The hierarchic control of nuclear differentiation - the ciliate model system

在这个项目中，我们将试图分析不同水平的控制参与核分化的stichotrichous纤毛虫Stylonychia，并试图解决的问题，他们如何相互影响。每当我们实验性地干扰一个途径时，我们将测试lES切除、DNA片段化和基因解读，并询问不同的其他途径如何受到影响。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。我们将通过特异性抑制剂或RNAi抑制DNA甲基转移酶，并测试抑制性组蛋白修饰是否仍然可以引入发育中的大核。将表征不同发育阶段期间各种组蛋白H3变体的表达，并试图通过吗啉代特异性地沉默它们的表达。组蛋白去乙酰化可以被曲古抑菌素A抑制，并测试DNA是否仍然可以重新甲基化。各种组蛋白修饰的分布和它们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，我们可能能够找到基因解扰的一般规则，实验测试它们，并尝试将IES切除的RNAi途径与基因解扰所需的模板引导途径联系起来。将通过高分辨率显微镜观察不同细胞核中各种序列类别的分布，并测试它们在核分化期间的重新定位。我们将看到DNA甲基化或组蛋白去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沉默/消除特定序列，以及在这个过程中如何标记潜在的活性基因。

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.