【摘要】：背景UHRF2是一個具有E3泛素連接酶的多結(jié)構(gòu)域核蛋白,參與細(xì)胞周期、DNA損傷修復(fù)、表觀遺傳學(xué)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(diào)控HBV的轉(zhuǎn)錄與復(fù)制,并調(diào)節(jié)與ccc DNA結(jié)合的組蛋白H3乙�；健＝诘难芯堪l(fā)現(xiàn)UHRF2與組蛋白H3K9ac和H3K14ac存在相互結(jié)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(diào)控機(jī)制進(jìn)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(diào)控作用關(guān)系;免疫共沉淀(Co-IP)實驗驗證UHRF2與TIP60和HDAC1的相互作用關(guān)系;免疫熒光染色(IF)實驗驗證UHRF2與TIP60和HDAC1的細(xì)胞亞定位關(guān)系;半衰期實驗驗證UHRF2對TIP60半衰期的調(diào)控作用關(guān)系;體內(nèi)泛素化實驗驗證UHRF2對TIP60的泛素化作用關(guān)系;免疫組織化學(xué)染色(IHC)實驗驗證UHRF2與H3K9ac、H3K14ac、TIP60和H2AK5a之間的調(diào)控關(guān)系。結(jié)果1)western blot實驗證實,在正常細(xì)胞中過表達(dá)UHRF2可上調(diào)H3K9ac及H3K14ac的表達(dá),而在腫瘤細(xì)胞中過表達(dá)UHRF2則抑制H3K9ac及H3K14ac的表達(dá)。干擾UHRF2與過表達(dá)UHRF2結(jié)果一致。2)western blot實驗證實,在HEK293、LO2和Hep G2細(xì)胞中缺失UHRF2的YDG結(jié)構(gòu)域或RING結(jié)構(gòu)域可廢除UHRF2對H3K9ac及H3K14ac的調(diào)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(jié)合作用,且UHRF2的YDG結(jié)構(gòu)域、PHD結(jié)構(gòu)域和RING結(jié)構(gòu)域?qū)HRF2與TIP60的結(jié)合作用至關(guān)重要,缺失這些結(jié)構(gòu)域可顯著降低UHRF2與TIP60的結(jié)合作用,而對UHRF2與HDAC1的結(jié)合無顯著影響。4)Western blot實驗證實,在正常細(xì)胞中過表達(dá)UHRF2可上調(diào)TIP60的表達(dá)及酶活性,而在腫瘤細(xì)胞中過表達(dá)UHRF2則可抑制TIP60的表達(dá)及酶活性,對于HDAC1的表達(dá)無顯著調(diào)控作用。且UHRF2對TIP60的調(diào)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(xì)胞(HEK293和LO2細(xì)胞)中過表達(dá)UHRF2可延長TIP60的半衰期,而在腫瘤細(xì)胞(Hep G2細(xì)胞)中過表達(dá)UHRF2則可加速TIP60的降解速率,且UHRF2蛋白RING結(jié)構(gòu)域的缺失可廢除這一調(diào)控作用。6)體內(nèi)泛素化實驗證實,在正常細(xì)胞(HEK293和LO2細(xì)胞)中過表達(dá)UHRF2可增加TIP60的泛素化,而在腫瘤細(xì)胞(Hep G2細(xì)胞)中過表達(dá)UHRF2則可抑制TIP60的泛素化。7)Western blot實驗證實,在HEK293、LO2細(xì)胞和Hep G2細(xì)胞中過表達(dá)TIP60可上調(diào)H3K9ac及H3K14ac的表達(dá),干擾TIP60則可抑制H3K9ac及H3K14ac的表達(dá)。8)Western blot實驗證實,過表達(dá)UHRF2的同時干擾TIP60,廢除了UHRF2對H3K9ac及H3K14ac的調(diào)控作用;同時過表達(dá)UHRF2和TIP60則可進(jìn)一步上調(diào)H3K9ac及H3K14ac的表達(dá);當(dāng)我們應(yīng)用TIP60抑制劑MG149處理細(xì)胞時,無論過表達(dá)野生型UHRF2還是UHRF2各結(jié)構(gòu)域突變體均無法繼續(xù)對H3K9ac及H3K14ac進(jìn)行調(diào)控。9)免疫組織化學(xué)實驗證實,在UHRF2高表達(dá)的肝癌組織中,TIP60、H2AK5ac、H3K9ac和H3K14ac呈現(xiàn)低表達(dá),而在癌旁組織中,UHRF2的高表達(dá)則伴隨著TIP60、H2AK5ac、H3K9ac和H3K14ac的高表達(dá)。結(jié)論在正常細(xì)胞(HEK293和LO2細(xì)胞)中,UHRF2延長TIP60的半衰期,可上調(diào)TIP60的蛋白表達(dá)量及酶活性,從而上調(diào)H3K9ac及H3K14ac的表達(dá)量,而在腫瘤細(xì)胞(Hep G2細(xì)胞)中,UHRF2可加速TIP60的降解速率,抑制TIP60的蛋白表達(dá)量及酶活性,進(jìn)而抑制H3K9ac及H3K14ac的表達(dá)。
[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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.2

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