Cullin4B参与调控DNA甲基化介导的转录抑制进而促进宫颈癌发生
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摘要
Cullin4B (CUL4B)属于cullin家族,是Cullin4B-Ring E3泛素连接酶复合体(CRL4B)的骨架蛋白。泛素-蛋白酶体途径被认为是生物体内最为主要的蛋白质选择性降解方式,参与调控了包括细胞周期、信号转导、病毒感染、生长发育等几乎所有的生命过程。参与调控泛素化过程的基因异常可以导致多种疾病,如肿瘤、神经系统发育障碍等。人类Cullin家族有8个成员,分别是CUL1,CUL2, CUL3, CUL4A, CUL4B, CUL5, CUL7和PARC,其中CUL4A和CUL4B同源性最高,而在植物、裂殖酵母、线虫、果蝇等低等真核生物中,CUL4仅有一种蛋白。由于CUL4A与CUL4B高度同源,因此早期研究者常将两者作为一体(CUL4)来研究。但是随着研究的逐渐深入,发现CUL4A和CUL4B在功能上并不完全重叠,如敲除小鼠Cul4b基因会导致胚胎致死,人类CUL4B突变导致X-连锁智力低下综合征,说明CUL4A并不能完全补偿CUL4B在发育中的作用。
有研究表明在裂殖酵母中,人类cullin4的同源蛋白pcu4蛋白的异常表达会导致异染色质形成障碍。在哺乳动物中CUL4B可以降解组蛋白H3K4甲基转移酶复合物中的核心组分WDR5,并且CUL4B可以结合多梳蛋白抑制复合物2(PRC2),通过单泛素化H2AK119从而发挥转录抑制作用。与CUL4A主要分布在细胞质不同,CUL4B具有独特的N端,其上含有核定位信号,从而使CUL4B主要分布于核内,也预示了CUL4B在细胞核内发挥重要作用。
本研究发现CUL4B可以与组蛋白甲基转移酶SUV39H1.异染色质蛋白HP1以及DNA甲基转移酶DNMT3A相互作用,从而在由DNA甲基化介导的基因转录沉默中发挥重要作用。抑制CUL4B表达不仅使H2AK119的单泛素化修饰降低,同时H3K9的三甲基化和DNA甲基化水平也会减少,从而使某些基因如抑癌基因IGFBP3发生去抑制作用。我们证实CUL4B可以促进细胞增殖、迁移和肿瘤发生,至少在一定程度上是由于CUL4B抑制IGFBP3的表达所引起的。在人的宫颈癌组织样本中,CUL4B表达水平显著增高,并与IGFBP3的表达呈显著负相关。我们的研究建立了组蛋白泛素化／组蛋白甲基化和DNA甲基化在转录抑制作用中的分子作用模型,并且认识了CUL4B在宫颈癌发生中的分子作用机制。
第一部分CUL4B相互作用蛋白的鉴定
为了更好地理解CUL4B作用,我们使用免疫亲和纯化和质谱连用的方法寻找与CUL4B相互作用的蛋白。取得了以下研究结果：
1、本实验室前期利用免疫亲和纯化结合质谱分析方法发现了一批CUL4B结合蛋白,其中有多种蛋白与表观遗传调控有关,本课题将对质谱检测到的DNMT3A,SUV39H1和HP1α/β/γ蛋白进行验证分析,这些蛋白均是介导DNA甲基化且与转录抑制相关的蛋白。首先我们采用Western blotting方法,用抗这些蛋白的特异性抗体对洗脱样品进行了分析,证实洗脱样品中存在DNMT3A, SUV39H1,PRMT5和HP1α/β/γ。
2、为了证实CUL4B与SUV39H1/HP1/DNMT3A存在于一个复合物中,我们通过快速液相色谱(FPLC)方法对核蛋白提取物进行蛋白组分分离,然后用Western blotting方法进行检测,发现CUL4B, DDB1和ROC1与SUV39H1/HP1/DNMT3A的主峰都出现在669-1000kDa区域。提示CUL4B与SUV39H1/HP1/DNMT3A存在于一个复合物中。
3、为进一步证明CUL4B与这些蛋白质间的相互作用,我们在HEK293T细胞和HeLa细胞中进行了免疫共沉淀(IP)分析。用抗CUL4B抗体进行IP,在沉淀蛋白质中我们用特异性抗体检测到SUV39H1, HP1α, HP1β,HP1γ, DNMT1, DNMT3A和DNMT3B,证实CUL4B可以与上述蛋白相结合。反之,我们分别用抗SUV39H1, HP1α, HP1β,HP1γ, DNMT1, DNMT3A和DNMT3B抗体进行IP,然后用抗CUL4B抗体进行IB检测,同样证实了这些蛋白可以与CUL4B相互结合。另外,在HEK293T和HeLa细胞中,SUV39H1, HP1α和DNMT3A也可以结合CRL4B复合物中的另外两个组分即DDB1和ROC1。从而进一步证实了CUL4B可以与SUV39H1/HP1/DNMTs相结合。
