Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS


SMINSULATOR Sintesi della relazione

Project ID: 260787
Finanziato nell'ambito di: FP7-IDEAS-ERC
Paese: France

Final Report Summary - SMINSULATOR (Unveiling the Roles of Chromatin Insulators in Higher-order Chromatin Architecture and Transcription Regulation one molecule at a time.)

Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently organized by long-range contacts. Topological associating domains (TADs) are chromosomal regions within which genomic loci have a higher propensity to interact. TADs range from tens of Kb to Mb in size, and are constitutive structuring elements of eukaryotic chromosomes. Many regulatory proteins are thought to play a role in the regulation and maintenance of TADs. One class of chromatin regulatory proteins called insulator factors are predominantly positioned at barriers between TADs and have been shown to mediate directly or indirectly long-range DNA loops. In Drosophila, several types of insulators factors exist, with BEAF, CP190, chromator, and dCTCF being the most prominent at TAD barriers.
In this project, we set to develop single-molecule methodologies to investigate the overall roles and mechanisms of insulator factors in higher-order chromatin structure. First, we studied the network of insulator cofactors required to mediate long-range DNA interactions. The network of protein-protein interactions and the ability of each factor to bind DNA specifically was mapped biochemically. A novel biophysical approach based on fluorescence cross-correlation spectroscopy was developed to specifically determine the minimal set of proteins essential for bridging long-range intermolecular DNA interactions. Finally, we demonstrated by biochemical and genome-wide approaches the role of different insulator factors in the formation of DNA loops.
Secondly, we investigated the dynamics of folding and refolding of a TAD during the cell cycle. This TAD contains sequences required for replication initiation (therefore called origin TAD). We showed that the sub-cellular localization of the origin TAD is correlated with its folding status. Concomitantly with its unfolding, the origin TAD is relocated and replication initiation begins. Remarkably, these results indicate a functional correlation between TAD sub-nuclear localization, condensation state, and functional activity.
Thirdly, we studied how TAD borders are organized in the cell and the roles of insulator proteins in mediating higher-order interactions between different TADs by novel super-resolution microscopy methods. Importantly, we were able to correlate this organization with the epigenetic state of TADs. These results allowed us to conclude that TADs are functionally organized at the single cell level. Finally, we developed several new single-molecule localization microscopy and DNA labeling technologies to investigate nuclear organization in eukaryotic cells.

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