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The Experimental Study of the Effects and Mechanism of Telomere Repeat Binding Factor 1 on p53 and ATM

Author: XuZuoZuo
Tutor: HuangHe
School: Zhejiang University
Course: Internal Medicine
Keywords: Phosphorylation sites Professional degree Mitosis Doctoral Dissertation Deletion mutant Cell cycle arrest Telomerase Centrosome Zhejiang University School of Medicine
CLC: Q343
Type: PhD thesis
Year: 2008
Downloads: 123
Quote: 0
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Abstract


Telomeres,the ends of eukaryotic chromosomes,are specific DNA-protein complexes that are essential for preserving the integrity of chromosomes during the cell cycle.The homeostasis of mammalian telomeres is regulated by a number of telomere-associated proteins.Telomere repeat binding factor 1(TRF1)plays crucial role in telomere biology.Overexpression of TRF1 shortens telomeres in human cells through inhibiting telomerase access and interacting with TIN2、POT1 and PTOP.In ANLL cells but not ALL cells,the protein expression of TRF1 is low.The p53 tumor suppressor is one of the most important proteins,because in response to exposure to DNA damage agents,it induces cell cycle arrest in G1 checkpoint or apoptosis.The consequences of its mutation are profound,either in the germlines of patients with Li-Fraumeni syndrome,or in mice with targeted gene knockouts.In response to DNA damage,multiple mechanisms regulate p53 activity and posttranslational modification,including phosphorylation of serine 15,in particular.Ataxia telangiectasia is a relatively rare genetic disorder caused by mutations in the ATM gene.ATM is an important gene in regulation apoptosis,cell growth,telomere length monitoring and DNA damage.ATM binds and negatively regulates the function of TRF1 presumably via phosphorylation of TRF1 Ser 219.Treatment of human gastric cancer MGC803 cells with arsenic trioxide could cause up-regulation of TRF1 and TRF2 mRNA and protein levels,and induced G2/M phase arrest and cell apoptosis.Results also confirmed that after trioxide treatment,p53 protein levels increased significantly and also could bind directly at the telomere t-loop junction.These findings indicate TRF1 is involved in p53 signal pathway.Analysis of mice with multiple intestinal neoplasia provided evidence that telomere shortening can dramatically impair tumor development in epithelial cells. The APC tumor suppressor gene is mutated in patients with familial adenomatous polyposis.Heterozygous APCmin+/-mice develop numerous gastrointestinal adenomas,in similar fashion to patients with APC mutations.Telomere dysfunction in late generation G4 TERC-/-APCmin+/-mice profoundly suppressed adenoma formation,accompanied by high protein level of p53.Evidence from TERC-/-fibroblasts suggests that activation of arrest may represent part of the mechanism by which p53 acts to limit proliferation of cells sustaining telomere dysfunction.In late generation,TERC-/-p21-/-mice show an enhanced tumor predisposition or an altered tumor spectrum compared to TERC-/- p53+/+mice.There is evidence that p53 cell cycle arrest function contributes to replicative senescence in human cells,based on elevated levels of p21.Furthermore, inactivation of p21 in human fibroblasts extends cellular lifespan,indicating that p21 is an important downstream target of p53 in this context.Future genetic experiments of this type will be highly informative for determining the relevance of the G1 checkpoint response in p53 action downstream of telomere attrition.The association between TRF1 and p53 hasn’t be reported.So in this work,our study focused on TRF1 action on p53 and ATM.We tried to elucidate the molecule mechanism regulated by the proteins interaction.The first part validated the interaction between TRF1 and p53.Using TRF1 as a bait,we identified p53 as its new interacting partner.To test if TRF1 interacts with p53,GST pulldown and immunoprecipitation experiments were performed.Significantly,these experiments demonstrated an association between TRF1 and p53 in vitro and in vivo.P53 interacted with TRF1 via the C terminal region.Like p53,TRF1 also localized to centrosome of mitotic cells.The co-localization of p53 and TRF1 to centrosome suggested their interaction and involvement in centrosome function.Elimination of TRF1 by small interfering RNA (siRNA)didn’t diminish the localization of p53 to centrosome.Our studies demonstrated that TRF1 isn’t essential for the localization of p53 to centrosome.The second part demonstrated the effects and mechanism of TRF1 on p53.To further elucidate p53-mediated cell cycle checkpoint cascade and apoptosis signal pathway regulating by TRF1,we constructed the mimicking phosphorylation mutant(GFP-p53S15D)and the non-phosphorylatable mutant(GFP-p53S15A)and carried out immunoprecipitation assay、Western blot and immunofluorescence. Immunoprecipitation and pulldown experiments demonstrated the association between GFP-p53S15A/GFP-p53S15Dand TRF1.Immunofluorescence study revealed co-distribution of TRF1 with p53S15after UV stress.Therefore,we suggested that TRF1 may involve in the stability and phosphorylation of p53.In addition,TRF1 enhanced the Ser 15 phosphorylation of p53 and protein level of p21.We further investigated transcriptional induction of the p21 gene by examining the effect of TRF1 on p21 gene promoter activity by dual-luciferase reporter assay.The p53 responsive element was required for activation of p21 promoter by TRF1.Taken together,the experiment results validated that TRF1 could enhance the Ser 15 phosphorylation and induce p53-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.The results also indicated TRF1 may regulate the cell cycle and the development of tumors via p53 signal pathway.The third part demonstrated the effects and mechanism of TRF1 on ATM.To further investigate the cell cycle regulated by Ser 219 phosphorylation of TRF1.We constructed the mimicking phosphorylation mutant(GFP-TRF1S219D)and the non-phosphorylatable mutant(GFP-TRF1S219A).The mutant genes were verified by direct sequencing and protein expression of GFP-tagged mutants was confirmed by immunoblotting.Immunofluorescence demonstrated GFP-TRF1S219Aand GFP- TRF1S219Dboth localized to telomere in HeLa cells.The protein level of ATM was increased when overexpression wide type TRF1 or mutants.Moreover,overexpression of GFP-TRF1 or GFP-TRF1S219Aresulted in an accumulation of HeLa cells in G2/M phase(P<0.05).Taken together,the experiment results validated that the Ser 219 phosphorylation of TRF1 by ATM could regulate the G2/M block induced by overexpression of TRF1. The regulation of cell cycle by TRF1 mutants demonstrated the association between TRF1 and mitosis,and indicated the important function of telomere proteins in tumor development.

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