摘要
伴随着人类基因组计划(Human Genome Project, HGP)的完成,诞生了一大批新基因。目前公认的人类编码蛋白质的基因数量大约在20,000-25,000个,其中大部分基因功能还不明确,这就为生物学各个领域的研究提供了巨大的未知空间。
本实验室与国家人类基因组北方研究中心合作,采用反向生物学策略,对大批量的人类未知功能基因进行了克隆与功能研究。通过对NCBI的RefSeq数据库进行搜索,并汇集其他来源的人类预测基因,总计未知功能基因序列约6,900条。经序列校正与分析,挑出近3,000个Est支持较好基因,确定其ORF并设计引物PCR扩增,连入真核表达载体pcDNA3.1B中,共计得到1,000多个克隆,形成一个人类未知功能基因ORF文库。
为了研究和筛选上述新基因与细胞凋亡、增殖、分化、转化、损伤修复等细胞重要生命活动的相关性,人类基因组北方中心,建立了多个细胞筛选平台,其中DCUN1D3就是利用其中的SRE细胞筛选平台、通过高通量筛选技术获得的。SRE(serum response element)存在于许多立即早期基因(immediate early gene, IEG)上游的启动子序列中,比如c-fos, fosB, junB, egr-1 and -2;与其相互作用的转录因子有SRF, ELK-1, SAP1, TFII and NET(TCF family);血清,溶血磷脂酸(LPA),LPS,佛波脂及紫外线等能够通过MAPK信号转导途径诱导活化SRE。而SRE所启动的立早基因与细胞增殖,细胞凋亡等细胞生命活动密切相关。为此,在前期工作中,我们利用整合内参荧光素酶PRL活性(Renilla Luciferase,海肾荧光素酶;Rinilla luciferase更多地被用作转染效率的内参,以消除细胞数量和转染效率的差异)[11]的实验体系,将目的基因表达质粒与上游包含血清反应元件(SRE)的萤火虫荧光素酶报告质粒共转,对能够启动立早基因上游调控元件SRE活性的基因进行了细胞高通量筛选,获得了一个能够活化SRE的新基因DCUN1D3。
1、生物信息学及表达谱、细胞内定位分析
(1)生物信息学分析:DCUN1D3基因是一个没有任何功能报道的新基因,其编码314个氨基酸,富含亮氨酸,包含一个未知功能结构域298 (DUF298)。生物信息学分析无跨膜区,无明显的信号肽,C末端含有一个四个碱性氨基酸组成的核定位信号。在MiniMotif数据库中对DCUN1D3进行可能的Motif搜索发现,在273位氨基酸附近有一个识别未配对胸腺嘧啶的Motif,而胸腺嘧啶二聚体的形成是紫外线损伤DNA的特异指标。在214位和250位氨基酸位点各有一个可能的ATM激酶磷酸化位点,ATM是有活性的共济失调毛细血管扩张突变激酶(ataxia telangiectasia mutated)的英文简称,作为监测DNA双链断裂的关键分子参与了一系列DNA损伤反应。同时2006年cell杂志的一篇文献提示在该基因第一个内含子上有一个潜在的p53结合位点。以上种种线索提示DCUN1D3与DNA损伤可能有联系。
(2)DCUN1D3基因的表达谱分析及细胞内定位
首先利用RT-PCR方法证实DCUN1D3基因在多种肿瘤细胞系中都有转录。进一步,我们又制备了DCUN1D3基因的原核表达蛋白,并利用其制备了DCUN1D3特异性抗体。免疫组化及Western blot结果证实了DCUN1D3蛋白的天然存在。而且,RT-PCR、免疫组化及Western blot结果表明,DCUN1D基因在多种正常组织和肿瘤组织以及多种肿瘤细胞系中广谱表达。更有意义的是,实时定量PCR及免疫组化的结果显示, DCUN1D3的表达水平在正常组织中往往高于其配对的肿瘤组织。细胞内定位研究结果显示, DCUN1D3在细胞质及细胞核均匀分布,以胞质和核周分布为主。
2、进一步证实了DCUN1D3活化血清反应元件(SRE)
其次,我们进一步验证了DCUN1D3对SRE的活化作用,并进一步探讨其活化SRE的可能机理。初步实验结果提示,DCUN1D3可能不是通过ELK1和SRF调节SRE活性的,并且DCUN1D3对于血清刺激的MAPK信号途径中ERK的通路活化没有协同作用。提示DCUN1D3是通过其它机制活化SRE。
3、DCUN1D3与DNA损伤的关系
生物信息学分析结果提示DCUN1D3可能与DNA损伤有联系。(1)首先,我们探讨了DCUN1D3在紫外线照射(UVC)和离子照射(ionizing irradiation)条件下的表达水平变化,结果表明紫外线照射和离子照射均可使DCUN1D3的mRNA和蛋白水平明显上调。(2)有文献提示在DCUN1D3基因第一个内含子上有一个潜在的p53结合位点,因此我们探讨了DCUN1D3的表达上调是否依赖p53?我们的实验结果发现,超表达p53蛋白并不能增强DCUN1D3的表达;此外,在DNA损伤条件下,比较表达野生型p53的细胞系与p53缺陷型细胞系中DCUN1D3表达水平的差异,其实验结果不能得出其p53依赖性表达的结论。(3)由于生物信息学分析结果提示,其C端存在一个核定位信号,因此我们又探讨了DCUN1D3蛋白在DNA损伤因素作用下,在细胞内是否出现核转位变化?免疫细胞化学结果显示,在UVC损伤条件下,可以明显观察到内源性DCUN1D3向细胞核转位;利用外源性GFP-DCUN1D3融合蛋白也能够观察到类似的结果。进一步,我们又构建了缺失C端20个氨基酸的GFP-DCUN1D3融合蛋白。实验结果表明缺失了C端的GFP-DCUN1D3蛋白变得不稳定并且丢失了入核能力。提示DCUN1D3蛋白的核转位是依赖其C端的核定位信号。(4)探讨了DCUN1D3蛋白在DNA损伤因素作用下对细胞周期及生存的影响。我们合成并筛选出了特异性沉默DCUN1D3基因的siRNA。将其转染入细胞,观察在DNA损伤条件下,DCUN1D3基因被沉默后对细胞周期和细胞存活的影响。结果显示, DCUN1D3 siRNA有意义地增加了S期细胞比率;同时细胞凋亡数目明显减少。同时检测了DNA损伤信号通路中一些至关重要的分子,如p53,cyclin D1等等。发现这些分子也都有一定程度的减少。提示DCUN1D3基因在UVC引发的DNA损伤修复通路中扮演了一个重要的角色。
4、寻找DCUN1D相互作用分子
