CRL4B催化H2AK119单泛素化并协同PRC2复合物促进肿瘤进程
摘要
蛋白泛素化修饰参与调控个体生长发育、细胞周期、信号转导、病毒感染等多种生物学过程。Cullin-RING家族是哺乳动物体内最大的E3泛素连接酶家族,其中Cullin蛋白在泛素连接酶复合体中起支架作用。Cullin4(CUL4)通过DDB1和底物受体蛋白识别并特异性结合多种底物蛋白,将泛素呈递给底物并通过多泛素化修饰导致底物蛋白最终被蛋白酶体降解,或通过单泛素化修饰调节蛋白功能。人类的CUL4包括CUL4A和CUL4B。CUL4A和CUL4B通过与DDB1和ROC1结合构成CRL4A和CRL4B复合物。尽管CUL4A与CUL4B有许多共同的功能,但CUL4B有独特的N末端核定位信号,其亚细胞定位主要定位于细胞核,提示CUL4B可能具有独特的核依赖作用。本实验室前期研究发现CUL4B基因功能丧失突变导致X连锁精神发育迟滞综合征,Cul4b基因全身性敲除小鼠在胚胎发育早期死亡。对CUL4B作用机制的研究发现,CUL4B复合物CRL4B可以降解Cyclin E、调控microRNA并参与DNA复制起始。
为明确CRL4B复合物的功能,本研究从分离并鉴定CUL4B相互作用蛋白入手,发现CRL4B可以通过DDB1-RbAp46/48的直接结合与PRC2复合物相互作用,协同完成基因启动子区域的组蛋白H2AK119ub单泛素化(H2AK119ub1)和H3K27三甲基化过程(H3K27me3),抑制抑癌基因表达；CUL4B能促进肿瘤细胞的增殖、迁移和裸鼠成瘤,且CUL4B在多种肿瘤组织中高表达。这些研究结果为开发以CUL4B及其相关通路为靶点的肿瘤治疗药物奠定了理论基础。
第一部分CRL4B催化H2AK119单泛素化,协同PRC2复合物发挥转录抑制作用
为了探究CUL4B的核依赖的功能、明确CUL4B的相互作用蛋白、深入了解CRL4B复合物的作用,我们采用免疫亲和纯化结合质谱分析CUL4B结合蛋白,并用Gal4融合蛋白——报告基因体系分析CUL4B在转录调控中的作用,取得了以下的研究结果：
(1)CRL4B与PRC2复合物通过DDB1-RbAp46/48的直接结合而形成大的复合物：通过免疫亲和纯化及质谱分析CUL4B的相互作用蛋白,我们在CUL4B结合蛋白中发现了转录抑制复合物PRC2的主要组分EZH2、EED、RbAp46/48的多肽。用Western blot分析证实在CUL4B结合蛋白中检测到PRC2组分EZH2、SUZ12、EED和RbAp46/48,但未检测到PRC1组分。随后,我们通过免疫共沉淀验证了CUL4B、DDB1与EZH2、SUZ12、EED和RbAp46/48的相互结合,但CRL4B与PRC1复合物没有结合。为明确CRL4B与PRC2复合物主要成员是否存在于一个复合物中,我们用FPLC对HeLa细胞中提取的核蛋白进行分离,用Western blot检测洗脱液中各蛋白分布。结果发现PRC2复合物及CRL4B复合物存在较大范围的重叠(669kDa-1300kDa),表明二者共同存在于一个大的复合物中。GST pull-down实验显示CRL4B是通过DDB1-RbAp46/48的直接作用与PRC2复合物结合的。在细胞内干扰DDB1或RbAp46/48表达能有效减少CUL4B与EZH2或SUZ12的相互结合。
(2)CRL4B协同PRC2复合物参与转录抑制：CUL4B亚细胞定位主要在细胞核。PRC2复合物是重要的转录抑制复合物,两者之间的相互结合提示CUL4B可能参入转录调控。因此,我们首先利用Ga14融合蛋白——报告基因体系分析CUL4B在转录调控中的作用,发现Ga14-CUL4B发挥转录抑制作用。为了明确CUL4B不同结构域在其发挥转录抑制中的作用,我们构建了缺失不同结构域的Gal4-CUL4B载体,分析发现CUL4B的DDB1结合区(DID)和Cullin功能支架区(Cullin)是CUL4B发挥转录抑制作用所必需的。