? MAPPING TECHNOLOGY DEVELOPMENT The spatial organization of the genome inside the nucleus - and its functional relevance - can only be fully understood when viewed in in relation to the major nuclear compartments, including the lamina, nucleoli, pericentric heterochromatin, nuclear pores, nuclear speckles, among others. Here we will develop and apply several innovative methods to visualize and map the associations of the genome with these compartments, and to unravel their functional relevance. We will optimize and apply two unique methods (TSA and DamID) that each combine direct microscopy visualization with genome-wide mapping of nuclear compartmentalization. The complementarity as well as the intrinsic dual-mode nature of these methods greatly facilitates data interpretation and validation. TSA is a new method for the visualization and mapping of cytological distances of genomic loci to specific nuclear compartments. We will optimize the dynamic range, sensitivity, and tunable spatial resolution of TSA. We will also develop targets for mapping genomic distances to all major nuclear compartments, including lamina, nucleoli, pericentric heterochromatin, nuclear pore, and nuclear speckles, as well as specific chromosomal sequences. DamID is a method to visualize and map molecular contact with nuclear compartments. We will implement DamID for systematic mapping of genome interactions with the major compartments, and improve the time resolution of DamID to study better the dynamics of these interactions in interphase and after mitosis. In addition, we will use our powerful high-throughput integrated reporter technology (TRIP) and Repli- Seq method to unravel the functional importance of genome compartmentalization. We will develop a series of novel TRIP-based assays to produce genome-wide maps of chromosomal position effects on promoter function, gene induction kinetics, mRNA splicing, mRNA nuclear export. In addition, we will generate replication-timing maps with improved time and genomic resolution. Overlaying these maps with the TSA and DamID data will provide important clues about the functional relevance of nuclear compartmentalization. The outcome of this work will be (i) a series of standardized protocols and reagents for the in situ visualization and mapping of genome compartment contacts and distances; (ii) a set of new TRIP reporter constructs to independently query several key parameters of mRNA production; (iii) a Repli-Seq protocol with improved temporal and genomic resolution; (iv) comprehensive series of genome-wide maps that describe molecular and cytological proximities to the major nuclear compartments, as well as several key functional parameters. These datasets form a firm basis for the computational modeling of the Data Analysis and Modeling (DAM) module. The technologies and datasets generated here will not only have impact on our understanding of nuclear organization but also on many other areas of genome biology.
Showing the most recent 10 out of 25 publications