DCUN1D3是如何在细胞内发挥其生物学功能的?寻找其相互作用分子将成为解答这个问题的关键。首先通过生物信息学分析其可能的相互作用蛋白,同时检索文献发现小鼠的Dcun1d3可与泛素连接酶SCF复合体的各个成员以及CAND1分子相互作用,而小鼠Dcun1d3基因与人DCUN1D3有97%的同源性。因此,后期通过免疫共沉淀和DCUN1D3-GST-pulldown的方法,我们证实了DCUN1D3能够与SCF复合体中的cullin1分子、CAND1分子存在相互作用。提示其功能可能与泛素连接酶关系密切。未来的研究方向将基于这些分子间的相互作用,阐释它们在DNA损伤反应中的机制。
综上所述,通过对新基因DCUN1D3的系统研究,第一次在国际上揭示了该基因与DNA损伤,尤其是紫外线损伤密切的关系。DNA损伤修复与基因组稳定性密切相关,因此我们的工作,为研究肿瘤癌变机制与开发具有临床应用价值的功能基因提供了重要的实验依据。
With the completion of Human Genome Project, a large number of novel genes emerge. The total number of protein-coding genes in human genome is about 20,000-25,000. Remarkably, most of these genes are functionally unclear or poorly studied, and many of them are functionally unknown completely. Therefore, it will provide a big unknown area for biomedical research which is full of opportunities and challenges.
Cooperating with CHGB (Chinese National Human Genome Center, Beijing), we have cloned a large number of novel human genes and studied the functions of them. Totally, we got 6,900 mRNA candidates which are functionally unknown by searching the human RefSeq database of NCBI, or provided by others. We used the EST database to proofread, extend and obtain the full length sequences of these candidate sequences, after which their ORFs were predicted. About 3,000 genes well-supported by Ests were selected, and their ORFs were amplified by PCR using designed primers. Subsequently, these ORFs were cloned into the mammalian expression vector pcDNA3.1B. Finally, over 1,000 clones were obtained, composing a ORFs library.
High-throughput cell-based screening has been widely applied in functional genome studies, through which functional clues could be obtained efficiently. During this process, an SRE (serum response element) screening system based on dual-luciferase reporter assay was applied to discover new regulators in SRE signaling pathway. Finally, DCUN1D3 was selected for further study.
1. Bioinformatic, expression profile and subcellular analysis
(1)Bioinformatic analysis:DCUN1D3 is a novel gene without any functional report, which encodes 314 amino acids. It is rich in leucine and contains a domain of unknown function 298 (DUF298). Bioinformatics predict no transmebrane region, no obvious signal peptide. But there is a nuclear localization signal at its C-terminal which consists of four basic amino acids. We searched in the MiniMotif database for possible motif of DCUN1D3 and found that there is one motif which recognizes the unpaired Thymidine at the 273rd amino acid. Two possible ATM phosphorylation sites are at the 214th and 250th amino acid respectively. Moreover, it was reported in Cell in 2006 that there is a potential p53 binding loci in the first intron of DCUN1D3 gene. All clues above hint that DCUN1D3 may have a close relationship with DNA damage.