为了明确CUL4B复合物与PRC2复合物相互作用对CUL4B转录抑制活性的影响,我们采用RNA干扰技术分别干扰CRL4B、PRC2和PRC1组分表达,发现干扰DDB1、EZH2、 RbAp46/48表达显著降低CUL4B的转录抑制活性,而干扰PRC1复合物的主要成员BMI1或RING1B表达则不影响CUL4B的转录抑制功能,表明CRL4B复合物的转录抑制作用是依赖于PRC2复合物而不依赖于PRC1复合物的。定量染色质免疫共沉淀(qCh1P)分析发现,表达Ga14-CUL4B不仅可以促进CUL4B、DDB1结合到Ga14启动子区域上,而且可以促进PRC2复合物组分EZH2结合到Ga14启动子区域,但并不影响PRC1组分在启动子区域的结合；与之相对应,两种复合物的组蛋白修饰产物H3K27me3和H2AK119ub1在启动子区域也明显增多。对比分析缺失DID或Cullin结构域的Ga14-CUL4B载体发现,与转染全长Ga14-CUL4B相比,转染缺失DID结构域的表达载体Ga14-ADID导致在Ga14启动子区域结合的DDB1和EZH2明显减少,同时H3K27me3和H2AK119ubl也明显减少；转染缺失Cullin结构域的Gal4-△Cullin载体,尽管不影响DDB1在启动子区域富集,但导致启动子区域的EZH2、H3K27me3和H2AK119ub1都明显减少。这些研究结果说明CRL4B发挥转录抑制作用不仅需要其自身的完整结构,而且依赖于PRC2复合物。
(3)干扰CUL4B表达不仅导致组蛋白H2AK119ub1减少,而且也导致H3K27me3水平下降：免疫荧光和Western blot结果显示,干扰CUL4B表达导致细胞内H3K27me3和H2A119ub1水平的明显下降,转录激活标志H3K4me3明显升高。对Cul4b全身性敲除小鼠E7.5胚胎的免疫组化分析也检测到H3K27me3和H2A119ub1水平的显著降低。
(4)CRL4B催化H2AK119单泛素化：以上研究结果提示CRL4B可能是通过单泛素化H2AK119而协同PRC2发挥转录抑制作用。为此,我们采用体外泛素化实验方法检测CRL4B复合物是否可以催化H2AK119单泛素化,结果证实了我们的推测,而且Cullin结构域是其发挥泛素化功能所必需的。为进一步证实单泛素化位点,我们单独或联合突变了H2A第118或／和119位赖氨酸,发现突变118位氨基酸不影响CRL4B对H2A的泛素化修饰,突变119位赖氨酸明显降低H2A泛素化水平,118和119位同时突变导致CRL4B对H2A的泛素化作用消失,说明CRL4B对H2A的单泛素化特异地发生在第119位的赖氨酸上,其作用类似于PRC1组分RING1B。干扰CUL4B或RING1B基因表达均可导致H2AK119ub1的水平降低,表明CRL4B复合物可能是RING1B泛素化H2AK119的替代酶。
(5)bAp48是CRL4B复合物催化H2AK119单泛素化的底物受体蛋白：为了确定CRL4B复合物泛素化H2A的底物受体蛋白,我们分别分析了与DDB1相互作用的WD40蛋白RbAp46/48和EED的作用。结果发现加入RbAp48能有效地提高CRL4B单泛素化H2AK119的效率,而加入EED没有明显作用,说明CRL4B单泛素化H2AK119是以RbAp46/48作为底物受体蛋白的,这一结果进一步通过干扰RbAp46/48影响CRL4B单泛素化H2A而得到证实。
综上,本部分结果表明CRL4B与PRC2复合物通过DDB1-RbAp46/48直接作用形成大的复合物,通过催化H2AK119单泛素化和H3K27三甲基化CRL4B与PRC2复合物协同抑制基因转录。
第二部分CRL4B协同PRC2抑制抑癌基因表达,促进肿瘤进程
EZH2在多组织肿瘤异常激活,通过抑制抑癌基因如(DH1、p16INK等的表达,EZH2促进肿瘤细胞增殖与转移。为了探究CRL4B与PRC2协同作用的生物学效应,本部分首先进行了CUL4B和EZH2共同调控的靶基因分析,并在此基础上分析CRL4B和PRC2复合物在靶基因上的占位情况以及CUL4B如何通过调控这些靶基因而在肿瘤发展中发挥作用。取得了如下结果：
