The. Sirt7 In Ra Induced P19 Cell Neural Differentiation In The Process Of Functional Studies

Neurogenesis is a complicated multiple steps event. In each step, certain specific genes are induced and others are repressed. It has been found that epigenetic regualtion is involved in regulating the expression of many genes during neurogenesis.1. SirT7 is a member of the Sirtuin family of protein deacetylases and derived from the yeast Silent information regulator 2(Sir2). Since the Sir2 family of proteins are also able to exert their enzymatic activity not only on histones but also on numerous other proteins, such as the transcriptional regulators, they are involved in many cellular processes, ranging in yeast from gene silencing, DNA repair, progression of the cell cycle, to the control of ageing. What the function of SirT7 is in RA-induced neural differentiation remains unclear. To investigate the mechanism of SirT7, RA (all-trans retinoic acid)-induced P19 embryonal carcinoma cells were used as a early stage neuronal differentiating model neural in vitro. In this study, we analyze the expression of SirT7 and discussed the possible regulation mechanism of it.P19 cells were exposed to RA for 4 days and cultured in medium without RA for another 3 or 4 days to differentiate into neuron-like cells with the formation of neurite networks. Most differentiated P19 cells showed positive immunostaining for neuron specific classâ…¢-tubulin (TuJ1), and a dramatically increased protein level of TuJ1 in Western blot was observed. Quantitative RT-PCR assay showed that SirT7 mRNA greatly increased after 2 days of RA treatment and the protein level was same as mRNA by Western Blot assay.2. SirT7 has two alternatively spliced transcripts in vivoWestern blot assay showed that SirT7 had two alternatively spliced transcripts, the V1 and V2. To investigate the promoter activity of two variants, the duel luciferase reporter assay was performed and showed that the promoter of the two vatiants both had high activity. Furthermore, we found the two transcripts by 5'-RACE experienment. Immunofluorescence assay showed that V1 located mainly in nucleolus and V2 in the whole cell.3. SirT 7 V2 promotes the expression of Mash1Basic helix-loop-helix (bHLH) proteins are expressed in most stages in neural lineages during development and play crucial roles in the determination of cell fates and differentiation. Neurogeninl (Ngn1) and Mash1 are two important members of bHLH proteins, which induce neurogenesis and prohibit differentiation of neural stem cells into astrocytes. Therefore, we focus our work on the effect of SirT7 V2 on the expression of Ngn1 and Mash1. The data showed that SirT7 V2 enhanced the expression of Mashl gene. Moreover, SirT7 V2 induced the promoter activity of Mashl by the duel luciferase reporter assay.4. SirT7 V2 promotes the phosphorylation of Akt-pT308Akt pathway has a critical role in mediating signaling transduction for cell proliferation, differentiation and survival. It may plays diverse roles in the determination of cell fates and differentiation. Some reported that its activation contributed to remain pluripotent of stem cells. In ischemia model, the activation of PI3K/Akt/Erk induced the progress of neurogenesis, immigration and differentiation. To investigate the mechanism of SirT7 V2 in the RA-induced differentiation of P19 cells, we made trials on the cell signal pathway and found that SirT7 V2 could enhance the phosphorylation of Akt-pT308, whereas it had no effect on the site of Akt-pS473. It suggested that SirT7 V2 play a role in the neural differentiation though Akt pathway.5. SirT7 V2 interacts with PP1 cTo find out the position of SirT7 V2 in the PI3K/Akt pathway, we established SirT7 V2 stable-expressed P19 cell line and searched for the proteins interacting with it by TAP-MS assay. It showed that SirT7 V2 interacted with PP1c and got further evidence by IP.PP1 is a major protein Ser/Thr phosphatase and is ubiquitously expressed in all eukaryotic cells. PP1 figures prominently in a wide range of cellular processes, including meiosis and cell division, apoptosis, protein synthesis, metabolism, cytoskeletal reorganization, and the regulation of membrane receptors and channels. Meanwhile, PP1c acts as a major phosphatase to dephosphorylate Akt. Our data suggested that SirT7 V2 played a part in the PI3K/Akt pathway by PP1c and thus modulate Akt functions.6. SirT7 V2 could counter the effect of PP1c to Akt-pT308PP1c in diverse dosages and SirT7 V2 were co-transfected in P19 cells. It showed that SirT7 V2 could counteract the effect of PP1c on the site of Akt-pT308. It gives us some evidences for the SirT7 V2 in regulating Akt-pT308.SirT 7 is a member of Sir2 family and is less reported these years. The dissertation firstly reported that SirT7 had two alternatively spliced transcripts and exterted some research on SirT7 V2 function in the process of RA-induced neural differentiation. These data enriched our knowledge on the regulatory mechanism of SirT7 during neural differentiation, which provided a novel insight into the function of SirT7. These findings are helpful for us to understand details of epigenetic regulations in neural development, and may provide novel clue for future therapy in neurological diseases.