Regulation And Function Of Retroelements

逆转录因子的调控和功能

• 批准号: 6992793
• 负责人: Henry L. LEVIN
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6992793
• 关键词: Escherichia coli; Schizosaccharomyces pombe; bacterial proteins; complementary DNA; fungal genetics; genetic regulation; genetic transcription; human immunodeficiency virus 1; messenger RNA; oligonucleotides; plasmids; protein purification; transposon /insertion element; virus RNA; virus genetics; virus integration; virus replication

Diseases caused by retroviruses such as HIV-1 and the adaptation of retroviruses for use in gene therapy has intensified the need to understand how these viruses replicate. Primary objectives of ours are to understand how reverse transcription of viral mRNA occurs and how the cDNA products are integrated into the genome of infected cells. As a result of their similarity to retroviruses, LTR-retrotransposons are important models for retrovirus replication. The retrotransposon we study is the Tf1 element of the fission yeast, Schizosaccharomyces pombe. Tf1 efficiently reverse transcribes its mRNA and its integrase (IN) inserts the cDNA into the genome of S. pombe. Our studies of reverse transcription and integration are greatly facilitated by the techniques of yeast genetics. This approach not only allows us to identify features of the transposon critical for activity but host genes required for transposition can also be identified. The ease of culturing yeast also allows us to apply biochemistry to investigate the mechanisms we identify by genetics. The integration of HIV-1 cDNA shows a significant preference for actively transcribed genes. Similarly, the insertion of murine leukemia virus shows a strong preference for sites within 5kb of transcription initiation. Very little is known about how these viruses interact with the structures of chromatin and recognize transcription units. Our recent observation that the integration of Tf1 occurs specifically at pol II promoters presents the real opportunity to study in S. pombe an integration mechanism that parallels that of retroviruses. This year we explored the ability of Tf1 to recognize pol II promoters by creating an integration assay for specific target plasmids. We generated a plasmid with the ade6 gene of S. pombe and included it in a strain that was induced for Tf1 transposition. Fifty separate insertions into the target plasmid were isolated and analyzed. Ninety-five percent of the inserts occurred within a 160 nucleotide window in the promoter of ade6. This target assay clearly reproduced the promoter preference we observed with insertions into genomic sites. The insertions within the 160 nucleotide window exhibited a strong pattern of periodicity. Four narrow clusters of inserts were spaced approximately 30 bp apart. The window of 160 nucleotides corresponded with the amount of DNA wrapped around a single nucleosome and consistent with the possibility, the 30 bp pattern corresponded to the amount of DNA bound to the histone fold pairs within a single nucleosome. The IN of Tf1 contains Zn finger and catalytic domains similar to INs of retroviruses. Unlike other INs, the Tf1 