4、为了理解CRL4B和SUV39H1/HP1/DNMTs复合物相互作用的分子机制,我们首先纯化了带有GST标签的GST融合蛋白即GST-CUL4B, GST-DDB1和GST-ROC1,同时在体外翻译了SUV39H1, HP1α, HP1β, HP1γ, DNMT1, DNMT3A和DNMT3B蛋白,通过GST pull-down实验证实了CUL4B和DDB1都可以直接结合SUV39H1, DNMT1, DNMT3A和DNMT3B。
综上,通过免疫印迹(IB)、免疫共沉淀(IP)和GST pull-down等一系列分析,证实CRL4B和SUV39H1/HP1/DNMTs存在直接的相互作用。为理解CUL4B参与转录调控提供了一个新的分子作用机制。
第二部分鉴定CRL4B/SUV39H1/HP1/DNMT3A复合物调控的靶基因
已有研究表明SUV39H1/HP1/DNMT3A复合物的主要功能是甲基化DNA从而发挥转录抑制作用,基于我们发现CRL4B和SUV39H1/HP1/DNMT3A复合物具有相互作用,于是我们检测了CRL4B/SUV39H1/HP1/DNMT3A可能共同调控的靶基因。取得了如下结果：
1、向HeLa细胞中转染CUL4B的shRNA和shSCR载体,构建稳定干扰CUL4B表达的HeLa细胞系和对照HeLa细胞系,然后用抗5甲基胞嘧啶(5-mC)抗体对shCUL4B细胞及对照细胞进行甲基化DNA免疫沉淀芯片(MeDIP-chip)分析,从而检测抑制CUL4B表达对基因组DNA甲基化状态的影响。用5-mC抗体沉淀的甲基化DNAs通过无偏差扩增、标记,然后与含有人类基因组中从转录起始点的-800到+200范围的启动子区域共22,532个位点的芯片进行杂交(错配率低于0.05的情况下)。与对照细胞相比,我们发现总共有4045个基因组区域(1878个基因)在shCUL4B细胞中其甲基化水平有显著的差异,其中包括1965个在shCUL4B细胞中的高甲基化位点(953个基因)和2080个低甲基化位点(925个基因)。
2、我们使用Molecule Annotation System软件对差异基因进行了信号通路分析(p值小于10-3)。与对照细胞相比,在shCUL4B细胞中总共将高甲基化和低甲基化的基因列出了主要的10个信号通路。在这些信号通路中选取了具有代表性的11个低甲基化的基因包括RPS6KA6,IGFBP3,AXIN1,SFRP5,FOXO3, WNT2B,IQGAP2,NKX3.1,RELN,KLF3和PER2,然后通过定量ChIP实验进一步分析这些基因启动子区域的甲基化水平。结果与MeDIP-chip分析结果基本一致。
3、在MeDIP-chip检测到的基因中,胰岛素样的生长因子结合蛋白3(IGFBP3)是一个已知的抗增殖、促凋亡和抑制转移的蛋白。研究发现在多种癌症中IGFBP3启动子区域均存在异常高甲基化现象,包括肺癌,肝癌,胃癌,结肠癌,乳腺癌和宫颈癌。于是我们进一步分析CRL4B/SUV39H1/HP1/DNMT3A复合物是否可以调控IGFBP3的表达。我们用抗CUL4B,DDB1,ROC1, SUV39H1,HP1α,HP1β和DNMT3A抗体进行了定量染色质免疫共沉淀(qChIP)实验,结果显示上述蛋白均可以结合在IGFBP3的启动子上。
4、用组蛋白修饰抗体进行ChIP实验检测结果显示H2AK119ub1和H3K9me2/3的修饰以及DNA甲基化水平在IGFBP3的启动子上明显富集。
5、在HeLa细胞中分别用抗CUL4B,DDB1,SUV39H1,HP1α,DNMT3A,5-mC和对照IgG抗体进行了染色质免疫共沉淀(ChIP)实验。结果表明CRL4B复合物和SUV39H1/HP1/DNMT3A复合物都结合于IGFBP3的启动子上,同时5甲基胞嘧啶也存在于相同的启动子区域。为了证实CRL4B和SUV39H1/HP1/DNMT3A是作为同一个复合物存在于IGFBP3的启动子上,我们进行了ChIP/Re-ChIP实验。首先用抗CUL4B,DDB1,SUV39H1,HP1α,DNMT3A和5-mC的抗体进行第一轮染色质免疫共沉淀实验,然后将第一轮ChIP沉淀下来的样品再用其它上述抗体进行第二轮染色质免疫共沉淀实验。结果显示CRL4B和SUV39H1/HP1/DNMT3A是作为一个复合物结合在IGFBP3的启动子上。
