The hierarchic control of nuclear differentiation - the ciliate model system

在这个项目中，我们将尝试分析不同水平的控制涉及到的核分化在刺毛纤毛虫，并试图解决的问题，他们是如何相互影响。每当我们通过实验干扰一个通路时，我们将测试lES切除，DNA片段化和基因解码，并询问其他不同的通路是如何受到影响的。首先，我们将测试不同营养核的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来抑制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。我们将对不同发育阶段的组蛋白H3变体的表达进行表征，并试图通过morpholinos特异性地沉默它们的表达。组蛋白去乙酰化可以被曲古抑素A抑制，并检测DNA是否仍然可以重新甲基化。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的几个大核目标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码的一般规则，实验测试它们，并尝试将lES切除的RNAi途径与基因解码所需的模板引导途径联系起来。通过高分辨率显微镜观察不同序列类在不同细胞核中的分布，并测试它们在细胞核分化过程中的再定位。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将被观察到。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，以及在此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.