IN possesses a chromodomain at its C-terminus. Chromodomains have been found to bind directly to histone H3 in specific nucleosomes. This suggests the possibility that Tf1 integration is mediated by an interaction between IN and specific nucleosomes at pol II promoters. Consistent with this model, we found that IN purified from bacteria bound specifically to histone H3 and not H2a, H2b, or H4. In addition, mutations in the chromodomain caused a significant reduction in transposition and a defect in recognition of the ade6 promoter. Other evidence that the chromodomain recognizes histones is that the insertions we isolated in the promoter of ade6 corresponded to a location where published data indicates a positioned nucleosome exists. To study the function of the chromodomain in vitro we developed biochemical assays for IN activity. We attached a six-his tag to the N-terminus of Tf1 IN and purified the protein from E. coli using Ni resin. The INs of retroviruses including that of HIV-1 are extremely insoluble and difficult to concentrate. We were surprised to find that the full-length IN of Tf1 was easy to purify and could readily be isolated in high concentrations. The INs of retroviruses possess processing activity that removes two terminal nucleotides from the 3? ends of the cDNA before insertion occurs. Based on the position of the minus strand primer, LTR-retrotransposons are not believed to possess processing activity. However our sequence data of Tf1 cDNA revealed 95% of the 3? ends had untemplated addtions. A 3? processing activity would remove these nucleotides and position the critical ?CA? dinucleotide at the ends of the cDNA where they must be located for integration to occur. We assayed the Tf1 IN for processing activity using oligonucleotides that mimic the ends of the cDNA. The IN had strong processing activity that removed two, three, four or five additional nucleotides from the 3? end of the oligos. This processing activity as observed in vitro suggests that the multiple nucleotides present the both ends of the cDNA could be removed by IN in vivo. This would allow integration of the most prevalent cDNA species we detected. We also tested the Tf1 IN for integration activity. We found the IN did have strong IN activity as indicated by the ability of the enzyme to insert oligonucleotides into each other. The resulting products were substantially larger than the original oligonucleotides. Oligonucleotides that mimicked the donor cDNA were altered to test whether Tf1 IN required specific sequences at the 3? ends. As is the case with other INs, Tf1 IN had a strong requirement for the dinucleotide CA at the 3? ends. The results of the processing and integration assays demonstrated that the IN of Tf1 has the same activities as retroviral INs and therefore is an excellent model for the IN of HIV-1. In addition, the high solubility of the Tf1 IN suggests that the protein may form crystals that could result in the first high resolution structure of an intact IN. This structure could contribute significantly to our understanding of retrovirus integration. We are pursuing this possibility in collaboration with the