(1)CRL4B协同PRC2复合物调控多种信号通路的基因表达：为了解CRL4B协同PRC2复合物发挥的生物学功能,我们首先用EZH2和CUL4B抗体在食管癌细胞株KYSE410细胞中进行ChIP-on-chip检测。分析发现,CUL4B与EZH2共同调控619个基因,GO与pathway分析发现这些基因的功能涉及多个重要的通路,其中包括多种抑癌基因,如p16、PTEN等。为验证ChIP-on-chip检测结果,我们从中选择了数个与肿瘤相关基因进行验证分析。结果显示,干扰CUL4B或EZH2基因表达导致18个靶基因转录上调；qChIP分析显示,CUL4B、 DDB1、EZH2结合在这些靶基因启动子区域,但未检测到RING1B在启动子区域的结合；同时,H3K27me3和H2AK119ub1也在启动子区域富集。此外我们发现干扰EZH2并不影响CUL4B在这些基因启动子区域的结合能力,而干扰CUL4B明显减少了EZH2在这些基因启动子区域的结合,进而导致启动子区域H2AK119ub1和H3K27me3的明显减少及H3K4me3的增多。进一步说明CUL4B促进PRC2复合物结合到靶基因启动子区域并促进PRC2催化H3K27甲基化。
(2)CRL4B协同PRC2调控p16和PTEN基因表达：在CUL4B和EZH2共同调控的靶基因中,我们选择了p16和PTEN两个重要的抑癌基因进一步分析CRL4B和PRC2复合物协同抑制靶基因表达的分子机制。我们首先利用多种细胞株检测CUL4B表达对p16和PTEN的影响。结果发现干扰CUL4B导致p16(?)PPTEN表达水平明显上调,证实CUL4B负调控这两个基因的转录。为了明确CRL4B和PRC2复合物在它们启动子区域的结合情况,我们进行ChIP/Re-ChIP实验,结果发现CRL4B和PRC2复合物在p16和PTEN启动子区域的共定位。通过qCIP分析了它们在p16和PTEN基因转录起始位点(TSS)上游-10kb到下游+10kb的结合情况,发现CUL4B、DDB1、EZH2、SUZ12在p16和PTEN启动子区域的结合峰明显重叠,而PRC1复合物的RING1B在PTEN基因启动子上没有结合,在p16启动子上结合峰位于TSS下游。干扰CUL4B或DDB1的表达导致EZB2在p16和PTEN上的结合大大降低,而干扰EZH2或SUZ12对CUL4B或DDB1的结合无明显影响,提示CRL4B优先于PRC2结合到p16和PTEN的启动子区域。
(3) CUL4B通过调控p16和PTEN促进肿瘤细胞增殖、克隆形成及迁移：为分析CUL4B在肿瘤进程中的作用,我们利用食管癌细胞株分析了CUL4B对细胞增殖和迁移的影响。发现低表达CUL4B引起G0/G1期细胞增多,进入S期的细胞减少,过表达则相反。克隆形成实验表明,过表达CUL4B促进肿瘤细胞克隆形成,而干扰CUL4B表达克隆形成减少。过表达CUL4B对肿瘤细胞克隆形成能力的影响可以通过过表达p16或干扰EZH2得到拯救,低表达CUL4B的克隆形成的减少可通过低表达p16得以拯救,说明CUL4B通过协同PRC2复合物抑制p16表达而促进肿瘤细胞增殖。另一方面,我们通过Transwell迁移小室实验分析过表达CUL4B对KYSE410细胞迁移能力的影响。发现CUL4B促进食管癌细胞迁移,而同时过表达PTEN或过表达显性负效应载体可以抑制CUL4B促进肿瘤细胞迁移的作用。我们分析NCI60数据库实体瘤细胞中CUL4B和上皮-间质细胞转化(EMT)标记物表达情况,发现在多种侵袭性高的细胞中CUL4B高表达,其表达与细胞侵袭能力正相关。
(4)CUL4B促进肿瘤生长：裸鼠皮下成瘤实验结果显示,干扰CUL4B明显抑制KYSE410及EC9706细胞株在裸鼠皮下肿瘤生长。对肿瘤进行Western blot及免疫组化检测,证实在CUL4B低表达的肿瘤组织中H2AK119ub1和H3K27me3显著降低,H3K4me3水平升高,靶基因p16和PTEN表达水平明显上调。
