Mechanisim Of PTEN On Regulation Of DNA Repair Gene RAD51 Expression And Phosphorylated PTEN Links Chromatin Remodeling To DNA Damage Response

PTEN(Phosphatase and TENsin homolog deleted on chromosome 10)was identified in 1997 as a tumor-suppressor gene by Li's and other two labs, which had both lipod-phosphatase and protein-phosphatase activity. PTEN is mutated/deleted in a wild variety of solid tumors that we have known, it's mutation/deletion frequency is equal to P53. Cells were treated with exogenous or endogenous gene toxicity, particularly when ionizing radiation, DNA double-strand breaks will occur. Two ways were involved in the DNA damage repair, which were known as NHEJ (non-homologous end joining) and HR (homologous recombination). RAD51 is the most important gene in the HR pathway. In previous study, we found that the mutation/deletion of PTEN could lead to a lower expression of RAD51 and an increation in spontaneous DNA break. RAD51 present on regulation of PTEN reports of relatively small, and different perspectives. In this study, we will deeply investigate the regulation of PTEN on RAD51 and its mechanism on the basis of the previous study in the laboratory.To make full DNA damage repair and cell cycle checkpoint function, cells must open heterochromatin cohesion, promoting DNA damage repair proteins near the sites, this change in chromatin structure called chromatin remodeling. Chromatin remodeling plays an important role in DNA replication, damage repair and gen transcription. There are few reports about chromatin remodeling in current researchs, because the research method is the main limitation. In this study, a large-scale chromatin remodeling AO3-1 cell model was used to observe wheth the target protein had the function of chromatin remodeling through the green fluorescent protein fusion technology. We first found that PTEN could function with chromatin remodeling, and on the basis of that, we also investigate the role of PTEN's domains and C terminal of the phosphorylation sites in chromatin remodeling as well as the mechanism of PTEN involved in chromatin remodeling from the perspection of protein complexes. We have made the following progress so far:1. Neutral single cell gel electrophoresis and immunofluorescence methods found that comparion with PTEN wild-type cells, PTEN null cells increase the tail moment, the number of spontaneousγ-H2AX foci increased and more significantly byγ-ray irradiation (p<0.01). Experiments show that PTEN deletion led to decreased cell genomic stability.2. When PTEN-deficient cells were treated with LY294002, a PI3K/AKT pathway inhibitor, an expression of RAD51 was found through real-time PCR. This is the initial proof that PI3K/AKT pathway is one way of PTEN regulating the expression of RAD51. PTEN wild-type cells transfected wih wild-type AKT (AKT WT) or constitutively active AKT (AKT AC) showed a reduced RAD51 expression while PTEN deficient cells transefected with wild-type PTEN (PTEN WT) or loss of kinase activity in AKT (AKT DN) found that RAD51 expression increased. SiRNA-mediated AKT knockdown leads to an upregulation of RAD51. These data prove tha PI3K/AKT signaling pathway is indeed involved in the regulation of PTEN on the role of RAD51.3. FOXO3a is one of the downstream substrates of PI3K/AKT signaling pathway. We used bioinformatics software Signal scan and TFSEARCH software analysis the promoter region of RAD51 gene and found that FOXO3a could bind to the region. Dual-luciferase reporter system was used to prove this point initially. Western blot analysis found that nuclear PTEN-deficient FOXO3a phosphorylation levels increased. Nuclear FOXO3a phosphorylation leve showed a reduction in PTEN-deficient cells transfected with PTEN WT or AKT DN. In contrast, nuclear FOXO3a phosphorylation leve showed an increase in PTEN wild-type cells transfected with AKT WT or AKT AC. SiRNA-mediated FOXO3a knockdown led to a reduction of RAD51 on both protein and mRNA levels in PTEN wild-type cells. In conclusion we propose PI3K/AKT/FOXO3a signaling pathway is involved in PTEN regulating RAD51 expression.4. We first find that PTEN can be involved in chromatin remodeling, the function depends on the PTEN protein phosphatase activity of amino-terminal, C terminal (incluing C2 domain, PDZ domain and C2+PDZ) could be an inhibitor in chromation remodeling. S380 phosphorylation and K402 acetylation sites are important to PTEN in the regulation of chromation remodeling.5. Using blue native-PAGE (BN-PAGE) combined with ESI-Q-TOF-MS analysis, we first found a series of interactions with the PTEN protein, including: H2, H3, H4, and Chk2 and so on. Western blot confirmed that PTEN is indeed withγ-H2AX, Chk2 interaction, suggesting that PTEN may be involved in DNA damage repair in the early identification, cell cycle control or DNA damage repair through phosphorylation by Chk2.In the study, we first found that PTEN can regulate RAD51 expression through PI3K/AKT/FOXO3a signaling pathway, PTEN can rely on its protein phosphatase activity involved in chromatin remodeling. Our results provide experimental evidence for better understanding the relationship between PTEN and development of tumor, and for tumor prevention and possible drug target.