6、我们通过定量RT-PCR和Western blotting方法检测了干扰CUL4B表达对IGFBP3表达的影响。在HeLa, SiHa和Ca Ski细胞中干扰CUL4B会引起IGFBP3转录水平及其蛋白水平的显著上调。我们在HeLa细胞中加入DNA甲基化抑制剂脱氧胞嘧啶的类似物5-aza-deoxycytidine (5-aza-dC)(常用来诱导基因表达和诱导细胞分化)后,IGFBP3的表达被诱导；但是,干扰CUL4B的细胞中,5-aza-dC处理并不明显改变IGFBP3的表达,这说明在宫颈癌细胞中CUL4B参与DNA甲基化介导的IGFBP3的转录沉默,也支持了CRL4B复合物和SUV39H1/HP1/DNMT3A在功能上的相关性。
7、在干扰CUL4B的HeLa细胞中,通过qChIP实验显示干扰CUL4B会导致DDB1, ROC1, SUV39H1, HP1α, HP1β和DNMT3A在IGFBP3启动子上的结合水平降低。同时在IGFBP3启动子上H2AK119ub1, H3K9me2, H3K9me3和5-mC的水平也有显著的下降。说明CUL4B是SUV39H1/HP1/DNMT3A复合物发挥作用的重要催化亚基。
第三部分CUL4B在宫颈癌发生中的作用
已有研究表明IGFBP3是一个抑癌基因,它的主要生物学功能是抑制细胞增殖、促进细胞凋亡和抑制细胞转移。以上研究表明CUL4B可以使IGFBP3的启动子发生甲基化并且抑制IGFBP3的转录,因此本部分将分析CUL4B是否通过对IGFBP3的调控从而促进宫颈癌的发生和发展。取得了如下结果：
1、与对照HeLa细胞相比,在HeLa细胞中稳定干扰CUL4B导致细胞生长抑制,然而同时干扰CUL4B和IGFBP3可以缓解由于CUL4B抑制而引起的生长抑制。
2、克隆形成实验证实干扰CUL4B明显减少克隆数目,但同时干扰CUL4B和IGFBP3可以使由于CUL4B抑制引起的克隆形成减少得以部分恢复。
3.TUNEL实验检测细胞凋亡表明在CUL4B干扰组有25.82%的细胞发生凋亡,而对照组只有1.42%的细胞发生凋亡,而同时干扰CUL4B和IGFBP3则凋亡表型得到了部分拯救(凋亡细胞数从25.82%降到了11.21%)。
4、软琼脂克隆形成实验表明干扰CUL4B显著减少克隆的大小和数目,说明CUL4B在不依赖支持物生长中发挥作用,预示了CUL4B可能与肿瘤细胞的恶性程度相关。另外,转移小室实验结果显示当干扰CUL4B后迁移的细胞数目与对照细胞相比有10倍的减少,但同时干扰CUL4B和IGFBP3会部分拯救由于CUL4B抑制引起的细胞转移能力下降。说明抑制CUL4B表达导致肿瘤细胞转移能力降低至少部分是由于其负调控IGFBP3所致。
5、我们收集了64例宫颈癌组织标本,其中有30例成对的癌与癌旁组织,对这些标本进行了免疫组化染色。结果显示CUL4B在宫颈癌组织中显著高表达,并且其表达水平与组织分级呈显著正相关,同时CUL4B的表达与IGFBP3的表达呈显著负相关。
综上所述,我们发现CRL4B可以与SUV39H1, HP1和DNMTs相互作用。CRL4B通过催化H2AK119的单泛素化继而催化H3K9的三甲基化和DNA的甲基化。我们发现CUL4B可以通过抑制IGFPB3从而促进细胞增殖、迁移和肿瘤的发生。宫颈癌组织样本分析发现CUL4B显著高表达,同时与IGFPB3的表达水平呈显著负相关。这些结果有助于深入理解CUL4B在表观遗传调控和肿瘤发生中的作用及其作用机制。
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 Proteins
In 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 Complex
Since 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 Carcinoma
As 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.
