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UHRF2通过其RING结构域与TIP60作用进而调控组蛋白H3K9和H3K14乙酰化水平

发布时间:2018-07-24 12:51
【摘要】:背景UHRF2是一个具有E3泛素连接酶的多结构域核蛋白,参与细胞周期、DNA损伤修复、表观遗传学和泛素-蛋白酶体系统。我们课题组在前期研究工作中证实UHRF2参与调控HBV的转录与复制,并调节与ccc DNA结合的组蛋白H3乙酰化水平。近期的研究发现UHRF2与组蛋白H3K9ac和H3K14ac存在相互结合作用。本课题基于上述研究成果,继续对UHRF2与组蛋白乙酰化之间的调控机制进行深入探索。方法本文主要通过western blot实验验证UHRF2对H3K9ac、H3K14ac、TIP60、H2AK5ac和HDAC1的调控作用关系;免疫共沉淀(Co-IP)实验验证UHRF2与TIP60和HDAC1的相互作用关系;免疫荧光染色(IF)实验验证UHRF2与TIP60和HDAC1的细胞亚定位关系;半衰期实验验证UHRF2对TIP60半衰期的调控作用关系;体内泛素化实验验证UHRF2对TIP60的泛素化作用关系;免疫组织化学染色(IHC)实验验证UHRF2与H3K9ac、H3K14ac、TIP60和H2AK5a之间的调控关系。结果1)western blot实验证实,在正常细胞中过表达UHRF2可上调H3K9ac及H3K14ac的表达,而在肿瘤细胞中过表达UHRF2则抑制H3K9ac及H3K14ac的表达。干扰UHRF2与过表达UHRF2结果一致。2)western blot实验证实,在HEK293、LO2和Hep G2细胞中缺失UHRF2的YDG结构域或RING结构域可废除UHRF2对H3K9ac及H3K14ac的调控作用。3)Co-IP和IF实验证实,UHRF2与TIP60和HDAC1存在共同定位和相互结合作用,且UHRF2的YDG结构域、PHD结构域和RING结构域对UHRF2与TIP60的结合作用至关重要,缺失这些结构域可显著降低UHRF2与TIP60的结合作用,而对UHRF2与HDAC1的结合无显著影响。4)Western blot实验证实,在正常细胞中过表达UHRF2可上调TIP60的表达及酶活性,而在肿瘤细胞中过表达UHRF2则可抑制TIP60的表达及酶活性,对于HDAC1的表达无显著调控作用。且UHRF2对TIP60的调控作用会随着蛋白酶体抑制剂MG132的加入而被废除。5)半衰期实验证实,在正常细胞(HEK293和LO2细胞)中过表达UHRF2可延长TIP60的半衰期,而在肿瘤细胞(Hep G2细胞)中过表达UHRF2则可加速TIP60的降解速率,且UHRF2蛋白RING结构域的缺失可废除这一调控作用。6)体内泛素化实验证实,在正常细胞(HEK293和LO2细胞)中过表达UHRF2可增加TIP60的泛素化,而在肿瘤细胞(Hep G2细胞)中过表达UHRF2则可抑制TIP60的泛素化。7)Western blot实验证实,在HEK293、LO2细胞和Hep G2细胞中过表达TIP60可上调H3K9ac及H3K14ac的表达,干扰TIP60则可抑制H3K9ac及H3K14ac的表达。8)Western blot实验证实,过表达UHRF2的同时干扰TIP60,废除了UHRF2对H3K9ac及H3K14ac的调控作用;同时过表达UHRF2和TIP60则可进一步上调H3K9ac及H3K14ac的表达;当我们应用TIP60抑制剂MG149处理细胞时,无论过表达野生型UHRF2还是UHRF2各结构域突变体均无法继续对H3K9ac及H3K14ac进行调控。9)免疫组织化学实验证实,在UHRF2高表达的肝癌组织中,TIP60、H2AK5ac、H3K9ac和H3K14ac呈现低表达,而在癌旁组织中,UHRF2的高表达则伴随着TIP60、H2AK5ac、H3K9ac和H3K14ac的高表达。结论在正常细胞(HEK293和LO2细胞)中,UHRF2延长TIP60的半衰期,可上调TIP60的蛋白表达量及酶活性,从而上调H3K9ac及H3K14ac的表达量,而在肿瘤细胞(Hep G2细胞)中,UHRF2可加速TIP60的降解速率,抑制TIP60的蛋白表达量及酶活性,进而抑制H3K9ac及H3K14ac的表达。
[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.
【学位授予单位】:重庆医科大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R730.2

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