Study The Function Of THAP11: A Candidate Gene For Polyglutamine Disorders And Tumor Suppressor

Background:Polyglutamine(polyQ) diseases are neurodegenerative diseases,which are caused by expanded CAG repeats in gene coding region resulting in translation products possessing expanded polyglutamine repeats.At present,a large number of pathogenic genes of polyQ diseases have not been revealed.THAP11 contains a section of polyglutamine fragment which is coded by CAG repeats.Might THAP11 be a candidate gene for polyglutamine disorders? So,we need to investigate the CAG repeats of THAP11 in normal population and neurodegenerative diseases patients.In addition,we need to study the function of THAP11 containing abnormal expanded CAG which might be founded in patients with neurodegenerative diseases in order to further confirm THAP11 a candidate gene for polyglutamine disorders.On the other hand,the other members of THAP family are involved in transcription suppression,cell apoptosis and proliferation suppression,and some proteins have been identified as tumor suppressor.So,might THAP11 also be a candidate gene for tumor suppressor? we also need to study the function of THAP11 in order to verify this assumption.Objective:1):To know the normal range of the CAG repeats of THAP11 through detecting the number of the repeats in normal Chinese Hans and investigate the range of the CAG repeats of THAP11 in Chinese Hans patients with neurodegenerative disorders in order to find whether the number of the CAG repeats is beyond the normal range,and explore the possibility of THAP11 to be a polyglutamine disorders candidate gene.2)To verify the possibility of a polyglutamine disorders gene after studying the function of THAP11,of which the CAG repeats is abnormal.3)To explore the possibility of THAP11 to be a tumor suppressor gene by studying the function of THAP11.Method:1)By extracting genomic DNA,PCR,sequencing,we investigated the CAG repeats of THAP11 in Chinese Hans patients with neurodegenerative disorders in order to find the character of the genotype and the distribution of THAP11 CAG repeats,and whether the CAG repeats is beyond the normal range.2) THAP11 mutation including expanded CAG repeats was cloned to pEGFP-N1 vector which express green fluorescent and pIND vector which belongs to the molting hormone induced system in order to observe the THAP11 location in cells and whether they have the specific pathological changes of protein aggregation.3)Through flow cytometry test to find whether the THAP11 mutant including abnormal expanded CAG repeats influenced the cell cycle,and further detect the effect of the THAP11 mutation on cell cycle protein by means of Western Blotting.4)By use of chromatin shape detection technique to detect the effects of THAP11 on chromatin shape,which may provides a clue for other effects,such as on gene transcription.We utilized Real time-PCR to detect the effects of THAP11 on the mRNA expression of oncogene c-myc in HepG₂ cells and simultaneously we also used Western blotting to detect the variation in protein expression,which may suggests the relationship between THAP11 and tumor.Result:1)Through investigating the CAG repeats of THAP11 in normal Chinese Hans,we find the CAG repeat polymorphism with respect to length and repeat substructure of CAG repeats with CAA interruption.The normal Chinese Hans CAG repeats number varies from 22 to 34,in which 29 is the major repeats number(69.9%) and the next is 28(14.5%).We also find that the major genotype of THAP11 is (CAG)₃CAA(CAG)₅CAA(CAG)₂CAA(CAG)₅CAA(CAG)₁₀(62.35%).The number of CAA interruptions rangs from 2 in 23 and 25 CAG repeats to 6 in 29 CAG repeats,of which 4 is the major type.Among these,the longest CAG repeat that is not break by CAA is 15.2)Compared with the control,we found that the CAG repeats of THAP11 in patients with neurodegenerative disorders have the repeats expansion,including 35 CAG repeats existing in two patients(1.66%) with heterogenesis chromatin and 38 repeats in one patient(0.83%) with hetergenesis chrornatin.Among these,the 38 CAG repeats genotype is(CAG)₃CAA(CAG)₅CAA(CAG)₂CAA(CAG) ₅CAA(CAG)₈CAA(CAG)₁₀.3)Through the detection of fluorescent location of abnormal THAP11 containing extended CAG in cells,we found that THAP11(35Q) protein was more dense than THAP11(29Q) in intranuclear,although they were whole cell location.THAP11(38Q) had a potein aggregation in intranuclear besides the same phenomena as THAP11(35Q).By use of the molting hormone induced system,we observed the dynamic processes of gradual changes in location from the initial whole cell to nuclear and the last a protein aggregation.4)Through detecting the influence of different polyQs of THAP11 on cell cycle,we found the normal THAP11(29Q) did not cause significant changes in the cell cycle,while THAP11(38Q) could cause significant G0/G1 arresting,of which the cell number accounts for 90.24%that was higher than the control(58.52%).Through the detection of cell cycle protein,we found that THAP11(29Q)had no effect on cell cycle protein,but THAP11(38Q) could cause the increased P21 and decreased CyclinD1,which also reflects G0/G1 arresting.5)Through the chromatin shape detection technique,we observed THAP11 could make chromatin more condensable,which suggests THAP11 might repress transcription and could be a tumor suppressor.Later,we found THAP11 expression is weaker in tumor tissue than in corresponding normal tissue,which further suggests THAP11 might a tumor suppressor.In addition,we found THAP11(38Q) could inhibit the c-myc mRNA and protein expression,which further confirms that THAP11 could be a candidate gene for tumor. Conclusion:1) we detected the normal variation range of CAG repeat of THAP11 gene in normal Chinese Hans,which varies from 22 to 34.In addition,we identified the genotype and distribution of THAP11.2) Through detecting the number of the CAG repeats of THAP11 in Chinese Hans patients with neurodegenerative disorders,we found a THAP11 containing 38 CAG repeats in one patient with neurodegenerative disorders,which primarily suggests that the patient might suffer from polyQs disorders. 3) we found that the abnormal THAP11 containing expanded CAG could generate a intranuclear inclusion that is a characteristic pathological changes of polyQs disease.More over,the abnormal THAP11 may cause cell cycle G0/G1 arresting,which further suggests THAP11 might be a candidate gene for polyQs disease.4) we found THAP11 might have transcription repression function.Compared with the normal,THAP11 expression is weaker in tumor tissue.More over,THAP11 can repress oncogene c-myc expression.All these results suggest that THAP11 could be a candidate gene for tumor suppressor.