【摘要】：背景UHRF2是一個具有E3泛素連接酶的多結構域核蛋白,參與細胞周期、DNA損傷修復、表觀遺傳學和泛素-蛋白酶體系統(tǒng)。我們課題組在前期研究工作中證實UHRF2參與調控HBV的轉錄與復制,并調節(jié)與ccc DNA結合的組蛋白H3乙�；健＝诘难芯堪l(fā)現UHRF2與組蛋白H3K9ac和H3K14ac存在相互結合作用。本課題基于上述研究成果,繼續(xù)對UHRF2與組蛋白乙�；g的調控機制進行深入探索。方法本文主要通過western blot實驗驗證UHRF2對H3K9ac、H3K14ac、TIP60、H2AK5ac和HDAC1的調控作用關系;免疫共沉淀(Co-IP)實驗驗證UHRF2與TIP60和HDAC1的相互作用關系;免疫熒光染色(IF)實驗驗證UHRF2與TIP60和HDAC1的細胞亞定位關系;半衰期實驗驗證UHRF2對TIP60半衰期的調控作用關系;體內泛素化實驗驗證UHRF2對TIP60的泛素化作用關系;免疫組織化學染色(IHC)實驗驗證UHRF2與H3K9ac、H3K14ac、TIP60和H2AK5a之間的調控關系。結果1)western blot實驗證實,在正常細胞中過表達UHRF2可上調H3K9ac及H3K14ac的表達,而在腫瘤細胞中過表達UHRF2則抑制H3K9ac及H3K14ac的表達。干擾UHRF2與過表達UHRF2結果一致。2)western blot實驗證實,在HEK293、LO2和Hep G2細胞中缺失UHRF2的YDG結構域或RING結構域可廢除UHRF2對H3K9ac及H3K14ac的調控作用。3)Co-IP和IF實驗證實,UHRF2與TIP60和HDAC1存在共同定位和相互結合作用,且UHRF2的YDG結構域、PHD結構域和RING結構域對UHRF2與TIP60的結合作用至關重要,缺失這些結構域可顯著降低UHRF2與TIP60的結合作用,而對UHRF2與HDAC1的結合無顯著影響。4)Western blot實驗證實,在正常細胞中過表達UHRF2可上調TIP60的表達及酶活性,而在腫瘤細胞中過表達UHRF2則可抑制TIP60的表達及酶活性,對于HDAC1的表達無顯著調控作用。且UHRF2對TIP60的調控作用會隨著蛋白酶體抑制劑MG132的加入而被廢除。5)半衰期實驗證實,在正常細胞(HEK293和LO2細胞)中過表達UHRF2可延長TIP60的半衰期,而在腫瘤細胞(Hep G2細胞)中過表達UHRF2則可加速TIP60的降解速率,且UHRF2蛋白RING結構域的缺失可廢除這一調控作用。6)體內泛素化實驗證實,在正常細胞(HEK293和LO2細胞)中過表達UHRF2可增加TIP60的泛素化,而在腫瘤細胞(Hep G2細胞)中過表達UHRF2則可抑制TIP60的泛素化。7)Western blot實驗證實,在HEK293、LO2細胞和Hep G2細胞中過表達TIP60可上調H3K9ac及H3K14ac的表達,干擾TIP60則可抑制H3K9ac及H3K14ac的表達。8)Western blot實驗證實,過表達UHRF2的同時干擾TIP60,廢除了UHRF2對H3K9ac及H3K14ac的調控作用;同時過表達UHRF2和TIP60則可進一步上調H3K9ac及H3K14ac的表達;當我們應用TIP60抑制劑MG149處理細胞時,無論過表達野生型UHRF2還是UHRF2各結構域突變體均無法繼續(xù)對H3K9ac及H3K14ac進行調控。9)免疫組織化學實驗證實,在UHRF2高表達的肝癌組織中,TIP60、H2AK5ac、H3K9ac和H3K14ac呈現低表達,而在癌旁組織中,UHRF2的高表達則伴隨著TIP60、H2AK5ac、H3K9ac和H3K14ac的高表達。結論在正常細胞(HEK293和LO2細胞)中,UHRF2延長TIP60的半衰期,可上調TIP60的蛋白表達量及酶活性,從而上調H3K9ac及H3K14ac的表達量,而在腫瘤細胞(Hep G2細胞)中,UHRF2可加速TIP60的降解速率,抑制TIP60的蛋白表達量及酶活性,進而抑制H3K9ac及H3K14ac的表達。
[Abstract]:Background UHRF2 is a multi domain nuclear protein with E3 ubiquitin ligase, involved in cell cycle, DNA damage repair, epigenetics and ubiquitin proteasome system. In our previous work, our group confirmed that UHRF2 participates in the regulation of HBV transcription and replication and regulates the level of histone H3 acetylation with CCC DNA. Recent research We found that UHRF2 and histone H3K9ac and H3K14ac interact with each other. Based on the above research results, we continue to explore the regulatory mechanism between UHRF2 and histone acetylation. Methods this paper mainly through the Western blot test to verify the regulatory role of UHRF2 on H3K9ac, H3K14ac, TIP60, H2AK5ac and HDAC1, immunization. The interaction between UHRF2 and TIP60 and HDAC1 was verified by the precipitation (Co-IP) experiment; the immunofluorescence staining (IF) test verified the relationship between UHRF2 and the cell Sublocation of TIP60 and HDAC1; the half life experiment verified the regulation of UHRF2 on TIP60 half life; in vivo ubiquitination test verified the ubiquitination of UHRF2 to TIP60; immunohistochemical staining. IHC test verified the regulatory relationship between UHRF2 and H3K9ac, H3K14ac, TIP60 and H2AK5a. Results 1) Western blot experiments confirmed that overexpression of UHRF2 in normal cells can up regulate the expression of H3K9ac and H3K14ac. The blot experiment confirmed that the YDG domain or RING domain missing UHRF2 in the HEK293, LO2 and Hep G2 cells can abolish UHRF2's regulation of H3K9ac and H3K14ac. The deletion of these domains can significantly reduce the combination of UHRF2 and TIP60, but the combination of UHRF2 and HDAC1 has no significant effect on.4) Western blot experiments confirmed that the overexpression of UHRF2 in normal cells can up regulate the expression of TIP60 and the activity of enzymes, while UHRF2 in the tumor cells can inhibit TIP60 expression and enzyme activity. Sex has no significant regulation on the expression of HDAC1. And the regulation of UHRF2 on TIP60 will be abolished with the addition of the proteasome inhibitor MG132 and the half-life experiment confirms that the overexpression of UHRF2 in normal cells (HEK293 and LO2 cells) can prolong the half-life of TIP60, while the overexpression of UHRF2 in the tumor cells (Hep G2 cells) can accelerate the decline. The degradation rate of IP60, and the deletion of the UHRF2 protein RING domain can abolish this regulatory effect.6) in vivo ubiquitination experiments confirmed that the overexpression of UHRF2 in normal cells (HEK293 and LO2 cells) increased the ubiquitination of TIP60, while the overexpression in the tumor cells (Hep G2 cells) could inhibit TIP60 ubiquitination. The overexpression of TIP60 in HEK293, LO2 and Hep G2 cells can up regulate the expression of H3K9ac and H3K14ac, and the interference of TIP60 can inhibit the expression of H3K9ac and H3K14ac. The expression of AC and H3K14ac; when we used TIP60 inhibitor MG149 to treat the cells, no matter over expressed wild type UHRF2 or UHRF2 domain mutants could not continue to regulate.9 in H3K9ac and H3K14ac. The high expression of UHRF2 is associated with high expression of TIP60, H2AK5ac, H3K9ac and H3K14ac in the para cancerous tissues. Conclusion in normal cells (HEK293 and LO2 cells), UHRF2 prolongs the half-life of TIP60 and up regulates the protein expression and enzyme activity of TIP60, thus increasing H3K9ac and H3K14ac expression. The degradation rate of fast TIP60 inhibited the protein expression and enzyme activity of TIP60 and inhibited the expression of H3K9ac and H3K14ac.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.2

