High resolution analysis of interchromosomal nuclear compartmentalization in situ and in vivo

为了在电子显微镜（EM）水平上研究细胞核中染色体的拓扑组织，在第一个资助期内制定了方案，以允许染色体和亚染色体区域的描绘。由于通过免疫染色观察BrdU结合后分离的染色体的方法显示出最好的结果，因此该方法将用于与其他标记物共同描绘染色体的实验，例如i)内源性rna；ii)异位表达的丝蛋白作为染色体间空间的标记；iii)由整合的lac-算子序列产生的染色体内区域，通过免疫电镜检测的lac-阻遏物- gfp融合蛋白可见。由于细胞核内和染色体间精细结构的完整性取决于水化状态，因此我们也将重点关注EM-cryotechnology的适应性，以最小的标本扰动获得最大的结构信息。预期的结果将对理解i)间期细胞核内的染色体组织和ii)高阶核结构和亚区隔化具有重要意义。更重要的是，这些分析将有助于评估基因组功能（如基因表达）与细胞核结构之间的关系。

In order to investigate the topological organziation of chromosomes in the cell nucleus at the level of electron microscopy (EM), protocols were elaborated within the fist funding period to allow delineation of chromosomes and subchromosomal regions. Since the method to visualize segregated chromosomes after BrdU incorporation via immunostaining revealed the best results, this method will be utilized in experiments co-delineating chromosomes with other markers, such as i) endogenous RNAs, nuclear bodies etc. as well as ii) ectopically expressed filament proteins as marker for interchromosomal space and iii) intra-chromosomal regions generated by integrated lac-operator sequences and visualized via lac-repressor-GFP fusion protein detected by immuno-EM. Since the integrity of the intra- and interchromosomal fine structure in the nucleus is dependent on the hydration state, we will also focus on the adaption of EM-cryotechnology to gain maximum structural information with minimum pertubation of specimen. The anticipated results will have important implications for the understanding i) of chromosome organization within interphase nuclei and ii) of the higher order nuclear architecture and subcompartmentilization. Even more importantly, these analyses will allow to assess the relationship between genome function, such as gene expression, and nuclear structure.