Research The Regulation Mechanisms Of Pluripotency Genes By AdOx

Embryonic stem cells (ESC) have the potential of developing into three germ layers and further differentiating into different cell types. This characteristic of embryonic stem cells is known as pluripotency. Embryonic stem cells have two characteristics known as self-renewal and pluripotency maintenance. The chromatin is in a highly "open" state of ESCs and histone methylation plays an important role in the process of early embryogenesis.Adenosine dialdehyde (AdOx), inhibiting the activity of hydrolase-products in methyl's transference, which indirectly repressing the process of methyl's transcription by leading accumulation of by-products in cells, is a commonly used methyltransferase inhibitors. P19 embryonal carcinoma cells have many features of embryonic stem cells, such as the potential of differentiating into different types of cell and strong self-renewal ability. P19 cells can differentiate into neurons with all-trans retinoic acid (atRA) treatment, which is a good model in investigating the mechanism of pluripotency maintenance in embryonic stem cells and the mechanism of neuronal differentiation.We use AdOx to treat P19 cells to investigate the role of protein methylations in the pluripotency maintenance of P19 cells and the mechanism of epigenetic regulation.1. AdOx reduces the neuronal differentiation potential of P19 cells. P19 cells were exposed to RA for 4 days and aggregated. Without RA for another 4 days and P19 cells differentiated into neuron-like cells. However, P19 cells, pretreated with 20μM AdOx for 1 day, were unable to aggregate and display any synapse-like structures. We could detect the neuron specific TuJ1 in normal P19 cells by immunostaining, but not in the AdOx pre-treated P19 cells. In contrast to some three fourth of the RA-induced cells expressed TuJ1 protein, only 8% of TuJ1-positive cells with less neurites were detectable with AdOx treatment. Similar data indicated the RA-induced TuJ1 expression was inhibited by AdOx as shown in Western blots and it was dosage dependent. Ngn1, Mash 1and NeuroD are key transcription factors of neuronal differentiation. We found that AdOx significantly repressed neuronal differentiation and the mRNA level of ngn1, mash land neurod by Real time RT-PCR assay. Id 1 is a transcription factor that can antagonist neuronal differentiation. To some extent, AdOx increased the mRNA level of id1.2. AdOx affects the self-renewal and pluripotency maintenance of P19 cell P19 cells have a strong ability of self-renewal and maintenance of pluripotency. We used flow cytometry to detect the impact of AdOx on the cell cycle of P19 cells. We found that AdOx seriously caused cell cycle arrest of P19 cells, and even 1μM AdOx can also cause significant cell cycle arrest. 1μM,5μM and 10μM AdOx mainly caused S phase arrest. And 20μM AdOx caused mainly G2/M phase arrest. It shows that AdOx seriously inhibited the self-renewal of P19 cells. Higher concentration of AdOx also caused the cell cycle arrest in HEK-293A and Hela cells.Real time RT-PCR showed that AdOx increased the mRNA level of pluripotency related genes including nanog, sox2 and fgf4 in P19 cells, but reduced the mRNA level of the key pluripotent gene oct3/4. The mRNA level of the up-regulated genes was declined soon after the withdrawal of AdOx. It suggested that the up-regulation of the pluripotency genes by AdOx is transient. Western Blot showed that 20μM AdOx increase the protein expression of Nanog but reduced the protein expression of Oct3/4 in P19 cells. In recovered P19 cells, the protein expression of Nanog did not increase more, and it means that AdOx disordered the expression of pluripotency genes in P19 cells. Real time RT-PCR showed that RA can reduce the mRNA level of pluripotency genes even in 20μM AdOx pre-treated P19 cells. However, the mRNA level of lin28 and fgf4 was higher in AdOx pre-treated P19 cells than in normal P19 cells. And this may interfer the neuronal differentiation potential in RA induced P19 cells.3. AdOx increase the mRNA level of several marker genes for specific differentiated lineages.AdOx seriously affected the pluripotency maintenance of P19 cells, but pluripotency, inhibited the neuronal differentiation potential of P19 cells.In order to investigate whether AdOx caused P19 cells to differentiate into other germ layers, we detected the mRNA level of developmental genes in AdOx treated P19 cells by Real time RT-PCR assay. Results showed that AdOx increased the mRNA level of developmental genes including two endodem markers (Gata6 and Sox 17), two mesoderm markers (Hand1 and Brachyury), one ectoderm marker (Pax6) and two trophectoderm markers (Bmp4 and Cdx2). It implicated that AdOx started