【參考文獻】

相關期刊論文 前9條

1 彭城;李國亮;張紅雨;阮一駿;;染色質三維結構重建及其生物學意義[J];中國科學:生命科學;2014年08期

2 周丹琳;胡斌;宣艷艷;段昌柱;;NIRF對HBV復制的影響及其對組蛋白H3乙�；降男揎梉J];中國細胞生物學學報;2013年03期

3 胡斌;周丹琳;宣艷艷;段昌柱;;NIRF在體內外可明顯抑制乙型肝炎病毒抗原的表達[J];中國生物化學與分子生物學報;2012年11期

4 楊艷;金方敏;白露;王筱繪;段昌柱;;HBc蛋白與NIRF蛋白細胞外相互作用鑒定[J];生物學雜志;2012年01期

5 鄭小梅;伍寧豐;;DNA甲基化作用的生物學功能[J];中國農業(yè)科技導報;2009年01期

6 王溪;朱衛(wèi)國;;組蛋白甲基化酶及去甲基化酶的研究進展[J];癌癥;2008年10期

7 PaolaGallinari;StefaniaDiMarco;PhillipJones;MichelePallaoro;ChristianSteinkühler;;HDACs,histone deacetylation and gene transcription: from molecular biology to cancer therapeutics[J];Cell Research;2007年03期

8 段昌柱;蒲淑萍;TSUTOMU MORI;HIDEO KOCHI;邱宗蔭;;NIRF對P53蛋白泛素化作用的研究[J];生物化學與生物物理進展;2006年02期

9 黃百渠,曾慶華,畢曉輝,王玉紅,李玉新;組蛋白和核小體在基因轉錄中的作用[J];科學通報;2000年19期

，

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