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

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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.