Functional Study Of Novel Human Gene DCUN1D3

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 DCUN1D3RT-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 nosignificant 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 damageBased 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 moleculesFinally, 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.