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Tosi, Alessandro (2013): Dissection of the topology, structure and function of the INO80 chromatin remodeler. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



Eukaryotic genomes are organized into highly condensed chromatin. This packaging obviously impedes essential DNA mediated processes. ATP-dependent chromatin remodelers are therefore required to establish a dynamic chromatin environment. The chromatin remodeler INO80 is involved in various fundamental nuclear processes such as DNA repair, DNA replication and transcription. INO80 is thought to contribute to these processes by controlling genome wide levels of the histone variant H2A.Z. The INO80 chromatin remodeler is a macro-molecular complex composed of >15 subunits and a molecular mass of ~1.3 MDa. INO80 is found in human, fly and yeast. INO80 contains core subunits, which are conserved across species, as well as species-specific proteins. Not much was known about the organization of the INO80 subunits and their contribution to chromatin remodeling. Therefore, a hybrid approach was applied on yeast INO80 combining chemical cross-linking and mass spectrometry (XL-MS) (in collaboration with Franz Herzog, Ruedi Aebersold’s group, ETH, Zurich), electron microscopy (EM) (in collaboration with Caroline Haas, Roland Beckmann’s group, Gene Center, Munich) and biochemical analysis. For this, firstly the purification of INO80 was established. In order to yield sufficient amounts of highly purified and monodisperse complex, INO80 was purified endogenously from yeast by a combination of affinity and chromatography methods. In addition, nanobodies targeting the INO80 complex were generated that could yield even larger amounts of INO80 in the future. EM analysis revealed that INO80 is an embryo-shaped particle with a dynamic head-neck-body-foot architecture that can undergo large conformational changes. XL-MS unraveled the interaction map of the INO80 complex. The analysis of INO80 deletion mutants verified the observed interactions in vivo and proved the modular architecture of INO80. Additionally, the gained knowledge allowed the design and purification of stable and novel sub-complexes that could improve crystallization behavior. An integration of the results from different techniques deepened our understanding of the molecular architecture of INO80. The enigmatic subunits Rvb1 and 2 assemble as a dodecamer composed of two hetero-hexameric rings within the head of the INO80 complex. Rvb1/2 is flanked by the Swi2/Snf2 ATPase of Ino80 and the actin related protein (Arp) 5 in the neck. The Nhp10-module localizes to the body and the Arp8-module to the foot. Biochemical analysis showed that the Nhp10-module is a high affinity DNA/nucleosome binder. The Nhp10-module might together with the Arp8-module target INO80 to chromatin. The Arp5-module is catalytically crucial for nucleosome remodeling and senses the histone entity in chromatin. In order to map interaction sites to the substrate, INO80-nucleosome complexes were analyzed by XL-MS and were visualized by EM. Two-dimensional class averages showed that the nucleosome bound to the central groove of INO80 and was flanked by the head and foot module. The nucleosome was oriented in respect to INO80 as the H2A/H2B dimer- the moiety to be exchanged- was in contact with subunits situated in the neck. All INO80 modules contribute to nucleosome binding and the observed flexibility proposes a mechanism of how INO80 may remodel its substrate. This study established a structural and functional framework of these large remodelers. The investigation of the interaction with the checkpoint kinase Mec1 will contribute to the understanding of the obscure signaling of INO80.