【作者】 林丕容；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 细胞与组织工程， 2010， 博士

【摘要】 Brpf1 (Bromodomain and PHD finger containing 1),也被称为Br140和Peregrin,是TrxG (Trithorax group)家族的新成员,是一个多结构域的蛋白。到目前为止,关于Brpf1的研究报导主要集中在染色质调控方面。研究表明BRPF1可以直接结合组蛋白,与活化的染色质相结合,募集组蛋白乙酰转移酶Moz乙酰化组蛋白,参与染色质重构。Rhox5是Rhox (reproductive homeobox genes on the X chromosome)同源异型框基因簇家族成员。Rhox5可特异性的在生殖系统中表达,例如在附睾、睾丸和卵巢中表达。研究表明Rhox5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥极有可能是与其他蛋白相互作用组成调控复合物来完成。在前期的实验中,以Rhox5蛋白为诱饵蛋白,筛选小鼠7天胚胎cDNA文库,获得了一个新的克隆2-31。以此为前提,本论文开展了以下三部分的工作：一、Brpf1及其新型转录本Brpf2的鉴定及表达谱分析从2-31克隆中抽提酵母质粒,转化大肠杆菌,最终经过序列测定后确定2-31克隆中所包含的基因序列为Brpf1基因的一种新的转录本,将其登陆GenBank,获得登录号DQ288860。使用RT-PCR和Northern blotting分析Brpf1和Brpf2的转录本在小鼠各组织中的表达情况,发现在大多数小鼠组织中(包括肝、胚胎、附睾、睾丸、卵巢、骨骼肌)中均检测到Brpf1和Brpf2两种转录本,而脾中仅检测到Brpf2转录本。表明Brpf1和Brpf2应该均具有生理学功能。在小鼠各组织中,Brpf1和Brpf2转录本在附睾、睾丸和卵巢中高表达,而在肝、胚胎、脾和骨骼肌中低表达,暗示Brpf1和Brpf2极有可能在生殖组织中发挥功能。生物信息学分析表明分析比对Brpf1和Brpf2的生物学特性,结果表明：Brpf1的mRNA全长4443bp,编码1246个氨基酸残基,其中包括2个PHD结构域、1个Bromodoamin结构域和1个PWWP结构域,预测分子量为140 kDa；而Brpf2的mRNA全长2747bp,但由于选择性剪接,导致终止密码子的提前出现,Brpf2蛋白仅编码442个氨基酸残基,丧失了参与染色质调控的功能性结构域Bromodoamin和PWWP结构域,仅保留了2个保守的PHD结构域,预测分子量为50 kDa。进一步扩增全长的Brpf1和Brpf2的mRNA序列,使用TNT体外转录翻译系统表达Brpf1和Brpf2的纯蛋白,其分子量为140 kDa和50kDa,与预测的分子量相符。二、Brpf1和Brpf2蛋白与Rhox5蛋白间的相互作用利用酵母双杂交实验确定单独的Brpf2并不能激活酵母双杂系统中的下游报告基因,交换载体,将Brpf2克隆至BD载体,而Rhox5克隆至AD载体,重复的双杂交实验亦证实Brpf2和Rhox5可以在酵母体内结合。将Rhox5蛋白与GST融合生成GST-Rhox5融合蛋白,融合蛋白在在终浓度为1 mmol/l的IPTG,37℃诱导培养6 h的条件下得到有效的表达,使用Glutathione-Sepharose 4B凝胶颗粒纯化后可以获得纯度较高的固着在Glutathione-Sepharose 4B凝胶颗粒上的GST蛋白和GST-Rhox5蛋白。体外的GST-pull down实验中,固着有GST-Rhox5蛋白的Glutathione-Sepharose 4B凝胶颗粒可以有效的pull down Brpf2蛋白,表明Brpf2可以在体外结合Rhox5蛋白。利用PCR方法扩增了Brpf1的CDS(coding sequence)序列,并分别将其克隆至BD载体和AD载体。酵母双杂交实验表明单独的Brpf1并不能激活酵母双杂系统中的下游报告基因,而Brpf1和Rhox5的共转化子则可以在SD/-Trp-Leu-His-Ade选择性培养基上生长,且表现β-半乳糖苷酶活性。这表明Brpf1和Rhox5可以在酵母体内结合。体外的GST-pull down实验中,固着有GST-Rhox5蛋白的Glutathione-Sepharose 4B凝胶颗粒可以有效的pull down Brpf1蛋白,表明Brpf1可以在体外结合Rhox5蛋白。三、Brpf1及Brpf2与Rhox5蛋白结合的关键结构Brpf2转录本编码的蛋白与Brpf1蛋白相比,丧失了Bromodomain和PWWP结构域,保留了两个保守的PHD结构域。鉴于两种蛋白Brpf1和Brpf2均可以结合Rhox5蛋白,因此推测有可能是两个PHD结构域的其中一个介导了两者与Rhox5蛋白的结合。为了验证Brpf1和Brpf2的PHD结构域是否是其与Rhox5蛋白结合的关键,分别构建了Brpf2 A、Brpf2 B和Brpf2 C突变体,其中Brpf2 A含有两个PHD结构域；Brpf2 B含有第一个PHD结构域,而Brpf2 C含有第二个PHD结构域。酵母双杂交实验和GST-pull down实验结果均表明Brpf1和Brpf2共有的第二个PHD结构域是其与Rhox5蛋白结合的关键结构,而且Brpf1和Brpf2与Rhox5蛋白间的结合极有可能是竞争性结合。同时,利用Rhox5蛋白的两个截短型突变体：不含homeodomain的Rhox5N和含有homeodomain的Rhox5C进行酵母双杂交实验和GST-pull down,结果表明Rhox5的homeodomain直接介导了其与Brpf1和Brpf2蛋白的相互作用。基于这样的实验结果,对于Brpf1和Brpf2与Rhox5蛋白功能性相互作用模式,我们可以提出这样的假设：Brpf1和Brpf2的第二个PHD介导了其与Rhox5蛋白的结合；当Rhox5与Brpf1结合时,Brpf1的Bromodomain和PWWP结构域被掩盖,不能与核小体结合；而当Brpf2与Brpf1竞争性结合Rhox5蛋白时,Brpf1构象发生改变,Bromodomain和PWWP结构域暴露,与组蛋白结合,使核小体构象发生改变,募集转录相关蛋白,促进基因转录。更多还原