(5)CUL4B在食管癌肿瘤组织中高表达：为明确CUL4B在临床肿瘤标本中的表达情况,我们对齐鲁医院病理科收集13对新鲜食管癌及癌旁标本中CUL4B的表达量进行了检测。实时定量及Western blot分析发现,相比正常癌旁组织,CUL4B在大多数食管癌肿瘤组织中高表达。
(6)CUL4B与食管癌病理分级正相关：为明确CUL4B在食管癌发生发展中的作用,我们选取了含182例肿瘤和正常组织的食管癌组织芯片进行免疫组化分析。结果发现CUL4B在食管癌肿瘤组织中的表达水平显著高于正常组织,且其表达水平与食管癌病理分级呈正相关。我们进一步验证了配对的肿瘤标本中p16、PTEN和CUL4B的表达相关性,发现CUL4B与p16、CUL4B与PTEN之间表达呈负相关。对31例新鲜肿瘤组织中mRNA表达水平分析表明CUL4B与p16、CUL4B与PTEN的表达水平呈显著负相关,相关系数分别为-0.7739和-0.6070(Pearson),-0.8435和-0.6234(Sperman)。
(7)CUL4B在多种肿瘤组织中高表达：为了验证CUL4B在肿瘤中高表达的普遍性,我们在多个食管癌样本肿瘤及癌旁中检测CUL4B表达。组织芯片免疫组化结果显示CUL4B在食管癌、结肠癌、肺癌、胃癌、卵巢癌的肿瘤中高表达。多组织肿瘤芯片结果也显示,CUL4B在食管癌、乳腺癌、结肠癌、肺癌及卵巢癌中过表达且表达量与病理分级正相关。商品化mRNA芯片显示,在多种肿瘤中EZH2和CUL4B有着相似的表达特性。
以上结果表明,CRL4B复合物促进PRC2复合物在启动子的结合,两者协同作用抑制一组抑癌基因表达。通过影响这些抑癌基因表达,CUL4B促进肿瘤细胞的增殖、克隆形成能力和肿瘤的侵袭与迁移,促进肿瘤细胞的成瘤能力。我们的结果为发现新的肿瘤临床治疗靶点提供了理论基础。
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 ONE
CRL4B monoubiquitinates H2AK119and cooperates with PRC2complex to repress transcription
We 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 TWO
CRL4B 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 CUL4B△Cullin 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.
为明确CRL4B复合物的功能,本研究从分离并鉴定CUL4B相互作用蛋白入手,发现CRL4B可以通过DDB1-RbAp46/48的直接结合与PRC2复合物相互作用,协同完成基因启动子区域的组蛋白H2AK119ub单泛素化(H2AK119ub1)和H3K27三甲基化过程(H3K27me3),抑制抑癌基因表达；CUL4B能促进肿瘤细胞的增殖、迁移和裸鼠成瘤,且CUL4B在多种肿瘤组织中高表达。这些研究结果为开发以CUL4B及其相关通路为靶点的肿瘤治疗药物奠定了理论基础。
第一部分CRL4B催化H2AK119单泛素化,协同PRC2复合物发挥转录抑制作用
为了探究CUL4B的核依赖的功能、明确CUL4B的相互作用蛋白、深入了解CRL4B复合物的作用,我们采用免疫亲和纯化结合质谱分析CUL4B结合蛋白,并用Gal4融合蛋白——报告基因体系分析CUL4B在转录调控中的作用,取得了以下的研究结果：
