| Tat, an regulatory protein encoded by HIV-1, plays a role in the transcription and replication of HIV. Tat can targets a variety of cell proteins to facilitate transcription of viral and disrupts cellular immunity by inducing lymphocyte apoptosis. It is secreted by infected cells and can enter into bystander cells as a paracrine molecule. DNA-PKcs is the catalytic subunit of DNA-PK, and belongs to the member of PI3K superfamily, it is also a crucial component in NHEJ pathway. When there is a break in the DNA, DNA-PKcs is recruited to the break site in a Ku-dependent manner, and is activated to start DNA damage repair response. In addition, DNA-PKcs can also play a role in V(D)J recombination, cell cycle regulation, apoptosis and autophagy.Our previous work demonstrated that Tat can depresses expression of DNA-PKcs, sensitizes cells to ionizing radiation, and impairs the cell cycle control by targeting the Tip60, Plkl and Cyclin Bl. Our group have demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence, and shorted the core region of DNA-PKcs from -106～3 to -63～-1. In addition, we found Tat could activate DNA-PKcs in a dose-dependent manner. On this basis, we have research it further, the main achievements are as following:1. Co-immunoprecipitation experiment and GST pull-down assay revealed Tat and DNA-PKcs strongly interact with each other. Both endogenous and exogenous Tat strongly stimulated DNA-PKcs auto-phosphorylation at Ser2056.2. It was reported that DNA-PKcs plays a fundamental role in CSR (class switch recombination).To determine whether Tat regulates CSR by targeting DNA-PKcs, We tested the role of Tat protein in CSR process. The results demonstrated that Tat can inhibits CSR at low concentrations (≤4 μg/ml) and stimulates CSR at high concentrations (≥8μg/ml).3. We have revealed the interaction between Tat and DNA-PKcs, but whether DNA-PKcs modulates HIV-1 transcription. The results revealed that high kinase activity and low protein level of DNA-PKcs promotes HIV-1 transcription, while low kinase activity and high protein level inhibit HIV-1 transcription.4. Co-immunoprecipitation results revealed that DNA-PKcs can form a large complex comprised of CDK9, Tat and Cyclin T1 via direct interacting with Tat and CDK9 but not Cyclin T1.Our study provides a new direction to search DNA-PKcs, and a new ideas or theoretical significance that Tat regulates host humoral immunity. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients, and provide a new strategy for development of novel drugs of anti-HIV-1 therapeutics.Tip60 (Tat Interact Protein) was first identified by Yeast Two-hybrid technique, which could interact with HIV1-tat protein, it was named Tip60 for its molecular weight of 60kd. Tip60 belongs MYST family involved in acetylating histone H2A, H3, H4, and a variety of related proteins, to regulate genes transcription and molecular response. Studies have confirmed Tip60 as a transcriptional regulatory factor could bind to the nuclear receptors and transcription factor to further activate or inhibit the expression of genes, such as the androgen receptor, AICD/Fe65, NCoR, c-MYC, E2F, in addition Tip60 also regulated activity and stability of proteins by acetylation. When there is a break in the DNA double strand, Tip60 can induce DNA damage repair response by acetylate the K3016 of ATM. In conclusion, we can see that Tip60 can acetylate a series of proteins to regulate their important cellμlar functions, such as DNA damage repair response, cell cycle, checkpoint activation, apoptosis and autophagy. In addition, Tip60 also plays a crucial role in the metastasis of tumor and embryonic development.Since homozygously knockout of Tip60 in mouse leads to embryonic lethality, we plan to establish Tip60 Knocked-out Cell using the most advanced technology of TALEN and CRISPR, for further investigating the role of Tip60 in cell cycle regulation, DNA damage repair, and autophagy regulation.TALEN technology experiment process:The target sequences of Tip60 were selected according to the bioinformatical analysis and the sequences were cloned into TALEN targeting vector respectively. After verification by sequencing, TALEN plasmids were directly transfected into HEK293 cells, and the cell genomic DNA were extracted 48h post transfection. The editing efficiency of TALEN was evaluated by PCR amplification and T7 endonucluease digestion.The TALEN plasmids with the highest efficiency were selected to screen Tip60 homozygously knockout cell. Transfected or infected cells were diluted to one cell per well of 96-well plate. After 4 weeks cultivation, the single-clone derived cells were sub-cultured and subjected to genomic DNA extraction, PCR amplification and T7 El digestion. Those cells with the highest T7 El digestion were further subjected to sequencing.CRISPR technology experiment process:The target sequences of Tip60 were selected according to the bioinformatical analysis and the sequences were cloned into CRISPR targeting vector respectively. After verification by sequencing, The gRNA lentivirus were packaged in 293 T cells and were used to infect U2OS cell line. Forty-eight hours later, the U2OS cells were screened with puromycin for three days and then infected with Cas9 adenovirus. The editing efficiency of CRISPR was evaluated by PCR amplification and T7 endonucluease digestion.The CRISPR gRNA lentivirus with the highest efficiency were selected to screen Tip60 homozygously knockout cell. Transfected or infected cells were diluted to one cell per well of 96-well plate. The Subsequent work for sequencing is the same as TALEN Technology. Those cells with the highest T7 EI digestion were further subjected to sequencing.In conclusion, we successfully obtained Tip60 heterzygously disrupted cell and paved the way to further investigate the detailed function of Tip60 in DNA damage repair, cell cycle regulation, autophagy and other signal pathways. |
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