other germ layers differentiation of P19 cells. Similar to the effects of AdOx on the pluripotency genes, elevated expression of the developmental marker genes were also transient that the up-regulated genes were reduced afer AdOx withdrawn. It shows that the AdOx treated P19 cells do not differentiate into any other germ layer at last. P19 cells which had been treated with 20μM AdOx for 1 day were exposed to RA for 2 days. We detected mRNA level of developmental genes by Real time RT-PCR assay. Results showed that the mRNA level of Gata6, Sox 17, Brachyury, Nestin, Bmp4 and Cdx2 were much higher than P19 cells without AdOx treatment. The higher mRNA level of these genes may interfere specific neuronal differentiation of P19 cells.4. Impact of AdOx on the histone methylationsHistone methylations are important for chromain structure maintenance and gene expression. AdOx is often used as a methylation inhibitor to inhibite several histone methylations.We detected the influence of AdOx on histone methylations in P19 cells by Western Blot assay. We found that several histone methylations were significantly inhibited in the whole chromatin level by AdOx, including H3K27me3. H3K9me3, H3K4me2, H4K20mel, H4K20me3, H3R17me2, H3R2me2 and H4R3me2. It shows AdOx not only repressed histone lysine methylation but also inhibited histone arginine methylation. AdOx changes the chromatin specific signatures in P19 cells.ChIP assay showed that the recruitment of H3K27me3, H3K9me3 and H3K9me2 at the regulatory region of pluripotency genes and developmental genes were reduced while didn't change much at the regulatory region of oct3/4. It suggested that the up-regulation of pluripotency related genes and developmental genes may caused by the declined of these repressed methylations. Meanwhile, the recruitment of H4K20me1, H4K20me2 and H4K20me3 at the regulatory regions of these genes were higher than IgG. It suggested that H4K20 methylations may participate in the regulation of gene expression. The promoter activity results showed that the reporter activity driven by nanog promoter was significantly enhanced while that driven by oct3/4 promoter was reduced in AdOx-treated P19 cells. The reporter assay results are coincident with the mRNA level changes, implicating that AdOx caused an opposite effect on the transcription of nanog and oc/3/4. We conducted ChIP assay to analyze the recruitments of H3K27me3, H3K9me3 and H3K9me2 at nanog and oct3/4. Our results indicated that all these repressive methylation marks on H3 were significantly reduced at the promoter regions of nanog after AdOx treatment, in contrast, these marks didn't change obviously on oct3/4. It is implicated that the decline of repressive methylation marks might be involved in the AdOx-mediated up-regualtion of Nanog, but not in the regulation of Oct3/4.5. The effects of AdOx on the mRNA level of E-Cadherin and N-Cadherin in P19 cells E-Cadherin and N-Cadherin are two conmon kinds of cadherins in cells. The results from Real time RT-PCR showed that AdOx caused the up-regulation of E-Cadherin while nearly had no significant effect on N-Cadherin, the neural differentiation-specific cadherin.6. The inhibition on neuronal differentiation by AdOx in P19 cells can not be recovered. After treating P19 cells with lower concentration of AdOx (5μM) for 1 day, P19 cells were cultured without AdOx for another 2 days, and then, P19 cells were exposed to RA. The results of Real Time RT-PCR assay showed that the mRNA level of key transcription factors including ngn1, mash1 and neurod were still be inhibited. It suggests that the inhibition on neuronal differentiation by AdOx in P19 cells can not be recovered. After treating P19 cells with 20μM AdOx for 1 day, P19 cells were cultured without AdOx even for 10 days, and we found that mRNA level of Gata6, Brachyury, Pax6, Nestin and Cdx2 was significiantly lower than that normally cultured P19 cells. It seems that AdOx treated P19 cells are no longer have differentiation potential.7. Protein arginine methyltransferase CARM1 might play a role in RA induced neuronal differentiationWestern Blot assay and Immunostaining showed that CARM1 might play a role in RA induced neuronal differentiation in P19 cells. In conclusion, AdOx causes the cell cycle arrest and interfers the pluripotency maintenance of P19 cells. AdOx causes the up-regulation of pluripotency genes and developmental genes may through repressive histone methylations in P19 cells. Repressive histone methylations participate in the regulaiton of nanog and oct3/4 gene expression. Futher studies on the regulatory functions of histone methylations in the pluripotency maintenance and neuronal differentiation shed lights on the finding of the important methyltransferases.