lab of David Davies. To explore the function of the chromodomain we expressed in E. coli Tf1 IN that lacked the chromodomain. This chromo minus IN (CH-) was assayed for integrase activities using the same artificial substrates described above. CH- possessed strong activities in both the processing and integration reactions. We were surprised to find that the enzyme lacking the chromodomain was about 10-fold more active that the intact IN. We also tested whether the chromodomain contributed to the recognition of the terminal dinucleotide at the 3? end of the donor DNA. The CH- protein exhibited a surprising relaxation of the sequence requirement at the ends of the donor DNA. Taken together, these results indicated that the chromodomain functioned as a negative regulator of integration and as a specificity factor for the donor DNA. One possibility that we are currently testing is that the chromodomain inhibits integration until it identifies a specific nucleosome at a pol II promoter. The chromodomain then binds H3 of that nucleosome. This alters the conformation of IN in a way that stimulates integration activity.

由HIV-1等逆转录病毒引起的疾病以及用于基因治疗中使用的逆转录病毒的适应性，加剧了了解这些病毒复制的需求。我们的主要目标是了解病毒mRNA的逆转录是如何发生的，以及如何将cDNA产物整合到感染细胞的基因组中。由于它们与逆转录病毒相似，LTR返回转座子是逆转录病毒复制的重要模型。我们研究的逆转录座子是裂变酵母菌的TF1元素。 TF1有效地逆转其mRNA及其积分酶（IN）将cDNA插入猪链球菌的基因组中。酵母遗传学技术极大地促进了我们对逆转录和整合的研究。这种方法不仅允许我们确定对活动至关重要的转座子的特征，而且也可以识别换位所需的宿主基因。培养酵母的易感性还使我们能够应用生物化学来研究我们通过遗传学识别的机制。 HIV-1 cDNA的整合表现出对主动转录基因的显着偏爱。同样，鼠白血病病毒的插入表现出对转录启动5KB的位点的强烈偏爱。对于这些病毒如何与染色质结构相互作用并识别转录单元的结构知之甚少。我们最近的观察结果，即TF1的整合在POL II启动子上特别发生，这是在S. Pombe中研究的真正机会，一种与逆转录病毒相关的整合机制。今年，我们通过为特定靶质粒创建集成测定法，探讨了TF1识别Pol II启动子的能力。我们用S. pombe的ADE6基因产生了质粒，并将其纳入了用于TF1转座的菌株中。分离并分析了50个单独的插入到靶质粒中。百分之九十五的插入物发生在ADE6启动子的160个核苷酸窗口中。该靶标测定清楚地复制了我们观察到的启动子偏好，并插入了基因组位点。 160个核苷酸窗口内的插入表现出强烈的周期性模式。四个狭窄的插入物分布约30 bp。 160个核苷酸的窗口与包裹在单个核小体周围的DNA量相对应，并且与可能性一致，30 bp模式对应于单个核小体内与组蛋白折叠对的DNA量相对应。 TF1的IN包含类似于逆转录病毒INS的Zn手指和催化域。与其他INS不同，TF1在其C-末端具有染色体。在特定核小体中，染色体构分已直接与组蛋白H3结合。这表明TF1整合是由POL II启动子IN和特定核小体之间的相互作用介导的。与该模型一致，我们发现在纯化的细菌中，特异性地结合了组蛋白H3而不是H2A，H2B或H4。此外，染色体群中的突变导致换座和缺陷，以识别ADE6启动子。其他证据表明，染色体域识别组蛋白的证据是，我们在ADE6启动子中分离的插入与已发表数据表明存在定位的核小体的位置相对应。为了研究体外染色体域的功能，我们开发了活性生化测定。我们在TF1的N端附加了一个六个HIS标签，并使用Ni树脂从大肠杆菌中纯化了蛋白质。包括HIV-1（HIV-1）的逆转录病毒的INS极其不溶性且难以集中。我们很惊讶地发现，TF1的全长很容易纯化，并且很容易在高浓度中隔离。逆转录病毒的INS具有处理活性，可以从3中去除两个末端核苷酸？发生插入之前的cDNA末端。根据减去链底漆的位置，LTR-返回转座子不被认为具有加工活性。但是，我们对TF1 cDNA的序列数据显示了3个？末端没有模拟的加法。 3？加工活性会去除这些核苷酸并定位关键？在cDNA的末端，必须将其放置以进行整合的二核苷酸。我们使用模仿cDNA末端的寡核苷酸来分析TF1进行处理。 IN具有强大的加工活性，从3中去除了两个，三个，四个或五个额外的核苷酸？寡聚的结尾。体外观察到的这种加工活性表明，存在的多个核苷酸可以通过体内在体内去除cDNA的两端。这将允许整合我们检测到的最普遍的cDNA物种。我们还测试了TF1的整合活动。我们发现，IN的活性确实具有很强的活性，这表明酶将寡核苷酸插入彼此的能力。所得的产品大大比原始的寡核苷酸大。模仿供体cDNA的寡核苷酸已改变以测试3在3中所需的特定序列中的TF1？结束。与其他INS一样，TF1在3处对二核苷酸CA有很强的要求吗？结束。处理和集成测定的结果表明，TF1的IN具有与逆转录病毒INS相同的活性，因此是HIV-1 IN的绝佳模型。另外，TF1在中的高溶解度表明蛋白质可能形成晶体，这可能导致完整的第一个高分辨率结构。这种结构可以极大地有助于我们对逆转录病毒整合的理解。我们正在与大卫·戴维斯（David Davies）的实验室合作追求这种可能性。为了探索我们在大肠杆菌TF1中表达的染色体构域的功能，其中缺乏染色体域。使用上述相同的人工底物分析了（CH-）中（CH-）中的Chromo减去（CH-）。在处理和整合反应中都具有强大的活性。我们很惊讶地发现缺乏染色体域的酶比完整的酶高10倍。我们还测试了染色体是否有助于3？供体DNA的结尾。 CH蛋白在供体DNA的末端表现出令人惊讶的序列需求的松弛。综上所述，这些结果表明，染色体构成是整合的负调节剂，也是供体DNA的特异性因子。我们目前正在测试的一种可能性是，染色体域抑制整合，直到它鉴定出Pol II启动子处的特定核小体为止。然后，染色体结合该核小体的H3。这以刺激整合活性的方式改变了构象。