The hierarchic control of nuclear differentiation - the ciliate model system

点击翻译按钮获取中文摘要

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.