mice. Western blot and immunohistochemical staining results showed upregulation of H3K4me3and downregulation of H2AK119ub1and H3K27me3. Tumor growth was greatly suppressed by CUL4B knockdown, indicating that CUL4B is required for tumor growth.(5) CUL4B is highly expressed in esophageal squamous carcinoma:We analyzed esophageal squamous carcinoma samples and adjacent non-cancer tissues from13cancer patients and found that CUL4B is overexpressed in tumor samples compared with the adjacent tissues.(6) CUL4B is positively related with tumor histological grades:We further analyzed a tissue microarray of182esophageal squamous carcinoma samples from commercial sources and Qilu Hospital by immunohistochemical staining and observed a statistically significant positive correlation between the level of CUL4B and tumor histological grades, tumor volumes and metastasis ability. There were also significant negative correlations between the expression level of CUL4B and those of p16and PTEN, substantiating the notion that CUL4B has strong oncogenic properties.(7) CUL4B is overexpressed in many types of cancer:Analysis of tissue microarray by immunohistochemical staining showed a statistically significant up-regulation of CUL4B in carcinomas compared to the adjacent normal tissues in many types of cancer. Similar expression patterns of CUL4B and EZH2were observed in cancers of breast, colon, kidney, lung, ovary, and thyroid by using the human cancer survey qPCR gene expression panel (Origene).In summary, we demonstrated that CRL4B and PRC2can co-occupy at the promoters of tumor suppressors and repress their expression. CUL4B promotes EMT and tumor cell metastasis. CUL4B is frequently upregulated in clinical cancer samples and its expression is positively correlated to tumor grade and progression. Together, our results demonstrated a potent oncogenic potential of CUL4B. |
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