有研究表明在裂殖酵母中,人类cullin4的同源蛋白pcu4蛋白的异常表达会导致异染色质形成障碍。在哺乳动物中CUL4B可以降解组蛋白H3K4甲基转移酶复合物中的核心组分WDR5,并且CUL4B可以结合多梳蛋白抑制复合物2(PRC2),通过单泛素化H2AK119从而发挥转录抑制作用。与CUL4A主要分布在细胞质不同,CUL4B具有独特的N端,其上含有核定位信号,从而使CUL4B主要分布于核内,也预示了CUL4B在细胞核内发挥重要作用。
本研究发现CUL4B可以与组蛋白甲基转移酶SUV39H1.异染色质蛋白HP1以及DNA甲基转移酶DNMT3A相互作用,从而在由DNA甲基化介导的基因转录沉默中发挥重要作用。抑制CUL4B表达不仅使H2AK119的单泛素化修饰降低,同时H3K9的三甲基化和DNA甲基化水平也会减少,从而使某些基因如抑癌基因IGFBP3发生去抑制作用。我们证实CUL4B可以促进细胞增殖、迁移和肿瘤发生,至少在一定程度上是由于CUL4B抑制IGFBP3的表达所引起的。在人的宫颈癌组织样本中,CUL4B表达水平显著增高,并与IGFBP3的表达呈显著负相关。我们的研究建立了组蛋白泛素化／组蛋白甲基化和DNA甲基化在转录抑制作用中的分子作用模型,并且认识了CUL4B在宫颈癌发生中的分子作用机制。
第一部分CUL4B相互作用蛋白的鉴定
为了更好地理解CUL4B作用,我们使用免疫亲和纯化和质谱连用的方法寻找与CUL4B相互作用的蛋白。取得了以下研究结果：
1、本实验室前期利用免疫亲和纯化结合质谱分析方法发现了一批CUL4B结合蛋白,其中有多种蛋白与表观遗传调控有关,本课题将对质谱检测到的DNMT3A,SUV39H1和HP1α/β/γ蛋白进行验证分析,这些蛋白均是介导DNA甲基化且与转录抑制相关的蛋白。首先我们采用Western blotting方法,用抗这些蛋白的特异性抗体对洗脱样品进行了分析,证实洗脱样品中存在DNMT3A, SUV39H1,PRMT5和HP1α/β/γ。
2、为了证实CUL4B与SUV39H1/HP1/DNMT3A存在于一个复合物中,我们通过快速液相色谱(FPLC)方法对核蛋白提取物进行蛋白组分分离,然后用Western blotting方法进行检测,发现CUL4B, DDB1和ROC1与SUV39H1/HP1/DNMT3A的主峰都出现在669-1000kDa区域。提示CUL4B与SUV39H1/HP1/DNMT3A存在于一个复合物中。
3、为进一步证明CUL4B与这些蛋白质间的相互作用,我们在HEK293T细胞和HeLa细胞中进行了免疫共沉淀(IP)分析。用抗CUL4B抗体进行IP,在沉淀蛋白质中我们用特异性抗体检测到SUV39H1, HP1α, HP1β,HP1γ, DNMT1, DNMT3A和DNMT3B,证实CUL4B可以与上述蛋白相结合。反之,我们分别用抗SUV39H1, HP1α, HP1β,HP1γ, DNMT1, DNMT3A和DNMT3B抗体进行IP,然后用抗CUL4B抗体进行IB检测,同样证实了这些蛋白可以与CUL4B相互结合。另外,在HEK293T和HeLa细胞中,SUV39H1, HP1α和DNMT3A也可以结合CRL4B复合物中的另外两个组分即DDB1和ROC1。从而进一步证实了CUL4B可以与SUV39H1/HP1/DNMTs相结合。
4、为了理解CRL4B和SUV39H1/HP1/DNMTs复合物相互作用的分子机制,我们首先纯化了带有GST标签的GST融合蛋白即GST-CUL4B, GST-DDB1和GST-ROC1,同时在体外翻译了SUV39H1, HP1α, HP1β, HP1γ, DNMT1, DNMT3A和DNMT3B蛋白,通过GST pull-down实验证实了CUL4B和DDB1都可以直接结合SUV39H1, DNMT1, DNMT3A和DNMT3B。
