【作者】 涂华玉；

【导师】 李卫；

【作者基本信息】 中国科学技术大学 ， 遗传学， 2011， 硕士

【摘要】 组蛋白翻译后修饰包括乙酰化、磷酸化、甲基化、泛素化和糖基化等。这些翻译后修饰在染色质的结构重塑、基因的转录调控、DNA的损伤修复等生命过程中发挥着极其重要的作用。作为染色质的核心蛋白，组蛋白H2B的泛素化与其他组蛋白修饰之间存在密切联系，是"组蛋白密码"多样性的重要一环。H2B的泛素化是一个由一系列事件构成的过程，许多因子都参与和影响着该过程。目前发现芽殖酵母中H2B泛素化需要Rad6、Bre1、Lge1以蛋白复合体的形式参与其催化过程，而Ubp8、Ubp10是细胞内参与H2B去泛素化的两种主要去泛素化酶，其去泛素化活性亦是相互补充的。研究表明Lge1在对募集Bre1和Ubp8至染色质的过程中发挥相反作用，它能促进Bre1的募集却抑制Ubp8的募集。组蛋白修饰在配子体的发生尤其早期减数分裂过程中扮演了重要的角色，H2B泛素化修饰已被证明参与其中，但是H2B的泛素化和去泛素化究竟发挥了什么样的作用，又是如何调节其下游的一系列表观遗传学过程进而影响减数分裂的还并不清楚。为了了解组蛋白H2B的泛素化修饰在减数分裂过程中的作用机制问题，我们围绕着H2B泛素化修饰相关的几种主要蛋白Bre1、Lge1、Ubp8、Ubp10从以下方面进行研究：1.我们建立H2B泛素化相关蛋白的C端融合GFP的酵母细胞系，然后通过荧光显微镜观察Bre1、Lge1定位于细胞核内。2.构建相应的基因特异性敲除的酵母菌株，观察其对减数分裂进程的影响，研究证实Bre1、Lge1基因的敲除会影响酵母的减数分裂，显著降低芽殖酵母的产孢率，而Ubp8、Ubp10去泛素化酶的双突变对酵母减数分裂影响并不显著。3.构建组蛋白H2B泛素化位点的突变体（H2B K123R）,以研究H2BK123位点泛素化在减数分裂中作用。4.构建了特异敲除H2B泛素化酶Rnf20（酵母中Bre1的同源物）的基因敲除载体，获得了第一代嵌合体小鼠，为进一步构建在睾丸和卵巢中特异敲除Rnf20的小鼠系做准备，从而通过H2B泛素化酶Rnf20敲除小鼠去研究其在睾丸和卵巢中的功能。通过上述实验，我们发现H2B的泛素化修饰影响了减数分裂的进程，为进一步阐明H2B的泛素化修饰在减数分裂过程中的作用机制问题奠定了基础。更多还原

【Abstract】 Histones are subject to multiple different types of posttranslational modifications,including acetylation, phosphorylation, methylation, ubiquitination and glycosylation.These different modifications play important roles in chromatin structureremodeling,transcriptional regulation and DNA damage repair. Histone H2Bubiquintylation involves lots of protein complexes including Rad6, Bre1 and Lge1 inbudding yeast. H2B deubiquintylation involves mainly Ubp8 and Ubp10 in buddingyeast. Lge1 can promote the recruitment of Bre1 and inhibit recruitment of Ubp8.H2B ubiquintylation has been shown to be important for gamete formation andmeiosis. However, the mechanism of how H2B ubiquintylation affects meiosis is stillunknown. In this thesis, I focused on studying the role of Bre1, Lge1, Ubp8 andUbp10 in yeast sporulation. The main findings include:1. I determined the subcellular localization of Bre1 and Lge1 by using yeast linesexpressing GFP fusion proteins. Bre1 and Lge1 mainly localized in nuleus.2. I found that knockout of Bre1 and Lge1 can significantly decrease the yeastsporulation efficiency. However, a double mutant of Ubp8 and Ubp10 did notsignificantly influence the yeast sporulation efficiency.3. I constructed a H2B K123R mutant yeast strain to study the role of H2Bubiquintylation in meiosis.4. I constructed a Rnf20 (homolog of Bre1 in mammals) conditional knockout vectorin mice and obtained F1 mosaic mice successfully, which can be mated to getgermline transmitted F2 and homozygous floxed F3 mice. In the future, we canselectively knockout Rnf20 in reproductive organs such as ovary and testis tostudy the role of Rnf20 in gametogenesis.As a summary, I made substantial progress on studying the role of H2Bubiquintylation in meiosis in budding yeast and mice. Further studies are needed tocompletely figure out how H2B ubiquintylation affect meiosis.更多还原