The hierarchic control of nuclear differentiation - the ciliate model system

核分化的层次控制——纤毛虫模型系统

• 批准号: 162309276
• 负责人: Professor Dr. Hans Joachim Lipps
• 金额: --
• 依托单位: Institut für Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/162309276?language=en
• 关键词: hierarchic; control; nuclear; differentiation; ciliate

In this project we will try to analyze the different levels of control involved in nuclear differentiation in the stichotrichous ciliate Stylonychia and try to address the question how they influence each other. Whenever we experimentally disturb one pathway we will test for lES excision, DNA fragmentation and gene unscrambling and ask how the different other pathways are affected. Initially we will test the DNA methylation pattem of the different vegetative nuclei and its dynamics during nuclear differentiation. We will inhibit DNA methyltransferases either by specific inhibitors or by RNAi and test whether repressive histone modifications still can be introducecd in the developing macronucleus. The expression of the various histone H3 variants during different developmental stages will be characterized and tried to specifically silence their expression by morpholinos. Histone deacetylation can be inhibited by Trichostatin A and tested whether DNA still can become de novo methylated. The distribution of various histone modifications and their dynamics during macronuclear differentiation will be analyzed in more detail in several macronuclear-destined sequences in the micronucleus and the developing macronucleus as well as in micronuclear-specific sequences which are eliminated during differentiation. By isolating many more excision intermediates we may be able to find general rules for gene unscrambling, test them experimentally and try to link the RNAi pathway for lES excision with the template guided pathway required for gene unscrambling. The distribution of various sequence classes in the different nuclei will be visualized by high resolution microscopy and their relocalization during nuclear differentiation will be tested. It will be seen whether inhibition of DNA methylation or histone deacetylation has any effect on the dynamics in nuclear architecture. In summary, we hope to get a deeper insight how a cell manages to silence/eliminate specific sequences during differentation and how potentially active genes are marked in the course of this process.

在这个项目中，我们将尝试分析斯毛毛纤毛虫柱甲藻核分化中涉及的不同控制水平，并尝试解决它们如何相互影响的问题。每当我们通过实验扰乱一条通路时，我们都会测试 LES 切除、DNA 片段化和基因解读，并询问其他不同通路如何受到影响。首先，我们将测试不同营养细胞核的 DNA 甲基化模式及其在核分化过程中的动态。我们将通过特定抑制剂或 RNAi 抑制 DNA 甲基转移酶，并测试是否仍然可以在发育中的大核中引入抑制性组蛋白修饰。将表征不同发育阶段期间各种组蛋白 H3 变体的表达，并尝试通过吗啉代特异性沉默其表达。 Trichostatin A 可以抑制组蛋白脱乙酰化，并测试 DNA 是否仍然可以从头甲基化。各种组蛋白修饰的分布及其在大核分化过程中的动态将在微核和发育中的大核中的几个大核预定序列以及在分化过程中被消除的微核特异性序列中进行更详细的分析。通过分离更多的切除中间体，我们也许能够找到基因解读的一般规则，通过实验测试它们，并尝试将 LES 切除的 RNAi 途径与基因解读所需的模板引导途径联系起来。不同细胞核中各种序列类别的分布将通过高分辨率显微镜可视化，并且将测试它们在核分化过程中的重新定位。将会看到 DNA 甲基化或组蛋白脱乙酰化的抑制是否对核结构的动力学有任何影响。总之，我们希望更深入地了解细胞如何在分化过程中设法沉默/消除特定序列，以及在此过程中如何标记潜在的活性基因。