Cell Dna Methylation And Demethylation Model Establishment And Applications

Heritable alterations in gene expression caused by mechanisms other than changes in the DNA sequence are called epigenetic alterations. DNA methylation is one of the most important molecular mechanisms mediating epigenetic regulation. DNA methylation is genome DNA 5-methylcytosine (5-mC), and it is a stable and widespread way of epigenetic modification. In mammals, it often occurs at the cytosine residue in CpG islands, which are short fragments of CpG-dense DNA. Such CGIs exist in the human genome, and many of them surround the 5’end of genes, particularly the promoter region or the first exon of a gene. DNA methylation regulates critical biological phenomena such as X-chromosome inactivation, genomic imprinting, chromatin structure and regulation of gene expression. What is more, it has been shown to be essential for cell differentiation and embryo development. Aberrations in DNA methylation play a causal role in a variety of diseases, including cancer. Cancer cells exhibit global DNA hypomethylation and specific promoter hypermethylation of tumor-suppressor genes, which can lead to instability of the genome (such as instability of the chromosome, activation of mobile genetic element and expression of proto-oncogene) and silence of tumor suppress gene.Based on the significant of the DNA methylation for the diseases, studies on DNA demethylation are very important. Current researches main focus on two aspects: (1) Passive DNA demethylation, such as using 5-aza-dc, TSA, RNAi, and using miRNA to down-regulate DNMT. But these methods have many disadvantages. First of all, they are not available for observing DNA demethylation at specific gene loci in a dynamic manner. Second, methyltransferase inhibitors are highly toxic in cultured cells. They can cause a wide range of cell biological effects, the loss of methylation of whole cell. So it is difficult to observe and study for a long time. At last, these methods are not available for high-throughput screening and they are costly. Based on those disadvantages of traditional methods, the study for DNA demethylation and the relationship between DNA methylation and histone modification are difficult. (2) Achieve DNA active demethylation. One study reported that chimeric DNA methyltransferases, fused with Gal4 DNA-binding domain (DBD), can target DNA methylation to specific DNA sequences and repress expression of target gene. However, Transient transfection with DNA plasmids is not able to establish the real status of chromosome DNA. Objective:(1) There are no general method can available for studying the molecular mechanism governing DNA demethylation in vitro. Therefore, we want to develop the construction of a stable cell model that can be used for studying DNA demethylation in vitro to achieve the observation of DNA demethylation at specific gene loci and can observe for a long time stably. (2) VP16 is a viral protein that has a strong activation domain and can activate transcription. In our experiment design, VP16 was mainly used as a strong activator in the reporter luciferase assay to select the cell clones. In addition to its role in transcriptional activation, VP16 has also been reported to enhance DNA demethylation in the promoter region after interaction with a host DNA-binding domain in Xenopus fertilized eggs. In this study, we demonstrated the strong negative association of DNA methylation status with reporter luciferase activity. We hypothesized whether that VP16 could induce the DNA demethylation in mammalian cells. (3) For detect the function of the cell model future, we choose a hydroxymethylcytosine enzyme TET1 and a chemical compound tea polyphenols (TP) as the research subjects. Using three cell models which described above, we verify whether they have effect on DNA methylation.Method:(1) We constructed a reporter gene vector with artificial CpG islands, PCBS-luc. It derived from the vector PCDNA 3.1 and contained a firefly luciferase reporter gene, an SV40 promoter, binding domain (BD) sites, and an artificial CpG island. PCBS-luc plasmids were linearized with restriction enzyme PstI and methylated in vitro using CpG Methylase M.SssI. HEK293 cells were co-transfected with methylated (or unmethylated) PCBS-luc plasmid and anti-hygromycin gene expression vector. Stable PCBS-luc cell lines were selected with hygromycin B and, surviving cell clones were picked and expanded. Using luciferase assay to screen the cell clones for the first, pick the cell clones with high or low luciferase value to do other detections. The stable integration of PCBS-luc into the HEK293 cells was confirmed by PCR. BSP was then used to detect the methylation level of the promoter region of PCBS-luc in the different cell lines. (2) We transfected PMIPVP16 into methylated cell line 7#, harvested the stable cells with puromycin and analyzed the methylation status changes of the promoter region. (3) We transfected PMIPTET1CD into methylated cell line 7#, and expressed BD-TET1CD fusion protein in cells. Through the combination of BD and binding site which stable integrated into the cell genome, inducted the TET1 hydroxylation catalytic domain (TET1CD) to the artificial CpG island, and made the methylation site conversion of the hydroxymethylation. Harvested the stable cells with puromycin and detected the changes of luciferase value and analyzed the methylation status changes of the promoter region. (4) We treated methylated cell line 9# and unmethylated cell line 8# with 20 ug/ml and 100 ug/ml tea polyphenols, respectively. We verified the methylation status of promoter region with BSP.Result:(1) We obtained 13 methylation cell clones and 8 unmethylation cell clones. Three cell lines (one completely methylated cell line 9, one partially methylated cell line 7 and one completely unmethylated cell line 8) were selected for BSP analysis to confirm their methylation status. (2) After verification using the PCBS-luc PCR product and measurement of the methylation status with BSP, we obtained methylated cell line 7 and unmethylated cell line 8, which had a stable methylation status for at least 15 generations. (3) We used this cell model to observe for the first time that VP16 could induce DNA demethylation in mammalian cells. (4) We reported that TET1CD has function of DNA demethylation on some specific sites, but the whole methylation status of the promoter region had no significant change. (5) In our experiment condition, we observed that 20 ug/ml TP may induce DNA methylation at some loci in our unmethylated cell line 8#. However,100 ug/ml TP had no effect on the unmethylated cell line 8#. Both 20 ug/ml and 100 ug/ml TP had no effect on the methylated cell line 9#.Conclusion:We have not only constructed the first cell model for studying DNA methylation and DNA demethylation but also demonstrated for the first time that VP16 can induce DNA demethylation in mammalian cells. In addition, we confirmed that the TET1 and the 20 ug/ml TP have effect on cell DNA methylation. These preliminary results are expected to provide a basis for subsequent experiment research and thinking.