(1)CRL4B与PRC2复合物通过DDB1-RbAp46/48的直接结合而形成大的复合物：通过免疫亲和纯化及质谱分析CUL4B的相互作用蛋白,我们在CUL4B结合蛋白中发现了转录抑制复合物PRC2的主要组分EZH2、EED、RbAp46/48的多肽。用Western blot分析证实在CUL4B结合蛋白中检测到PRC2组分EZH2、SUZ12、EED和RbAp46/48,但未检测到PRC1组分。随后,我们通过免疫共沉淀验证了CUL4B、DDB1与EZH2、SUZ12、EED和RbAp46/48的相互结合,但CRL4B与PRC1复合物没有结合。为明确CRL4B与PRC2复合物主要成员是否存在于一个复合物中,我们用FPLC对HeLa细胞中提取的核蛋白进行分离,用Western blot检测洗脱液中各蛋白分布。结果发现PRC2复合物及CRL4B复合物存在较大范围的重叠(669kDa-1300kDa),表明二者共同存在于一个大的复合物中。GST pull-down实验显示CRL4B是通过DDB1-RbAp46/48的直接作用与PRC2复合物结合的。在细胞内干扰DDB1或RbAp46/48表达能有效减少CUL4B与EZH2或SUZ12的相互结合。
(2)CRL4B协同PRC2复合物参与转录抑制：CUL4B亚细胞定位主要在细胞核。PRC2复合物是重要的转录抑制复合物,两者之间的相互结合提示CUL4B可能参入转录调控。因此,我们首先利用Ga14融合蛋白——报告基因体系分析CUL4B在转录调控中的作用,发现Ga14-CUL4B发挥转录抑制作用。为了明确CUL4B不同结构域在其发挥转录抑制中的作用,我们构建了缺失不同结构域的Gal4-CUL4B载体,分析发现CUL4B的DDB1结合区(DID)和Cullin功能支架区(Cullin)是CUL4B发挥转录抑制作用所必需的。为了明确CUL4B复合物与PRC2复合物相互作用对CUL4B转录抑制活性的影响,我们采用RNA干扰技术分别干扰CRL4B、PRC2和PRC1组分表达,发现干扰DDB1、EZH2、 RbAp46/48表达显著降低CUL4B的转录抑制活性,而干扰PRC1复合物的主要成员BMI1或RING1B表达则不影响CUL4B的转录抑制功能,表明CRL4B复合物的转录抑制作用是依赖于PRC2复合物而不依赖于PRC1复合物的。定量染色质免疫共沉淀(qCh1P)分析发现,表达Ga14-CUL4B不仅可以促进CUL4B、DDB1结合到Ga14启动子区域上,而且可以促进PRC2复合物组分EZH2结合到Ga14启动子区域,但并不影响PRC1组分在启动子区域的结合；与之相对应,两种复合物的组蛋白修饰产物H3K27me3和H2AK119ub1在启动子区域也明显增多。对比分析缺失DID或Cullin结构域的Ga14-CUL4B载体发现,与转染全长Ga14-CUL4B相比,转染缺失DID结构域的表达载体Ga14-ADID导致在Ga14启动子区域结合的DDB1和EZH2明显减少,同时H3K27me3和H2AK119ubl也明显减少；转染缺失Cullin结构域的Gal4-△Cullin载体,尽管不影响DDB1在启动子区域富集,但导致启动子区域的EZH2、H3K27me3和H2AK119ub1都明显减少。这些研究结果说明CRL4B发挥转录抑制作用不仅需要其自身的完整结构,而且依赖于PRC2复合物。
(3)干扰CUL4B表达不仅导致组蛋白H2AK119ub1减少,而且也导致H3K27me3水平下降：免疫荧光和Western blot结果显示,干扰CUL4B表达导致细胞内H3K27me3和H2A119ub1水平的明显下降,转录激活标志H3K4me3明显升高。对Cul4b全身性敲除小鼠E7.5胚胎的免疫组化分析也检测到H3K27me3和H2A119ub1水平的显著降低。
(4)CRL4B催化H2AK119单泛素化：以上研究结果提示CRL4B可能是通过单泛素化H2AK119而协同PRC2发挥转录抑制作用。为此,我们采用体外泛素化实验方法检测CRL4B复合物是否可以催化H2AK119单泛素化,结果证实了我们的推测,而且Cullin结构域是其发挥泛素化功能所必需的。为进一步证实单泛素化位点,我们单独或联合突变了H2A第118或／和119位赖氨酸,发现突变118位氨基酸不影响CRL4B对H2A的泛素化修饰,突变119位赖氨酸明显降低H2A泛素化水平,118和119位同时突变导致CRL4B对H2A的泛素化作用消失,说明CRL4B对H2A的单泛素化特异地发生在第119位的赖氨酸上,其作用类似于PRC1组分RING1B。干扰CUL4B或RING1B基因表达均可导致H2AK119ub1的水平降低,表明CRL4B复合物可能是RING1B泛素化H2AK119的替代酶。
