The Function Resreaches Of Telomere Binding-protein TPP1

One of hot topics in the fied of modern biology is about the study of telomere and telomerase,which are both closely related to the human cells' aging and tumor's formation and development.Telomere is a special DNA-protein complex at the end of eukaryotic linear chromosomes and has a unique cap structure at the end of chromosomes.Its main biological functions:1.To prevent chromosomes from chemical modification or degradation by ribozyme;2.To protect chromosomes from end-to-end fusion,recombinant and chromosome deficiency;3.To play an important role in maintaining normal replication of genetic material and the ability of proliferation.Telomere length will gradually shorten along with each division of normal cells.Seriously shortened telomeres are regarded as the singal of cell senescence,and when their length reachs a limit,they will cause cell senescence and apotosis.Therefore,scientists term the telomeres as " life clock".When the adjustment mechanism is abnormal,it always results in a series of diseas such as tumour and autoimmune disease,and the regulation of telomere length depends on both telomerase and telomere binding proteins.Telomerase is a kind of revertase and comprised of RNA and proteins termed ribonucleoprotein.It can use itself RNA as template to synthesize the telomere DNA and adds them to the end of telomere to maintain the telomere length.The presense of telomerase solves the problem of telomere shortening,at the same time the excessive expression of telomerase is directly related to cell immortalization and canceration.Telomere binding proteins are divided into two kinds:Single-stranded DNA binding protein and Double-stranded DNA binding protein.Current reports suggest that POT1 is the only single-stranded DNA binding protein,specificity combined with single-strand DNA.POT1 can also indirectly combine with TRF1 and TRP2 which both locate on double-stranded DNA through the interaction with TPP1 and Tin2.Telomere protein complexes with RAP1 as the core protein on the ends of telomere participate in the regulation of telomere length and protection together.At present the konwn main functions of Shelterin:1.To form the protection structure of the end of telomere,2.To take part in the formation of the T-loop of the end of telomere,3.To deeply regulate the synthesis of telomere DNA and the telomere length through controlling the activity of telomerase.The six core protein respectively own their different biological functions,they form a complex telomere protein network,participate in regulating the structure and function of telomeres.In this paper,we studied TPP1 functions about regulating the POT1 and affecting the location of TRF2:At first we constructed the fusion protein "POT1-TPP1-C100",and after silencing expression of endogenous protein TPP1,we confirmed that POT1 is able to interact with Tin2 under the mediation of TPP1-C100by co-immunoprecipitation.This fusion protein was stably expressed in tumor cell termed HepG2,and by IF and IF-FISH,we demonstrated that after silencing the expression of endogenous TPP1,POT1 is able to locate on the end of telomere under the medication of TPP1-C1OO.We successfully cloned POT1-TPP1-C100 that can stably express in HepG2 cells,and after knowdowning the expression of endogenous protein TPP1,the POT1-TPP1-C100 was able to enter the nucleus and locate on telomere.Evidences indicate that POT1 can prevent RPA from combining with telomeres to suppress telomere DNA damage response(DDR)which relys on ATR signaling pathways.We observed 53PB1 activation of HepG2 cells which overexpress exogenous protein of POT1-TPP1-C100 by immunofluorescence.After inhibiting endogenous TPP1 expression,we could see POT1 positioning at the end of the telomere,but there are still a lot of 53PB1 forming TIFs at the telomeres.We use Western Blot to test the activation volume of ?_H2AX of POT1-TPP1-C100 after silencing the expression of endogenous TPP1.The activation level of?_H2AX increased obviously;Respectively using ATM and ATR siRNA to silence the expression of ATM and ATR,showed that the activation level of ?_H2AX had reduced,showing that inducing the telomere DNA damage by suppressing TPP1 expression depends on both the ATM and ATR.Our experimental results suggest that TPP1 regulates POT1 functions of POT1 entering nucleus,positioning on telomeres and POTI after arriving at telomeres eventually playing the inhibition role of the ATR mediated telomere DNA damage response,and other processes.TPP1 is an integral part of a composite components for maintaining telomere protein complexes(Shelterin)stability,and ultimately playing a role on telomeres protection.TRF2 participates in maintaining the morphology and structural integrity of telomere,protecting normal functions of telomere and inhibiting activity of ATM kinase.Inhibiting or inactivating the activity of telomere can lead to dysfunction,causing telomere DNA damage and aggregating DNA damage factors,such as 53PB1,RAD17,?_H2AX and so on.In the process of immunofluorescence we found that after inhibition of the expression of endogenous TPP1,TRF2 didn't locate at telomere in the contral group of HepG2 cells and cells with high expression of exogenous POT1,but disperse in the nucleus.We also observed that the pathway of telomere DNA damage dependes on ATM signal through Western blot.After knockdowning the expression level of endohenous POT1,TRF1,TPP1,Tin2,we found that the silence of TPP1 and Tin2 can reduce TRF2 telomere positioning,but the other two proteins didn't have the same influence.Then we respectively knockdowned the endogenous TPP1 and Tin2 in the HepG2 cells which were able to respectively express the TPP1 and Tin2.We found that silencing the expression of Tin2 reduces the TRF2 telomere location in the high expression of heterologous TPP1,but silencing TPP1 has no affected.It is interesting that knockdowning either Tin2 or TPP1 affected the TRP2 telomere location in the HepG2 cells expressing exogenous Tin2.Our study demonstrates that TPP1 could indirectly regulate the TRF2 telomere location through connecting with Tin2.Moreover we also observed that TRF2 locates on the telomere in the HepG2 cells expressing exogenous POT1-TPP1-C100.We highly expressed this TPP1-C100 in HepG2 cells and observed how TPP1-C100 influences TRF2 telomere location after silencing the endogenous TPP1 in these cells.The result shows that only TPP1-C100 can effectively regulate TRF2 location.Conclusions:1,separate telomere position of POT1 cannot effectively inhibit telomere DNA damage response which depends on the ATR signal pathway,it must form a dimer with TPP]to play a role of inhibition.2,TPP1 can indirectly regulate the telomere position of TRF2 through interacting with Tin2,and the regulatory regions is in the interaction with Tin2 TPP1 C-terminal interacting with Tin2.The above-mentioned results further clarify TPP1 function in protection mechanism of telomere and laid a foundation for its biological significance in the research of tumor cells.