(2)Expression profile and subcellular localization of DCUN1D3
RT-PCR and real-time PCR results indicate that DCUN1D3 has a wide expression pattern in normal tissues, tumor tissues and cancer cell lines. To confirm its protein expression, we prepared DCUN1D3 prokaryotic protein and immunized the rabbits with it. After that, we got the specific antibody for DCUN1D3. The western blot result has a similar tendency with the RT-PCR result in several cell lines. The real-time PCR result and the immunohistochemistry results in paired normal and tumor tissues indicate that DCUN1D3 has a higher expression level in some normal tissues and their tumor counterparts. In addition, we also constructed the GFP-DCUN1D3 plasmid to observe the subcellular localization of the fusion GFP-DCUN1D3 protein. The GFP-DCUN1D3 protein expressed both in cytoplasm and in nuclear, mainly in the cytoplasm and the perinuclear.
2、Further confirmation of DCUN1D3 activation on SRE In the high throughput screening process, DCUN1D3 can activate the SRE, whereas no
significant activation of the vector reporter control, the AP-1, the CRE , the NF-κB, the ELK1 and the STAT1 reporters. So DCUN1D3 specifically activate the SRE. Furthermore, DCUN1D3 can also activate the reporter containing the c-fos gene and its upstream regulatory sequence. SRF (serum response factor) and the TCF (ternary complex factor) are the major transcription factors binding the SRE. So we validate if DCUN1D3 can trans-activate SRF using trans-dual luciferase reporter assay. The results showed that DCUN1D3 can only activate SRF weakly. Suggest that DCUN1D3 activates SRE through other transcription factors. Next we studied that the effect of over-expression on MAPK activation after serum stimulation. The results indicated that over-expression of DCUN1D3 can not enhance the Erk activation. Above all, the specific mechanisms for DCUN1D3 on SRE activation need further discussion.
3、DCUN1D3’s relationship with DNA damage
Based on the bioinformatics analysis, we studied systematically the behavior of DCUN1D3 under various DNA damage conditions. First of all, we observed the expression level of DCUN1D3 after UVC and ionizing irradiation and the results indicated that DCUN1D3 expression level was upregulated at both mRNA and protein level. Next we monitored DCUN1D3’s expression level under different doses of UVC. DCUN1D3 was upregulated under both high dose and low dose of UVC. We got similar results in different cell lines. Furthermore, we want to know if DCUN1D3’s UVC induction is p53-dependent. We treated the HCT116 cell lines (with wild type p53 and deletion of p53) under UVC and ionizing irradiation conditions. Meanwhile, Adenovirus mediated p53 transfer was used. However, we can not conclude that its induction under DNA damage was p53 dependent. Besides its expression, the subcellular localization of DCUN1D3 is also an interesting issue. We found that after UVC irradiation DCUN1D3 protein accumulated in the nucleus gradually. This accumulation may depend on its NLS at the C-terminal amino acids. So we constructed the mutant plasmid of GFP-DCUN1D3 with the deletion of 20 amino acids at the C-terminal. As expected, the deletion mutant lost the ability to accumulate in the nuclear and was unstable to be degraded in the cytoplasm.
Next step we did experiments about phenotype change. Three chemical-synthesized siRNAs were screened and the effective one was selected for further study. The siRNA was transfected into the cancer cells and then we observed the cell cycle checkpoint and the cell survival change. A significant increase in percentage of S phase cells is in the DCUN1D3 siRNA transfected group. And the cell death was inhibited to some extent. These results indicate that DCUN1D3 plays a very important role in UVC induced DNA damage and DNA repair. We also detected some key molecules in the DNA damage including p53, p21wip/cip etc. We found that the expression of these molecules also decreased to some extent, which correspond well with the cell phenotype change.
4、Searching for DCUN1D3’s interaction molecules
Finally, here comes the question that how DCUN1D3 plays and functions in cell? Finding its interaction molecules will be the key. We sought for these molecules in many ways. Firstly, we validated some possible molecules from the bioinformatics prediction. Secondly, we want to find the different bands on the PAGE of the immunoprecipitation. At last and also the most important, we have got the most possible interaction molecules from the paper in Yale University. It is reported that the mouse DCUN1D3 can interact with the components of ubiquitin ligase SCF and CAND1, what is more, the mouse DCUN1D3 shares a 99% identity with the human DCUN1D3. So we designed Co-IP and GST pulldown experiments to confirm that DCUN1D3 can interact with cullin1 in the SCF complex and with CAND1. Future study will base on these interactions to clarify their cooperation and working division in the DNA damage response.
Above all, for the first time in the world, we report the DCUN1D3 gene’s relationship with DNA damage, especially with UVC damage through systematically study. DNA damage leads to the genome instability. So our work will help to elucidate the mechanisms in carcinogenesis and to develop clinical-based functional gene therapy.
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