综上,通过免疫印迹(IB)、免疫共沉淀(IP)和GST pull-down等一系列分析,证实CRL4B和SUV39H1/HP1/DNMTs存在直接的相互作用。为理解CUL4B参与转录调控提供了一个新的分子作用机制。
第二部分鉴定CRL4B/SUV39H1/HP1/DNMT3A复合物调控的靶基因
已有研究表明SUV39H1/HP1/DNMT3A复合物的主要功能是甲基化DNA从而发挥转录抑制作用,基于我们发现CRL4B和SUV39H1/HP1/DNMT3A复合物具有相互作用,于是我们检测了CRL4B/SUV39H1/HP1/DNMT3A可能共同调控的靶基因。取得了如下结果：
1、向HeLa细胞中转染CUL4B的shRNA和shSCR载体,构建稳定干扰CUL4B表达的HeLa细胞系和对照HeLa细胞系,然后用抗5甲基胞嘧啶(5-mC)抗体对shCUL4B细胞及对照细胞进行甲基化DNA免疫沉淀芯片(MeDIP-chip)分析,从而检测抑制CUL4B表达对基因组DNA甲基化状态的影响。用5-mC抗体沉淀的甲基化DNAs通过无偏差扩增、标记,然后与含有人类基因组中从转录起始点的-800到+200范围的启动子区域共22,532个位点的芯片进行杂交(错配率低于0.05的情况下)。与对照细胞相比,我们发现总共有4045个基因组区域(1878个基因)在shCUL4B细胞中其甲基化水平有显著的差异,其中包括1965个在shCUL4B细胞中的高甲基化位点(953个基因)和2080个低甲基化位点(925个基因)。
2、我们使用Molecule Annotation System软件对差异基因进行了信号通路分析(p值小于10-3)。与对照细胞相比,在shCUL4B细胞中总共将高甲基化和低甲基化的基因列出了主要的10个信号通路。在这些信号通路中选取了具有代表性的11个低甲基化的基因包括RPS6KA6,IGFBP3,AXIN1,SFRP5,FOXO3, WNT2B,IQGAP2,NKX3.1,RELN,KLF3和PER2,然后通过定量ChIP实验进一步分析这些基因启动子区域的甲基化水平。结果与MeDIP-chip分析结果基本一致。
3、在MeDIP-chip检测到的基因中,胰岛素样的生长因子结合蛋白3(IGFBP3)是一个已知的抗增殖、促凋亡和抑制转移的蛋白。研究发现在多种癌症中IGFBP3启动子区域均存在异常高甲基化现象,包括肺癌,肝癌,胃癌,结肠癌,乳腺癌和宫颈癌。于是我们进一步分析CRL4B/SUV39H1/HP1/DNMT3A复合物是否可以调控IGFBP3的表达。我们用抗CUL4B,DDB1,ROC1, SUV39H1,HP1α,HP1β和DNMT3A抗体进行了定量染色质免疫共沉淀(qChIP)实验,结果显示上述蛋白均可以结合在IGFBP3的启动子上。
4、用组蛋白修饰抗体进行ChIP实验检测结果显示H2AK119ub1和H3K9me2/3的修饰以及DNA甲基化水平在IGFBP3的启动子上明显富集。
5、在HeLa细胞中分别用抗CUL4B,DDB1,SUV39H1,HP1α,DNMT3A,5-mC和对照IgG抗体进行了染色质免疫共沉淀(ChIP)实验。结果表明CRL4B复合物和SUV39H1/HP1/DNMT3A复合物都结合于IGFBP3的启动子上,同时5甲基胞嘧啶也存在于相同的启动子区域。为了证实CRL4B和SUV39H1/HP1/DNMT3A是作为同一个复合物存在于IGFBP3的启动子上,我们进行了ChIP/Re-ChIP实验。首先用抗CUL4B,DDB1,SUV39H1,HP1α,DNMT3A和5-mC的抗体进行第一轮染色质免疫共沉淀实验,然后将第一轮ChIP沉淀下来的样品再用其它上述抗体进行第二轮染色质免疫共沉淀实验。结果显示CRL4B和SUV39H1/HP1/DNMT3A是作为一个复合物结合在IGFBP3的启动子上。