【Abstract】 Brpfl (Bromodomain and PHD finger containing 1), also called Br140 or Peregrin, is a novel TrxG (Trithorax group) member with a central role during development. Brpf1 is a multidomain protein. It contains a unique combination of domains typically found in chromatin-associated factors, including PHD domain, bromodomain and PWWP domain. Brpf1 functions in association with the histone acetyltransferase Moz and promotes histone acetylation. Brpfl recruits Moz to distinct sites of active chromatin and remains at chromosomes during mitosis by binding to histone directly with its bromodomain and PWWP domain.Rhox5 is the founding member of the reproductive homeobox on the X chromosome (Rhox) gene cluster. It is selectively expressed in male and female reproductive tissues; moreover, mutational inactivation of Rhox5 in mice caused aberrant germ cell apoptosis, decreased sperm cell count, impaired sperm motility and subfertility. These observations together indicate a crucial role of RHOX5 in the development of reproductive tissues and spermatogenesis. However, the molecular mechanisms underlying RHOX5 functions remained unclear. In our previous research, we screened mouse 7 days embryo cDNA library using Rhox5 protein as a bait and discovered a novel clone 2-31. In this thesis, we carried out the following assays:1. Identification of a noval transcript of Brpf1, Brpf2, and study on its expression mapThe plasmid was extracted from yeast clone No.2-31 and then transfected into E.coli. After sequencing, the sequence was blast in NCBI and the results indicated that it was a novel transcript of Brpfl gene. This sequence was submitted to the GenBank and got the GenBank accession No. DQ288860。In order to map the expression level of Brpf1 and its noval transcript Brpf2, total RNA was isolated from seven types of mouse tissues separately. RT-PCR and Northern blotting results indicated that the expression of both Brpf1 and Brpf2 could be detected in most of the mouse tissue including liver, embryo, epididymis, testis, ovary and muscle. However, only Brpf2 transcript could be detected in spleen. This suggested that both Brpf1 and Brpf2 transcripts have some kind of physiological functions. Among the seven types of tissues, both Brpfl and Brpfs represented a higher expression level in epididymis, testis and ovary, while a lower expression level in liver, embryo, spleen and muscle. This suggested that Brpfl and Brpf2 may play an important role in the reproductive tisses. In order to understand the function of Brpf2, we analyzed the biological property of Brpf1 and Brpf2 by bioinformatics methods. The results indicated that the full length of Brpf1 mRNA is 4443 bp, encoding 1246aa. It is a multidomain protein, including two PHD domain, bromodomain and PWWP domain. The predicted Molecular Mass is 140 kDa. However, the full length of Brpf2 mRNA is 2747 bp and it only encodes 442 aa, partly owing to the creation of a new stop condon. Different from Brpf1, Brpf2 only has two PHD domains and the predicted Molecular Mass is 50 kDa. In order to confirm the actual Molecular Mass of Brpfl and Brpf2 protein, we amplified the full length of Brpf1 and Brpf2 mRNA and then obtained the pure protein of Brpfl and Brpf2 