(5)bAp48是CRL4B复合物催化H2AK119单泛素化的底物受体蛋白：为了确定CRL4B复合物泛素化H2A的底物受体蛋白,我们分别分析了与DDB1相互作用的WD40蛋白RbAp46/48和EED的作用。结果发现加入RbAp48能有效地提高CRL4B单泛素化H2AK119的效率,而加入EED没有明显作用,说明CRL4B单泛素化H2AK119是以RbAp46/48作为底物受体蛋白的,这一结果进一步通过干扰RbAp46/48影响CRL4B单泛素化H2A而得到证实。
综上,本部分结果表明CRL4B与PRC2复合物通过DDB1-RbAp46/48直接作用形成大的复合物,通过催化H2AK119单泛素化和H3K27三甲基化CRL4B与PRC2复合物协同抑制基因转录。
第二部分CRL4B协同PRC2抑制抑癌基因表达,促进肿瘤进程
EZH2在多组织肿瘤异常激活,通过抑制抑癌基因如(DH1、p16INK等的表达,EZH2促进肿瘤细胞增殖与转移。为了探究CRL4B与PRC2协同作用的生物学效应,本部分首先进行了CUL4B和EZH2共同调控的靶基因分析,并在此基础上分析CRL4B和PRC2复合物在靶基因上的占位情况以及CUL4B如何通过调控这些靶基因而在肿瘤发展中发挥作用。取得了如下结果：
(1)CRL4B协同PRC2复合物调控多种信号通路的基因表达：为了解CRL4B协同PRC2复合物发挥的生物学功能,我们首先用EZH2和CUL4B抗体在食管癌细胞株KYSE410细胞中进行ChIP-on-chip检测。分析发现,CUL4B与EZH2共同调控619个基因,GO与pathway分析发现这些基因的功能涉及多个重要的通路,其中包括多种抑癌基因,如p16、PTEN等。为验证ChIP-on-chip检测结果,我们从中选择了数个与肿瘤相关基因进行验证分析。结果显示,干扰CUL4B或EZH2基因表达导致18个靶基因转录上调；qChIP分析显示,CUL4B、 DDB1、EZH2结合在这些靶基因启动子区域,但未检测到RING1B在启动子区域的结合；同时,H3K27me3和H2AK119ub1也在启动子区域富集。此外我们发现干扰EZH2并不影响CUL4B在这些基因启动子区域的结合能力,而干扰CUL4B明显减少了EZH2在这些基因启动子区域的结合,进而导致启动子区域H2AK119ub1和H3K27me3的明显减少及H3K4me3的增多。进一步说明CUL4B促进PRC2复合物结合到靶基因启动子区域并促进PRC2催化H3K27甲基化。
(2)CRL4B协同PRC2调控p16和PTEN基因表达：在CUL4B和EZH2共同调控的靶基因中,我们选择了p16和PTEN两个重要的抑癌基因进一步分析CRL4B和PRC2复合物协同抑制靶基因表达的分子机制。我们首先利用多种细胞株检测CUL4B表达对p16和PTEN的影响。结果发现干扰CUL4B导致p16(?)PPTEN表达水平明显上调,证实CUL4B负调控这两个基因的转录。为了明确CRL4B和PRC2复合物在它们启动子区域的结合情况,我们进行ChIP/Re-ChIP实验,结果发现CRL4B和PRC2复合物在p16和PTEN启动子区域的共定位。通过qCIP分析了它们在p16和PTEN基因转录起始位点(TSS)上游-10kb到下游+10kb的结合情况,发现CUL4B、DDB1、EZH2、SUZ12在p16和PTEN启动子区域的结合峰明显重叠,而PRC1复合物的RING1B在PTEN基因启动子上没有结合,在p16启动子上结合峰位于TSS下游。干扰CUL4B或DDB1的表达导致EZB2在p16和PTEN上的结合大大降低,而干扰EZH2或SUZ12对CUL4B或DDB1的结合无明显影响,提示CRL4B优先于PRC2结合到p16和PTEN的启动子区域。
