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Higher order chromatin organization and function: a three-dimensional in situ analysis of structure, transcription and replication


Mapping and sequencing of individual human genes have advanced at a rapid pace. In order to fully understand gene regulation and safely control the expression of important human disease related genes in somatic gene therapy, it is not sufficient to know the regulatory and coding sequences of each gene and of individual factors involved in their expression. It is equally essential to understand the dynamic, three-dimensional nuclear organization and compartmentalization of higher order chromatin structures in relation to other nuclear domains representing the machineries for transcription, splicing, replication and repair. No single laboratory has the intellectual and methodological scope to tackle this problem on its own. In this proposal the expertise required for an interdisciplinary and integrated European effort is brought together by 10 laboratories from 5 European countries.

We will combine multi-colour fluorescence in situ hybridization (FISH) of chromatin structures (from the level of individual chromatin loop domains to the level of entire chromosome territories) together with the immunocytochemical localization of essential protein complexes, as well as in situ assays of nascent RNA synthesis. 3D-analyses will be undertaken at the light microscopic level, including newly developed microscope types with improved resolution and advanced 3D-image analysis procedures. 3D electron microscopic studies will supplement these light microscopic investigations. At the level of entire chromosomes we will perform measurements of the volume, shape and surface of their respective interphase territories and study the 3D-positioning of centromeric and telomeric subregions, as well as of selected individual genes. Model studies of normal human cell nuclei include the overall organization of the active and inactive X-chromosome territories and the three-dimensional topology of the major histocompatibility complex (MHC) in relation to the 3D-distribution of nascent RNA, replication clusters and specific protein domains. For comparison, the 3D-topology of aberrant higher order chromatin structures, such as homogeneously stained regions (HSR) and double minute chromosomes (DMC), will be studied in tumour cell nuclei.

The applicants expect that their combined efforts will (i) establish tools for 3D-gene mapping and 3D-genome analysis and (ii) provide a better understanding of the influence of higher order chromatin structures in situ on gene regulation and function in human normal and tumour cell nuclei. Such an approach will contribute to provide the necessary basis for an understanding of the topology and dynamics of specific, nuclear 3D-structures during development, differentiation and malignancy.

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Ruprecht-Karls-Universität Heidelberg
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80333 München

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