using a TNT T7 Quick Coupled transcription/translation reaction kit. The results demonstrated that the actual Molecular Mass of Brpfl and Brpf2 protein are 140 kDa and 50 kDa respectively.2. Study on the interaction of Brpf1/Brpf2 and Rhox5In order to confim the interaction between Brpf2 and Rhox5, the CDS sequence of Brpf2 was amplified and subcloned into the pGBKT7 vector. Yeast two hybrid assay indicated that Brpf2 could interact with Rhox5 directly in yeast. GST-RHOX5 fusion proteins were expressed in E. coli RosettaTM2 (DE3) cells induced with 1 mM IPTG at 37℃for 6 h and were purified effectively using the Glutathione-Sepharose beads. In Vitro GST-pull down assay, the beads coupled with purified GST-Rhox5 fusion protein can pull down the Brpf2 protein, which suggested that Brpf2 could bind Rhox5 in vitro.In order to confim the interaction between Brpfl and Rhox5, the CDS sequence of Brpfl was amplified and subcloned into the pGBKT7 and pGADT7 vector. Yeast two hybrid assay indicated that Brpf2 could interact with Rhox5 directly in yeast. In Vitro GST-pull down assay, the glutathione-Sepharose beads coupled with purified GST-Rhox5 fusion protein can pull down the Brpfl protein, which suggested that Brpf1 could bind Rhox5 in vitro.3. The critical structure for the interaction between Brpf1/Brpf2 and Rhox5 proteinCompared with Brpfl transcript, Brp2 loses the functional domain Bromodomain and PWWP domain. However, it keeps the two conserved PHD domain. Based on our previous study, both Brpfl and Brpf2 protein could interact with Rhox5 protein. Therefore, we postulate that one of the two PHD domains mediate their interaction with Rhox5.In order to verify whether the PHD domain mediates the Brpfl/Brpf2 interation with Rhox5, three types of Brpf2 trunctated mutants named Brpf2 A, Brpf 2B and Brpf2C were constructed. Brpf2 A includes two PHD domains; Brpf2 B includes the first PHD domain, while Brpf2C includes the second PHD domain. Two-ways yeast two-hybrid assay and GST-pull down assay suggested the second PHD domain is the critical domain for Brpfl/Brpf2 and Rhox5 interaction.In the meantime, we used two types of Rhxo5 tructated mutants, Rhox5N (N-terminal of Rhox5 protien, without homeobox domain) and Rhox5C (C-terminal of Rhox5 protein, including homeobox domain). Furthure yeast two-hybrid assay and GST-pull down assay suggested that the homeodomain of Rhox5 is the critical domain for Brpfl/Brpf2 and Rhox5 interaction.Based on the above results, we postulate:The second PHD domain of both Brpfl and Brpf2 mediates their interaction with Rhox5. When Rhox5 interacts with Brpf1, the bromodomain and PWWP domain of Brpfl will be shaded, which make Brpfl loses the ability of binding with nucleosomes. Wheras, when Brpf2 interacts with Rhox5, Brpf1 protein will be released. Therefore, the bromodomain and PWWP domain can intact with histone directly recruit the other transcriptional factors and then activate the gene expression.更多还原