6、我们通过定量RT-PCR和Western blotting方法检测了干扰CUL4B表达对IGFBP3表达的影响。在HeLa, SiHa和Ca Ski细胞中干扰CUL4B会引起IGFBP3转录水平及其蛋白水平的显著上调。我们在HeLa细胞中加入DNA甲基化抑制剂脱氧胞嘧啶的类似物5-aza-deoxycytidine (5-aza-dC)(常用来诱导基因表达和诱导细胞分化)后,IGFBP3的表达被诱导；但是,干扰CUL4B的细胞中,5-aza-dC处理并不明显改变IGFBP3的表达,这说明在宫颈癌细胞中CUL4B参与DNA甲基化介导的IGFBP3的转录沉默,也支持了CRL4B复合物和SUV39H1/HP1/DNMT3A在功能上的相关性。
7、在干扰CUL4B的HeLa细胞中,通过qChIP实验显示干扰CUL4B会导致DDB1, ROC1, SUV39H1, HP1α, HP1β和DNMT3A在IGFBP3启动子上的结合水平降低。同时在IGFBP3启动子上H2AK119ub1, H3K9me2, H3K9me3和5-mC的水平也有显著的下降。说明CUL4B是SUV39H1/HP1/DNMT3A复合物发挥作用的重要催化亚基。
第三部分CUL4B在宫颈癌发生中的作用
已有研究表明IGFBP3是一个抑癌基因,它的主要生物学功能是抑制细胞增殖、促进细胞凋亡和抑制细胞转移。以上研究表明CUL4B可以使IGFBP3的启动子发生甲基化并且抑制IGFBP3的转录,因此本部分将分析CUL4B是否通过对IGFBP3的调控从而促进宫颈癌的发生和发展。取得了如下结果：
1、与对照HeLa细胞相比,在HeLa细胞中稳定干扰CUL4B导致细胞生长抑制,然而同时干扰CUL4B和IGFBP3可以缓解由于CUL4B抑制而引起的生长抑制。
2、克隆形成实验证实干扰CUL4B明显减少克隆数目,但同时干扰CUL4B和IGFBP3可以使由于CUL4B抑制引起的克隆形成减少得以部分恢复。
3.TUNEL实验检测细胞凋亡表明在CUL4B干扰组有25.82%的细胞发生凋亡,而对照组只有1.42%的细胞发生凋亡,而同时干扰CUL4B和IGFBP3则凋亡表型得到了部分拯救(凋亡细胞数从25.82%降到了11.21%)。
4、软琼脂克隆形成实验表明干扰CUL4B显著减少克隆的大小和数目,说明CUL4B在不依赖支持物生长中发挥作用,预示了CUL4B可能与肿瘤细胞的恶性程度相关。另外,转移小室实验结果显示当干扰CUL4B后迁移的细胞数目与对照细胞相比有10倍的减少,但同时干扰CUL4B和IGFBP3会部分拯救由于CUL4B抑制引起的细胞转移能力下降。说明抑制CUL4B表达导致肿瘤细胞转移能力降低至少部分是由于其负调控IGFBP3所致。
5、我们收集了64例宫颈癌组织标本,其中有30例成对的癌与癌旁组织,对这些标本进行了免疫组化染色。结果显示CUL4B在宫颈癌组织中显著高表达,并且其表达水平与组织分级呈显著正相关,同时CUL4B的表达与IGFBP3的表达呈显著负相关。
综上所述,我们发现CRL4B可以与SUV39H1, HP1和DNMTs相互作用。CRL4B通过催化H2AK119的单泛素化继而催化H3K9的三甲基化和DNA的甲基化。我们发现CUL4B可以通过抑制IGFPB3从而促进细胞增殖、迁移和肿瘤的发生。宫颈癌组织样本分析发现CUL4B显著高表达,同时与IGFPB3的表达水平呈显著负相关。这些结果有助于深入理解CUL4B在表观遗传调控和肿瘤发生中的作用及其作用机制。
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 Proteins
In 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 Complex
Since 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 Carcinoma
As 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.
引文
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