(3) CUL4B通过调控p16和PTEN促进肿瘤细胞增殖、克隆形成及迁移：为分析CUL4B在肿瘤进程中的作用,我们利用食管癌细胞株分析了CUL4B对细胞增殖和迁移的影响。发现低表达CUL4B引起G0/G1期细胞增多,进入S期的细胞减少,过表达则相反。克隆形成实验表明,过表达CUL4B促进肿瘤细胞克隆形成,而干扰CUL4B表达克隆形成减少。过表达CUL4B对肿瘤细胞克隆形成能力的影响可以通过过表达p16或干扰EZH2得到拯救,低表达CUL4B的克隆形成的减少可通过低表达p16得以拯救,说明CUL4B通过协同PRC2复合物抑制p16表达而促进肿瘤细胞增殖。另一方面,我们通过Transwell迁移小室实验分析过表达CUL4B对KYSE410细胞迁移能力的影响。发现CUL4B促进食管癌细胞迁移,而同时过表达PTEN或过表达显性负效应载体可以抑制CUL4B促进肿瘤细胞迁移的作用。我们分析NCI60数据库实体瘤细胞中CUL4B和上皮-间质细胞转化(EMT)标记物表达情况,发现在多种侵袭性高的细胞中CUL4B高表达,其表达与细胞侵袭能力正相关。
(4)CUL4B促进肿瘤生长：裸鼠皮下成瘤实验结果显示,干扰CUL4B明显抑制KYSE410及EC9706细胞株在裸鼠皮下肿瘤生长。对肿瘤进行Western blot及免疫组化检测,证实在CUL4B低表达的肿瘤组织中H2AK119ub1和H3K27me3显著降低,H3K4me3水平升高,靶基因p16和PTEN表达水平明显上调。
(5)CUL4B在食管癌肿瘤组织中高表达：为明确CUL4B在临床肿瘤标本中的表达情况,我们对齐鲁医院病理科收集13对新鲜食管癌及癌旁标本中CUL4B的表达量进行了检测。实时定量及Western blot分析发现,相比正常癌旁组织,CUL4B在大多数食管癌肿瘤组织中高表达。
(6)CUL4B与食管癌病理分级正相关：为明确CUL4B在食管癌发生发展中的作用,我们选取了含182例肿瘤和正常组织的食管癌组织芯片进行免疫组化分析。结果发现CUL4B在食管癌肿瘤组织中的表达水平显著高于正常组织,且其表达水平与食管癌病理分级呈正相关。我们进一步验证了配对的肿瘤标本中p16、PTEN和CUL4B的表达相关性,发现CUL4B与p16、CUL4B与PTEN之间表达呈负相关。对31例新鲜肿瘤组织中mRNA表达水平分析表明CUL4B与p16、CUL4B与PTEN的表达水平呈显著负相关,相关系数分别为-0.7739和-0.6070(Pearson),-0.8435和-0.6234(Sperman)。
(7)CUL4B在多种肿瘤组织中高表达：为了验证CUL4B在肿瘤中高表达的普遍性,我们在多个食管癌样本肿瘤及癌旁中检测CUL4B表达。组织芯片免疫组化结果显示CUL4B在食管癌、结肠癌、肺癌、胃癌、卵巢癌的肿瘤中高表达。多组织肿瘤芯片结果也显示,CUL4B在食管癌、乳腺癌、结肠癌、肺癌及卵巢癌中过表达且表达量与病理分级正相关。商品化mRNA芯片显示,在多种肿瘤中EZH2和CUL4B有着相似的表达特性。
以上结果表明,CRL4B复合物促进PRC2复合物在启动子的结合,两者协同作用抑制一组抑癌基因表达。通过影响这些抑癌基因表达,CUL4B促进肿瘤细胞的增殖、克隆形成能力和肿瘤的侵袭与迁移,促进肿瘤细胞的成瘤能力。我们的结果为发现新的肿瘤临床治疗靶点提供了理论基础。
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 ONE
CRL4B monoubiquitinates H2AK119and cooperates with PRC2complex to repress transcription
We 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 TWO
CRL4B 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 CUL4B△Cullin 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.
引文
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